bluuebunny/crossref_metadata_2025 huggingface dataset:
Characterizing the plantazolicins: structure and discriminating activity of a novel class of natural products
Authors: Molohon Katie J , Melby Joel O , Lee Jaeheon , Evans Bradley S , Dunbar Kyle L , Bumpus Stefanie B , Kelleher Neil L , Mitchell Douglas A
Published: June 2021

The soil‐dwelling, plant growth‐promoting bacterium Bacillus amyloliquefaciens FZB42 is a prolific producer of complex natural products. A new FZB42 metabolite, plantazolicin (PZN), has been described as a member of the growing thiazole/oxazole‐modified microcin (TOMM) family. TOMMs are biosynthesized from inactive, ribosomal peptides and undergo a series of cyclodehydrations, dehydrogenations, and other modifications to become bioactive natural products. Using high‐resolution mass spectrometry, chemoselective natural product modification, genetic interruptions, and other spectroscopic tools, we have determined the molecular structure of PZN. In addition to two conjugated polyazole moieties, the amino‐terminus of PZN has been modified to Nα,Nα‐dimethylarginine. Interestingly, PZN exhibited a highly selective antibiotic activity toward Bacillus anthracis, but no other tested human pathogen. By altering oxygenation levels during fermentation, PZN analogues were produced that bear variability in their heterocycle content, which yielded insight into the order of biosynthetic events. Lastly, genome‐mining has revealed the existence of four additional PZN‐like biosynthetic gene clusters. Given their structural uniqueness and intriguing antimicrobial specificity, the PZN class of antibiotics may hold pharmacological value.
Nuclear Translocation of Phosphorylated Akt is Critical in Ellipticine‐Induced Apoptosis in Human Lung Epithelial Cancer Cells A549
Authors: Fang Kang , Wang Jing-Ping , Liang Huan-Chang , Yu Ya-Chu , Lin Kai-Han
Published: June 2021

Topoisomerase II inhibitor ellipticine and its analogues were reported as promising antitumor agents. We have reported previously that ellipticine induced nuclear translocation of p53 and Akt followed by autophagic apoptosis in human non‐small cell lung cancer cells A549. The effect can be reverted by autophagy inhibitors. In this work, we further demonstrated that transfection of p53 siRNA suppressed ellipticine‐induced apoptosis by blocking Akt nuclear translocation. In addition, introduction of either point‐mutated Akt473 or Akt308 inhibited p53 translocation as indicated by confocal microscopy and western blotting. On the other hand, in p53‐null H1299 cells with stably expressed wild type p53, cells become sensitive to ellipticine treatment accompanied with nucleus translocation of p53 and Akt. In addition, as cells were pretreated with autophagy inhibitor before being treated with ellipticine, Akt phosphorylation was suppressed with decreased apoptotic cell death. We concluded nuclear translocation of phosphorylated Akt is essential in leading to ellipticine‐induced cell death in human non‐small cell lung cancer cells.

The work is supported by grants from the National Science Council, Taiwan (NSC100‐2311‐B‐003‐003) and National Taiwan Normal University (99‐D3)
Structural insights into regulation of vertebrate homolog N‐acetylglutamate synthase/kinase from Maricaulis maris
Authors: Shi Dashuang , Li Yongdong , Cabrera-Luque Juan , Jin Zhongming , Yu Xiaolin , Zhao Genxiang , Allewell Norma M , Tuchman Mendel
Published: June 2021

Novel bifunctional N‐acetylglutamate synthase/kinases (NAGS/K) that catalyze the first two steps of arginine biosynthesis and are homologous to vertebrate N‐acetylglutamate synthase (NAGS), an essential cofactor‐producing enzyme in the urea cycle, were identified in Maricaulis maris and several other bacteria. The crystal structures of M. maris NAGS/K (mmNAGS/K) with and without L‐arginine bound indicate that mmNAGS/K functions as a tetramer, in contrast to the hexameric structure of Neisseria gonorrhoeae NAGS. Each subunit has an amino acid kinase (AAK) domain, which is likely responsible for N‐acetylglutamate kinase (NAGK) activity and has a arginine binding site, and an N‐acetyltransferase (NAT) domain that contains the putative NAGS active site. The angle of rotation between AAK and NAT domains varies among crystal forms and subunits within the tetramer. A rotation of 26° is sufficient to close the predicted AcCoA binding site, thus reducing enzymatic activity. Since mmNAGS/K has the highest degree of sequence homology to vertebrate NAGS of NAGS and NAGK enzymes whose structures have been determined, the mmNAGS/K structure was used to develop a structural model of human NAGS that is fully consistent with the functional effects of the 14 missense mutations that were identified in NAGS‐deficient patients.
The Effects of Leucine on Regulation of Glutamate Dehydrogenase by Purine Nucleotides
Authors: McMullan Rachel , Bell Ellis
Published: June 2021

The activity of glutamate dehydrogenase (GDH) is regulated by a complex interplay of homotropic ( cofactors ) and heterotropic (including purine nucleotides and leucine) allosteric effects. The aim of this study was to examine the allosteric regulation of GDH in an ”in vitro” context to more closely parallel cellular environments. Most previous studies of this complex enzyme have involved investigating the effects of single regulatory molecules on activity but the aim of the current project is to assess whether particular regulatory ligands modulate the effects of others. We examined the effects of pH, GTP, ADP and cofactor choice (NADP vs NAD) on leucine regulation of GDH activity. Kinetics studies showed that the activation by leucine on GDH had an optimum pH of 8, that cofactor NAD had greater activity than cofactor NADP, that GTP inhibited in the presence of both cofactors but was reversed differentially by leucine, and activity increased as cofactor saturation increased. To investigate the mechanism of these allosteric effects, CD in conjunction with thermal melts was used to analyze the stability of GDH in the presence and absence of leucine. The results analyzed using a 4 parameter Hill equation indicate that leucine significantly raises the Tm and the cooperativity of folding increases suggesting tighter coupling between different regions of the molecule.
Effect of the protein restriction on mitochondrial heart
Authors: Lagranha Claudia Jacques , Nascimento Luciana , Freitas Cristiane , Silva Alessandra , Silva-Filho Reginaldo , Fernandes Mariana P
Published: June 2021

