bluuebunny/crossref_metadata_2025 huggingface dataset:
Distribution of mitochondrial chaperonins in lung cells
Authors: Cappello Francesco , Corrao Simona , Anzalone Rita , Magno Francesca , Loria Tiziana , Iacono Melania Lo , Farina Felicia , Zummo Giovanni , de Macario Everly Conway , Macario Alberto JL , La Rocca Giampiero
Published: June 2021

Hsp60 and Hsp10 are considered typical mitochondrial chaperonins, residing inside the organelle, but works in the last years showed they can also be outside mitochondria and cells. The exact extramitochondrial locations of the chaperonins in the various normal cells and tissues under diverse conditions remain to be ascertained. Consequently, we undertook to examine the subcellular distribution of Hsp60 and Hsp10 in lung cells in order to provide a baseline for comparison between normal and pathological tissues. We used a battery of methods (light and electron microscopies, immunocytochemistry, subcellular fractionation‐purification plus electrophoresis, Western blotting) to study two cell types, lung fibroblasts and epithelial cells, unstressed and exposed to the stressor cigarette smoke extract, which is pertinent to lung pathology. Here we focus on Hsp10, which was found in the mitochondria, cytosol, and nucleus, with their levels changing after stress. Nuclear Hsp10 has not been reported before and was demonstrated in various ways, including immunoelectromicroscopy with specific antibodies.

Grant Funding Source: University of Palermo
Identification of genes regulating recombination of repetitive DNA
Authors: Chadha Akash , Lee Thomas , Scheifele Lisa
Published: June 2021

Transposons mold the genome by transposition and through subsequent recombination between the dispersed repeats. Recombination between Ty1 elements can produce chromosome aberrations in yeast. We previously developed a unique set of Retrotransposon Overdose (RO) strains which contain 3 to 10‐fold more Ty1 elements (Scheifele, PNAS 2009). We used RO strains to screen for genes controlling Ty1 recombination and identified DNA replication, repair, and cohesion genes that, when deleted, produce growth defects specific to RO strains. To confirm that these growth defects are due to increased recombination, we used a strain of yeast with a marked Ty1 element, individually deleted the genes identified in our screen, and measured the rate of marker loss; deletion of genes that control Ty1 recombination should increase loss of the genetic marker. Further, by comparing the frequency of marker loss, we can determine which genes have the greatest role in preventing recombination between Ty1 elements. While most of the gene deletions increased Ty1 recombination, the magnitude of their effects varied. In addition, we observed variation in the type(s) of recombination affected, with differential effects on recombination between transposons, between direct repeats, and between lys2 heteroalleles. This specificity suggests that there may be distinct mechanisms to regulate recombination between repetitive DNA sequences.
Characterization of mutant alleles of RTT105 in Saccharomyces cerevisiae
Authors: Melber Andrew J , Keeney Jill B.
Published: June 2021

Saccharomyces cerevisiae, commonly known as baker's yeast, contains several retrotransposons within its genome, including the Ty1 element. There are many host genes that have been shown to