Protein restriction during pregnancy has been associated with the development of adult diseases such as cardiovascular disease and obesity. Both epigenetic modifications and programming of the mitochondrial function have been recently proposed to explain how altered intrauterine metabolic environment may produce such disease. Given that our aim was evaluate the effect of maternal low‐protein diet in the mitochondrial heart from offspring. Treatment was performed during pregnancy and lactation, and was used only male offspring. At 90 days of age the animals from control and treated group were sacrificed, and heart was quickly removed and saved to biochemical analysis. We evaluate in mitochondrial heart: swelling, UCP3 expression, oxidative stress (Malondialdeyde‐MDA) and antioxidant enzymes (superoxide dismutase‐SOD and catalase activity‐CAT). Our results suggest that protein restriction induce significant alteration in oxidative stress biomarker and modulation of mitochondrial function. Taken together we believe that alteration in mitochondrial function induced by protein restriction during critical period of development could be associated with disease in adulthood. The present study receives financial support from FACEPE.
Biochemical Characterization of Pathogenic Mutations in Human Mitochondrial tRNA‐Ala and Alanyl‐tRNA Synthetase
Authors: Chihade Joseph , Her Koua , Steinbach Musetta , France Katherine
Published: June 2021

To understand the biochemical basis of pathogenic mutations in human mitochondrial (Hs mt) tRNAAla and Hs mt alanyl‐tRNA synthetase (AlaRS), we have examined the enzyme's tRNA substrate recognition. Hs mt tRNAAla lacks the G3:U70 wobble pair which is a conserved identity element in non‐mitochondrial alanine tRNAs. Aminoacylation by Hs mt AlaRS is insensitive to changes in the tRNA acceptor stem, including the G5:U68 wobble pair, and also insensitive to changes in the D‐stem‐loop, the T‐stem‐loop, and the anticodon loop. Aminoacylation rates are affected by changes in the variable loop‐anticodon stem region. Pathogenic tRNAAla mutations apparently do not directly interfere with AlaRS substrate recognition, but may promote RNA misfolding. Initial characterization of mutant tRNAs using in‐line probing shows evidence of aberrant folds. We are currently investigating the effects of recently discovered (Götz, et al., (2011) Am. J. Hum. Genet. 88, 635) pathogenic mutations in Hs mt AlaRS on RNA recognition and enzymatic function.
Development of a multiplex immunoassay for receptor signaling protein analysis
Authors: Wiese Rick , Harbison Stephen , Hayes David
Published: June 2021

Cellular signaling analysis is integral to a thorough understanding of living systems. Cellular analysis methods traditionally have involved the examination of single targets, but new techniques allow for multiplex analysis of proteins. MILLIPLEX® EpiQuant™ technology is an answer to multiplex limitations such as antibody cross‐reactivity and the inability to accurately quantify target protein levels. EpiQuant technology uses computational algorithms to identify unique peptide sequences in proteins. Antibodies generated against these sequences have pre‐defined target specificity. Total protein and protein phosphorylation measurements are made at the peptide level, using EpiQuant antibodies, following proteolytic fragmentation of samples. This process has a number of benefits including: a synthetic peptide can be used as a quantitative standard, cross‐reactivity with similar peptide sequences in other proteins can be predicted and avoided and multi‐site phosphorylated and total protein analysis of a single protein can be conducted in a single well. These attributes result in a superior methodology for cellular signaling multiplex analysis. We have utilized the EpiQuant technology to develop a multiplex assay consisting of 13 receptor signaling related protein targets, as well as, phosphorylation sites on these proteins, yielding a 27‐plex immunoassay. These total and phosphorylated protein targets include HER2, HER3, VEGFR1 and VEGFR2, among others. Here we demonstrate the use of this panel for the analysis of various stimulated tissue culture lysates.
Functionalized‐single‐walled nanotube (f‐SWNT)‐assisted in vitro delivery of the oncogene suppressor genes to cancer cells
Authors: Radominska-Pandya Anna , Karmakar Alokita , Bratton Stacie M , Xu Yang , Mahmood Meena , Ghosh Anindya , Biris Alexandru S
Published: June 2021

In this work, we have examined the possibility of delivering f‐SWNT‐DNA complexes containing p53 genes into MCF‐7 breast cancer cells. The major goal of these studies has been to develop protocols for efficient DNA binding and delivery into cancer cells by using the well‐characterized p53 gene as a model for cancer cell destruction. These studies have resulted in the successful generation of f‐SWCNTs with circular plasmid DNA attached to the outer surface, which, when introduced in vitro to cancer cell cultures, were found to induce apoptosis in a significantly larger percentage of the cells as compared to the nanotubes without plasmid DNA attached to their outside surface. The methods presented here can now be applied as delivery platforms for other genes of interest for various biomedical applications. We hypothesize that specific tumor suppressor genes can be specifically targeted to cancer cells using nanoparticles both in vitro and in vivo and can become an effective tool in cancer treatment. (NIH‐GM075893 to AR‐P; ASTA 08‐CAT‐03 to ASB).
A Role for Leukemia‐associated RhoGEF in the Final Stages of Cytokinesis
Authors: Martz Matthew Karl , Beeharry Neil , Yen Tim , Wedegaertner Philip
Published: June 2021

Mitosis requires the concerted effort of multiple RhoGEFs to properly undergo cytokinesis and complete cellular division. Here we show that Leukemia‐associated RhoGEF (LARG), a RhoA‐specific RGS‐RhoGEF, is required for abscission, the final stage of cytokinesis in which the intercellular membrane is cleaved between daughter cells. LARG co‐localizes with alpha‐tubulin in early mitosis followed by a translocation to the midzone and eventual co‐localization with RhoA at the midbody of the cytokinetic furrow. LARG translocates to particulate fractions during mitotic exit, paralleling that of Myosin 2, and returns to soluble fractions following completion of cytokinesis. Live cell imaging of HeLa cells siRNA depleted of LARG reveals a failure in abscission and retention of the intercellular bridge, resulting in a 6‐fold increase in abscission‐related apoptosis. In contrast to studies of other RhoGEFs, particularly Ect2 and GEF‐H1, LARG depletion does not result in cytokinetic furrow regression nor does its depletion affect internal mitotic timing. Abscission‐related apoptosis resulting from LARG depletion can be rescued by expression of siRNA‐resistant LARG. These results show that LARG is a novel and temporally distinct RhoGEF required for the completion of abscission.
Deacetylated αβ‐tubulin dimer acts as a positive regulator for Rheb GTPase
Authors: Lee Mi Nam , Koh Ara , Park Dohyun , Jang Jinhyuk , Kwak Dongoh , Jeon Hyeona , Kim Jaeyoon , Jung Heeyoon , Suh Pann-Ghill , Ryu Sung Ho
Published: June 2021

Ras homologue enriched in brain (Rheb) acts as a molecular switch to regulate diverse cellular functions such as cell growth and proliferation, cellular apoptosis, misfolded protein metabolism, and vesicle formation. Whereas the functional importance of Rheb is increased due to the continuous findings of its novel cellular functions, the regulatory mechanism of Rheb has not been fully understood. In this study, we suggest that αβ‐tubulin as a positive regulator for Rheb, identifying soluble αβ‐tubulin as a novel direct Rheb‐binding protein. Modulation of α‐tubulins levels by either its over‐expression or silencing affected the Rheb‐mediated mTORC1 activity due to the change in the GTP‐bound Rheb level. Contrary to acetylated tubulins, deacetylated form of tubulins showed a high binding affinity to Rheb and increased GTP‐Rheb levels, suggesting that deacetylated soluble αβ‐tubulin might function as a generating factor for GTP‐bound form of Rheb. Acetylated αβ‐tubulins were accumulated in mitosis, which resulted in the decrease of both Rheb‐tubulin interaction and GTP‐bound Rheb level. Constitutively active Rheb‐overexpressing cells showed an accelerated mitotic progression, which suggests that the αβ‐tubulins‐mediated regulation of GTP‐bound status of Rheb might be an important for the proper mitotic progression. Therefore, we propose that soluble deacetylaed αβ‐tubulins as a determinant for GTP‐bound Rheb level and as a temporal regulator of Rheb function which depends on its posttranslational modification subjected to cellular status such as cell cycle.
Perilipin 2 localization to the surface of lipid droplets
Authors: Logsdon Michelle M. , Brown Deborah A. , Listenberger Laura L.
Published: June 2021

Perilipin 2 binds to the surface of lipid droplets in most eukaryotic cells. However, the conditions that allow perilipin 2 to distinguish the lipid droplet surface from other intracellular organelles remain undetermined. Here, we describe the development of an in vitro assay to identify features of the lipid droplet that mediate perilipin 2 binding. We show that protease treatment of isolated lipid droplets does not prevent perilipin 2 binding, and that perilipin 2 can bind directly to synthetic liposomes (which lack proteins or a neutral lipid core). Moreover, the binding of perilipin 2 to synthetic liposomes is influenced by the phospholipid composition of these vesicles. Future experiments will further probe the characteristics of the lipid droplet surface that are required for perilipin 2 binding and determine if similar features of the lipid droplet surface mediate the binding of other lipid droplet‐associated proteins. This work was funded by the NIHGMS (LL and DB), the Research Corporation for Science Advancement (LL), and through the generous support of Dr. Elizabeth Nabel (ML).
Dynamic regulation of lipid droplets in the microalgae Chlamydomonas reinhardtii
Authors: TSAI CHIA-HONG , Benning Christoph
Published: June 2021

Under nitrogen deprivation the microalgae Chlamydomonas reinhardtii stores triacylglycerols (TAGs) in the lipid droplets. Upon resupply with nitrogen, TAGs are metabolized to generate energy and membrane building blocks needed by the cell. To understand how the lipid droplets remain intact in the presence of lipases and to determine factors required for the transition from lipogenesis to lipolysis, antibodies against the most abundant perilipin‐type protein in the Chlamydomonas lipid droplet (MLDP) were produced. Using these antibodies we were able to demonstrate the phosphorylation of MLDP and the possible recruitment of different sets of proteins to lipid droplets during lipogenesis and lipolysis. We also applied an antibody‐based dot‐blot technique to screen for the mutants unable to hydrolyze TAGs during lipolysis. One of the compromised‐hydrolysis‐of‐TAG mutants, cht7 showed 10‐fold higher TAG levels than the wild type after the induction of lipolysis. In addition, cell growth of cht7 under this condition was strongly delayed. The mutant phenotype suggests a direct coupling of lipolysis with cell cycle progression. Likely candidate genes responsible for the phenotype in this mutant were identified.

This research is sponsored by United States Air Force Office of Scientific Research.
Regulation of the alpha beta hydrolase domain (ABHD) protein family in murine and human obesity
Authors: Brown J. Mark , Martinez Kristina , Betters Jenna , Shores Nathan , Rudel Lawrence L , McIntosh Michael K , Thomas Gwynneth
Published: June 2021

In the search for factors that regulate obesity and diabetes, we have discovered a novel lipid‐signaling pathway involving the enzyme alpha beta hydrolase domain 5 (ABHD5), which plays a critical role in determining hepatic insulin sensitivity. While studying ABHD5, we noticed that several members of the ABHD family of enzymes have highly conserved GXSXG esterase motifs, as well as evolutionarily conserved acyltransferase motifs (HxxxxD), implicating the ABHD protein family in lipid synthesis, lipid catabolism, and signal transduction. Therefore, the goal of these studies was to examine the transcriptional regulation of ABHD enzymes in rodent and human models of altered triglyceride metabolism. We show that most ABHD enzymes are ubiquitously expressed, and several ABHD enzymes (1, 2, 3, 4, 5, 6, 11, 12, 13, and 14b) are regulated by high fat diet feeding in mice. Furthermore, most ABHD enzymes are upregulated in a time dependent fashion during human adipocyte differentiation. Finally, we have collected > 50 human liver biopsies from patients undergoing elective bariatric surgery as well as lean controls, and found that both ABHD6 and ABHD11 are significantly correlated with plasma lipid levels. These studies form the basis for ongoing efforts in our laboratory to characterize the biochemical and physiological role of individual ABHD enzymes in the development of the metabolic syndrome. These studies are funded in part by the American Heart Association (BGIA‐78400720)
Fatty Acid Uptake in the Early Branching Eukaryotic Pathogen Trypanosoma brucei
Authors: Paul Kimberly Sue , Vigueira Patrick , Winston Brette , McCall Barbara , Cain Sarah , Evans Holly , McCallister Jennifer
Published: June 2021

Trypanosoma brucei is a parasite that causes African Sleeping Sickness in humans. T. brucei synthesizes its own fatty acids (FAs), but also takes up FAs from its environment. Little is known about FA uptake in T. brucei. We used BODIPY‐conjugated fluorescent FAs and flow cytometry to monitor FA uptake in T. brucei. Uptake was linear with respect to time and concentration, and saturated only at high concentrations and long time points (80 μM, 40 min), suggesting a high uptake capacity. BODIPY‐FA uptake exhibited chain‐length specificity: C12 and C16 were taken up, while C2 and C5 were not. Surface trypsinization reduced C12 uptake by ~50%, suggesting involvement of surface protein(s). The T. brucei genome possesses few homologs to the known machinery, but they have 6 acyl‐CoA synthetases (ACS). ACS1‐4 are in a tandem array, are ~90% identical, and have overlapping chain‐length specificities (Jiang 2001 Biochem J 358:757). We knocked down ACS1‐4 using a panACS RNAi construct and observed a 40% and 70% reduction in C12 uptake after 2 and 6 days of induction, respectively. In contrast, panACS RNAi caused a 30% increase in C16 uptake after 4 days, suggesting compensatory upregulation of a distinct uptake pathway. panACS RNAi reduced cell growth 25% after 6 days. We propose there are at least two pathways for FA uptake: an ACS‐dependent pathway, and a second pathway, which may be mediated by cell‐surface proteins. (R15AI081207‐01A1)
Regulatory activation is accompanied by movements in the Cterminus of the Na‐K‐Cl cotransporter (NKCC1)
Authors: Monette Michelle Y , Forbush Biff
Published: June 2021

The Na‐K‐Cl cotransporter (NKCC1) is expressed in almost all cell types and is crucial in the regulation of cell volume and intracellular chloride concentration. To study the structure and function of NKCC1, we tagged the transporter with cyan (CFP) and yellow (YFP) fluorescent proteins at two sites within the C‐terminus and measured fluorescence resonance energy transfer (FRET) in stably expressing human embryonic kidney cell lines. Both singly‐ and doubly‐tagged NKCC1s were appropriately produced, trafficked to the plasma membrane, and exhibited 86Rb transport activity. When both fluorescent probes were placed within the same C‐terminus of an NKCC1 transporter, we recorded an 11% FRET decrease upon transporter activation with the phosphatase inhibitor, calyculin A. This result clearly demonstrates that regulatory activation in response to phosphorylation of the N‐terminus is accompanied by movement in the C‐terminus of NKCC1. When we introduced CFP and YFP separately in different NKCC1 constructs and cotransfected these in HEK cells, we observed FRET between dimer pairs, and the fractional FRET decrease upon transporter activation was 46%. Quantitatively, this indicates that the largest FRET‐signaled movement is between dimer pairs, an observation supported by further experiments in which the doubly‐tagged construct was cotransfectionally diluted with untagged NKCC1. Our results demonstrate that regulation of NKCC1 is accompanied by a large movement between two positions in the C‐termini of a dimeric cotransporter. We suggest that the NKCC1 C‐terminus is involved in transport regulation and that dimerization may play a key structural role in the regulatory process. This work was supported by NIH Grant GM083340 to BF.
Identification of the Structural Domains of Trypanosoma brucei Tim17 that is required for its function
Authors: Weems Ebony I. , Chaudhuri Minu
Published: June 2021

The import of nuclear encoded proteins is critical for mitochondrial functions. The protein translocators; TOM and TIMs, respectively, are multi‐protein complexes found in the outer and inner membranes of the mitochondria. There are three homologous components, Tim17, Tim22, and Tim23 that form two TIM complexes in higher eukaryotes. In contrast, Trypanosoma brucei, a parasitic protozoan that causes African trypanosomiasis, possesses a single protein, which closely resembles the Tim17 observed in other organisms. How T. brucei Tim17 (TbTim17) performs the functions of three different proteins in other eukaryotes remains elusive. Bioinformatic analysis revealed that although the secondary structure of TbTim17 is overall conserved, there is a significant divergence in its primary sequence that includes a segment near the N‐terminal region and a few stretches in the loop regions in between the predicted transmembrane domains. These divergent regions may perform trypanosome‐specific functions of TbTim17. To elucidate the important structural domain of TbTim17, we knockdown the expression of the endogenous TbTim17 by targeting the 3′UTR of its transcripts. TbTim17 RNAi inhibited cell growth and reduced the expression of TbTim17 protein more than 60%. Expression of a wild type TbTim17 fully complemented the knockdown effect of TbTim17. This in vivo system will be used to evaluate the effect of mutations on TbTim17 function.
Nucleotide sugar transporters of Trypanosoma brucei: glycosylation and infectivity
Authors: Liu Li , Xu Yu-Xin , Burleigh Barbara A. , Bangs James D. , Hirschberg Carlos B.
Published: June 2021

Trypanosoma brucei is the pathogenic agent of African trypanosomiasis. Sugars of surface glycoproteins play pivotal roles in parasite infectivity. A key step in glycosylation is transport of nucleotide sugars from the cytosol into the lumen of the Golgi apparatus by Nucleotide Sugar Transporters (NSTs). Mutations/deletions in these transporters have been shown to result in developmental diseases and impaired cell walls of yeast and pathogenic fungi. So far, no information about TbNSTs has been reported. Our goal is to understand how TbNSTs affect the pathogenicity of T. brucei by regulating the sugar modifications of its surface glycoproteins. We have identified four TbNSTs and characterized their substrate specificity by in vitro biochemical transport assays and phenotypic corrections of transport‐defective mutants such as K. lactis, MDCK cells and S. cerevisiae. Inhibition of TbNST4 gene expression in the procyclic‐form by RNAi resulted in underglycosylated procyclin, a major coat glycoprotein. Immunofluorescence staining also showed significant reduction of the surface procyclins in TbNST4‐silenced cells suggesting a transport defect and a change of surface glycoproteins. We are currently generating conditional TbNST knockout bloodstream‐forms to determine the parasite's virulence in a mouse infection model. Supported by NIH grants GM 30365 (CBH, LL and YXX), AI 35739 (JDB) and AI 090366 (BAB).
Unusual chemical and enzymatic stability of polysialic acid containing N‐glycolylneuraminic acid
Authors: Davies Leela , Pearce Oliver , Tessier Matthew , Assar Siavash , Smutova Victoria , Pshezhetsky Alexey , Woods Robert , Varki Ajit
Published: June 2021

The sialic acid N‐glycolylneuraminic acid (Neu5Gc) is present only at trace levels in the brains of all vertebrates tested to date, suggesting a detrimental effect of Neu5Gc. As a candidate mechanism for this potential toxicity, we hypothesized a reduced enzymatic breakdown of polysialic acid (PSA), a polymer of α2–8 linked N‐acetylneuraminic acid (Neu5Ac) found on the neural cell adhesion molecule (NCAM) and critical in many neurodevelopmental processes. We demonstrate that cultured neuroblastoma cells are able to incorporate Neu5Gc into PSA. Chemically synthesized polymers containing Neu5Gc exhibit resistance to the mammalian sialidase NEU1, an effect abolished by replacing the N‐glycolyl group with N‐propyl. Additional enzymatic and acidic conditions are being evaluated. Molecular modeling predicts similar tertiary structures of α2–8 linked Neu5Ac and Neu5Gc, but the presence of a glycolyl group creates a potentially stabilizing hydrogen bond with the adjacent carboxylate. This intrinsic stability of α2–8 linked Neu5Gc in PSA provides a potential explanation for the evolutionarily conserved absence of Neu5Gc from the vertebrate brain.
Biological features of human catalytic antibody light chains showing anti‐cancer activity
Authors: Uda Taizo , Hifumi Emi
Published: June 2021

The authors have reported about the human catalytic antibody light chains possessing catalytic activity, which was mostly encoded in Subgroup II in kappa type. In this study, the highly purified human light chains were submitted to investigate the function of the toxicity against cancer cells.

We designed two primer sets and performed a semi‐nested PCR using cDNA prepared from human leukocyte. As the results, eighteen clones of subgroup II germline gene of human kappa light chain were established. The mutants of C220A corresponding to the 18 clones were made. And the wild types and mutants were expressed in E. coli and the light chains recovered were highly purified by two‐step purification system. Out of 18 clones, #1 clone showed the cytotoxicity to both human lung (A548) and human stomach cancer cells (SNU‐1) but not to pancreas cancer cells (PANC‐1). Interestingly, the dimeric form showed the stronger effect than the monomeric form (C220A). The #1 clone could hydrolyze QAR‐MCA synthetic peptide showing the amidase activity. In addition, #1 clone has a unique amino acid sequence compared with that of other clones. Although it is still unclear that the relationship between catalytic activity and cytotoxicity of human light chain (#1 clone) at this moment, the catalytic triad structure of the clone may contribute the anti‐cancer effect.
Human Milk Oligosaccharides In Banked Donor Milk Compared To Mother's Milk In The NICU
Authors: Marx Carolin , Bridge Renee , Wolf Alison , Rich Wade , Kim Jae H , Bode Lars
Published: June 2021

Human Milk Oligosaccharides (HMO) are the third most abundant component of human milk. More than a hundred structurally distinct HMO have been identified, and the HMO composition varies between mothers as well as over the course of lactation. Newborn infants receive donor milk (DM) when their mother's milk (MM) is inadequate or unavailable. Our objective was to compare HMO content between DM and MM. We used HPLC‐FL analysis of fluorescently labeled HMO to analyze the variation in HMO amount and composition of 30 different batches of DM provided by the Mother's Milk Bank in San Jose, California, and compared it to MM samples donated by mothers of infants in our NICU. Concentrations and variations of lacto‐N‐tetraose, lacto‐N‐neotetraose, lacto‐N‐fucopentaose I and disialyllacto‐N‐tetraose were significantly lower in DM than in MM. Recent in vitro and animal studies have started to link individual HMO to infant health and disease, and our new data may suggest that HMO in DM does not always provide the same benefits as MM.
Maternal fucosyltransferase 2 genotype alters milk immunoglobulin A levels specific to some strains of norovirus
Authors: Taft Diana Hazard , Jiang Jason , Huang Peng-Wei , Davidson Barbara , Morrow Ardythe Luxion
Published: June 2021

Secretory immunoglobulin A (sIgA) found in mother's milk reflects her immune response to a lifetime of infectious challenge and helps protect infants from many infectious diseases, including noroviruses (NVs). Some NV strains preferentially infect secretors (Se) ‐ individuals with a functional copy of the fucosyltransferase 2 (FUT2) gene, those lacking a functional copy are non‐secretors (Non‐Se). We tested the hypothesis that maternal secretor status alters the levels of strain‐specific NV antibodies in milk. We analyzed the relative levels of milk sIgA against two strains of NV ‐ strain 387, which preferentially infects Se, and strain 207 which does not preferentially infect Se individuals. Strain‐specific and total sIgA were analyzed using ELISA in week 4 milk samples from 79 mothers. Maternal FUT2 genotype was analyzed by TaqMan assay to identify the 428G>A null mutation. The relationship between maternal secretor status and total, anti‐387, and anti‐207 sIgA was analyzed using Kruskal‐Wallis tests with Bonferroni correction. Non‐Se mothers had significantly lower levels of anti‐387 sIgA (p<0.001) in their milk. There were no significant differences in the amount of total or anti‐207 sIgA levels in milk based on maternal FUT2 genotype. This study indicates that milk sIgA levels can be influenced by genetics as well as history of infection.

Grant Funding Source: NICHD P01 HD 13021
Reducing the glycemic load of mixed meals reduces postprandial glycemia and insulinemia over the entire day but does not affect satiety
Authors: Liu Ann G , Most Marlene M , Brashear Meghan M , Johnson William D , Cefalu William T , Greenway Frank L
Published: June 2021

Low glycemic index/glycemic load diets may aid in body weight regulation through promotion of satiety. We evaluated the effects of mixed meals differing in glycemic index and carbohydrate content on postprandial serum glucose and insulin response, hunger and satiety over the course of a 12 hr day. In this randomized crossover trial, 26 overweight or obese adults received four diets in random order (HGI‐HC: high glycemic index, high carbohydrate; HGI‐LC: high glycemic index, low carbohydrate; LGI‐HC: low glycemic index, low carbohydrate; LGI‐LC: low glycemic index, low carbohydrate). Participants received breakfast, lunch, and dinner over the course of a 12 hr day. Primary outcomes were postprandial serum glucose and insulin quantified as area under the curve. Hunger, fullness, and satiety were assessed by visual analog scale. The HGI‐LC, LGI‐HC, and LGI‐LC diets significantly reduced glucose and insulin area under the curve compared to the HGI‐HC diet (P < 0.001 for all comparisons). There were no significant differences in ratings of hunger, fullness, or satiety between the different dietary treatments. Reducing the glycemic index or carbohydrate content of mixed meals is an effective method of controlling postprandial glycemia and insulinemia. This work was funded by General Mills.
Electron microscopy study of the alteration of P‐fimbriated E. coli adhesion to human uroepithelial cells
Authors: Kimble Lindsey L , Mathison Bridget D , Kaspar Kerrie L , Khoo Christina , Chew Boon P
Published: June 2021

Prevention of urinary tract infections by cranberry juice is attributed to inhibition of P‐fimbriated E. coli adherence to uroepithelial cells (UEC). This mechanism likely involves competitive binding of papG adhesins by bioactive compounds found in cranberry. We examined the effect of cranberry extract and procyanidins on E. coli fimbriation and adherence to human HTB‐4 UEC. E. coli were pre‐incubated with cranberry extract (2–10 mg PACs/mL), myricetin (0.001–0.01 mg/mL), procyanidin B2 (0.005–0.05 mg/mL) or human urine for 30 min and negatively stained for electron microscopy. Additionally, pre‐incubated E. coli were added to UEC cultures and incubated for 1 h. After washing, UEC were fixed, dehydrated and gold coated for imaging by scanning electron microscopy. E. coli grown on CFA and TSA both produced visible fimbriation; it appears that the fimbrae attached singly or in a branched manner to UEC. Pre‐incubation with cranberry extract, myricetin and procyanidin B2 inhibited E. coli adherence to UEC by altering fimbriae number, structure and distribution. Urine from subjects who consumed cranberry beverage also inhibited E. coli adhesion to UEC compared to subjects given a control beverage. Therefore, cranberry inhibits E. coli adherence to UEC likely by altering bacterial fimbriation. Funded by Ocean Spray Cranberries Inc.
Effect of fenugreek methanolic extract in inducing apoptosis through triggering proapoptotic genes
Authors: Alshatwi Ali A , Hasan Tarique N , Shafi Gowhar , Syed Naveed A , Khoja Kholoud K , Al-Hazzani Amal A , Alsaif Mohammed A , Lei David K. Y.
Published: June 2021

Fenugreek (Trigonella foenum graecum) is an active constituent of many traditional medicines to alleviate many diseases including cancer, diabetes, high cholesterol, wounds, inflammation, and gastrointestinal ailments. Since, the mechanism of action of several anticancer drugs is based on their ability to induce apoptosis, the objective of the study was sought to characterize the downstream apoptotic genes regulated by fenugreek methanolic extract (FME) in SiHa cells. FME effectively kills SiHa cells through induction of apoptosis. IC50 was recorded at 75μg/mL while apoptosis was confirmed by TUNEL assay and q‐PCR assays. Cells were exposed to 75μg/mL FME for 24 and 48 hours, and apoptosis was found in 26.2% and 35.1% cells respectively. Further, expressions of proapoptotic genes e.g. Caspase 3, 8, 9, p53, Fas, FADD, Bax and Bak were studied by q‐PCR. Caspase 3, 8, 9, p53, Bax and Bak were found to be increased significantly whereas expressions Fas and Fadd did not show any significant changes. In summary, the induction of apoptosis by FCE is affected by its ability to increase the expression of pro‐apoptotic genes such as Caspase 3, 8, 9, p53, Bax and Bak and it holds promise for consideration in complementary therapy in cervix cancer patients under chemotherapeutic interventions upon further investigation.
Uptake and metabolism of α‐mangostin by human cell lines: HepG2 liver cells, HT‐29 colon cells, and THP‐1 macrophage‐like cells
Authors: Orozco Fabiola Gutierrez , Chitchumroonchokchai Chureeporn , Riedl Kenneh M , Schwartz Steven J
Published: June 2021

Xanthones present in the pericarp of mangosteen fruit (Garcinia mangostana L.) possess a distinct isoprenylated tricyclic structure and have been reported to exhibit anti‐inflammatory and anti‐proliferative/ pro‐apoptotic activities. However, information on the absorption, metabolism and in vivo efficacy of xanthones remains elusive. Recent results from our lab show that ingested xanthones and their phase 2 metabolites are present in several tissues of mice. This study investigated the uptake and metabolism of α‐mangostin (α‐MG) by human liver, colon, and macrophage‐like cell lines. Free and conjugated (glucuronidated/sulfated) α–MG were analyzed by RP‐HPLC. Our results suggest that degree of uptake, metabolism and intracellular retention of α–MG is dependent on cell type. HepG2 readily accumulated free α–MG in cells. The conjugated α–MG was the majority detected in HT‐29 cells. α–MG content in THP‐1 cells was low and conjugates were predominant. Conversion of α–MG to other xanthones also was observed. LC‐MS‐MS analysis confirmed γ‐MG and 9‐ hydroxycalabaxanthone in HepG2 medium and cells. Other xanthones present in HepG2 and HT‐29 cells remain to be identified. These data suggest cell‐specific metabolism of α–MG. Mass spectrometric analysis and study of anti‐inflammatory activity of α–MG and metabolites are ongoing.

Grant Funding Source : OSU Food Innovation Center, Nutrient and Phytochemical Analytic Shared Resource and Conacyt Fellowship (Mexico)
Improving Bioavailability of 5‐Hydroxy Tangeretin by Food Grade Nanoemulsions
Authors: zheng jinkai , Li Yan , Charoensinphon Noppawat , Nutakul Wasamon , Dong Ping , Qiu Peiju , McClements David Julian , Xiao Hang
Published: June 2021

Our previous studies demonstrated potent anti‐cancer effects of 5‐hydroxy tangeretin (5HT, a citrus flavonoid). However, high hydrophobicity of 5HN results in poor bioavailability, which has greatly limited its utilization as a nutraceutical. Herein, we investigated the possibility of encapsulating 5HN within food‐grade nanoemulsions to improve its bioavailability. Three food‐grade emulsions with different droplet sizes (r = 67, 125 and 203 nm) were prepared to encapsulate 5HN by a high energy method. These delivery systems were then used to treat human colon cancer cells. HPLC analysis revealed that the nature of the delivery system had a strong influence on the cellular uptake of 5HN. There was poor uptake of 5HN from both bulk water and bulk oil, while cellular uptake of 5HN was much greater for all three emulsions, with the amount of 5HN uptaken increasing with decreasing droplet sizes. Consistent with these results, the viability of colon cancer cells was inhibited to a much greater extent by 5HN encapsulated in the emulsion systems with the smaller droplet sizes. This is presumably because a greater amount of the 5HN was uptaken into the cells where it could exhibit its inhibitory effect against the cancer cells. Our results suggest that nanoemulsions may increase the bioavailability of highly hydrophobic bioactivities such as 5HN, and facilitate their utilization in functional foods and pharmaceuticals.
Analysis of study length truncation in vitamin A compartmental analysis in human and rats
Authors: Gannon Bryan M , Howe Julie A , Green Michael H , Tanumihardjo Sherry A
Published: June 2021

Previous studies indicate that isotope dilution tests may not fully reveal total body vitamin A reserves, due to lack of time for dose to fully equilibrate with body stores. A healthy female was given an oral dose of 13C4‐retinyl acetate (17.5 umol), and blood samples were taken at baseline and over the course of 6 years. Serum retinol was analyzed for 13C/12C ratio by GC‐C‐IRMS, indicating fraction of dose in the plasma. Fraction of dose data are mathematically modeled using compartmental analysis. Different models were created to simulate a range of study‐lengths and total body reserves (TBR) and other kinetic parameters were calculated for each model. As length of study was increased, TBR was found to increase until it reached a maximum at around 500 days. Disposal rate was found to decrease as length of study increased until it reached a minimum at around 500 days. In agreement with non‐modeled isotope dilution, assumed absorption of dose is directly proportional to amount of total stores, indicating that even with compartmental analysis, accurate assessment of absorption is necessary for proper modeling and TBR calculation. To verify these conclusions with a larger sample size, data from an isotope dilution study in rats is being treated to a similar truncation of study length. These results indicate that there are slower turning over pools of vitamin A that would not be detected with a shorter study length.

Grant Funding Source : NIH‐NIDDK 61973
Do Intakes of 250–500mg/d of EPA and DHA Increase Blood Biomarkers?
Authors: Patterson Ashley C. , Chalil Alan , Charkhzarin Payman , Aristizabal Henao Juan J. , Streit Isaac , Ciobanu Flaviu , Stark Ken D.
Published: June 2021

Intakes of 250–500 mg/d of eicosapentaenoic acid (EPA, 20:5n‐3) and docosahexaenoic acid (DHA, 22:6n‐3) have been proposed for health benefits. There is little data examining the response of omega‐3 blood biomarkers to these dietary intakes as most intervention studies use higher doses of EPA+DHA. Presently, we examine the blood biomarker response of men (n=11) and women (n=10) after the consumption of 250mg/d of EPA+DHA and 500mg/d EPA+DHA for 4 week periods. Erythrocytes and plasma were collected at baseline, week 4 and week 8 and fingertip prick whole blood was collected weekly. Intake of EPA+DHA at baseline was 72±51mg/d as determined by food frequency questionnaire validated for n‐3 HUFA (highly unsaturated fatty acids, ≥20 carbons, ≥3 carbon‐carbon double bonds). Baseline %n‐3 HUFA in total HUFA was 24.0±2.0 and 22.3±3.3 in venous erythrocytes and plasma, respectively. Initial results show the %n‐3 HUFA in total HUFA in fingertip prick whole blood increased from 22.1±2.1% at baseline to 24.3±1.6% at week 1 and to 25.2±1.9% at week 2 (p<0.01). These results will indicate if 250–500mg/d of EPA+DHA can increase blood levels of EPA+DHA to levels associated with the prevention of sudden cardiac death. Supported by a doctoral research award (ACP) from the Canadian Institutes of Health Research.
The Candidate Tumor Suppressor Gene Ecrg4 Inhibits Proliferation of the Inflammed Mucosal Epithelium
Authors: Kurabi Arwa , Dang Xitong , Pak Kwang , Coimbra Raul , Eliceiri Brian , Baird Andrew , Ryan Allen
Published: June 2021

Mucosal membranes play an important role in innate and adaptive immunity acting as physical and biological barriers that prevent and regulate the response to infections. Using whole‐genome gene arrays, we identified a small number of regulatory genes that are differentially expressed during otitis media (OM), a mucosal epithelial proliferative response to infection in the middle ear (ME). One such gene is Esophageal cancer related gene 4 (Ecrg4), a candidate tumor suppressor that we found is normally expressed in the quiescent ME. We found that Ecrg4 expression is strongly down‐regulated during the proliferative phase of infection but recovers as mucosal hyperplasia recedes. When tested in vitro, mucosal epithelial proliferation and migration was reduced by Ecrg4 over‐expression when hyper‐proliferating mucosal epithelial cells were harvested from infected ME. When the analogous experiment was performed in vivo, we observed a decrease in the thickness of the mucosal epithelial layer and a reduction in inflammatory infiltrate entering the ME cavity 48 hrs after infection. Together these data suggest that the down‐regulation of Ecrg4 gene expression after infection plays a determinant function in mucosal epithelial hyperplasia and are the first that point to the value of a therapeutic strategy targeting Ecrg4 in the inflammation‐immunity cascade that precipitates mucosal epithelial proliferation.
Overexpression of β–catenin and EphB4 in Helicobacter pylori infected Mexican children with gastritis
Authors: Villarreal-Calderon Rodolfo , Mackinnon Alexander , González-Luévano Arturo , Aragón-Flores Mariana , Suster Saul
Published: June 2021

Chronic inflammation and infection are major gastric carcinogenic risk factors. As Helicobacter pylori (HP) infection is common in children, determining if HP infection is an early event in gastric carcinogenesis identifies a potential therapeutic target for reducing gastric cancer (GC) in these patients. Gastritis activity, atrophy, follicular pathology (FP), intestinal metaplasia (IM) and expression of 9 biomarkers (Ephrin Type‐B Receptor 4 [EphB4], Activation‐Induced Cytidine Deaminase, Caudal Type Homeobox 2 [Cdx2], p53, Macrophage Migration Inhibitory Factor, Matrix Metalloproteinase 3, α‐Methylacyl‐CoA Racemase, β‐catenin, Ecadherin) involved in gastric carcinogenesis and progression were examined in antral biopsies from 36 HP+ and 46HP‐ Mexican children (mean age 8.1y) with chronic gastritis. EphB4 expression (p=0.008), β‐catenin staining intensity (p=0.002), activity, atrophy and FP (p=0.0002, p<0.0001 and p=0.0007) significantly correlated with HP+ biopsies. 6 children (3 HP+) demonstrated aberrant Cdx2 expression without histological evidence of IM. Antral biopsies demonstrating preneoplastic markers may identify high risk patients who would benefit from anti‐HP therapy and closer follow up surveillance. R.V.C. recognizes generous support from FASEB and ASIP Minority Access to Research Careers Summer Research Opportunities Program in Pathology.
VEGF induces RANKL in Rheumatoid Arthritis synovial fibroblasts
Authors: Kim Kyoung-Woon , Kim Hae-Rim , Kim Bo-Mi , Cho Mi-La , Lee Sang-Heon
Published: June 2021

BACKGROUD

VEGF is the most endothelial cell‐specific angiogenic factor characterised to date. Several recent reports have demonstrated that VEGF is also implicated in the pathogenesis of RA, and serum levels of VEGF correlate well with RA disease activity, particularly with swollen joint counts.

OBJECTIVE

This study aims to determine the effect and mechanism of VEGF on bony destructive process in RA through RANKL regulation from synovial fibroblasts.

METHOD

Synovial fibroblasts were isolated from synovial tissues of RA patients. After RA synovial fibroblasts were treated with rhVEGF, the expression of RANKL mRNA was determined using real‐time PCR. After inhibition of pro‐inflammatory cytokines or intracellular signal molecules, the change of the VEGF‐induced RANKL expression was determined using real‐time PCR.

RESULTS

The expression of RANKL mRNA in RA synovial fibroblasts was increased after VEGF stimulation in a dose dependent manner. VEGF‐induced RANKL expression was also diminished after inhibition of Src, PKC and ERK.

CONCLUSION

Our results show that VEGF upregulates RANKL expression in RA synovial fibroblasts. These effects are mediated by the Src/PKC and ERK pathways.

This work was supported by the National Research Foundation of Korea Grant funded by the Korean Government (NRF‐2010‐R1A4A007‐0024198)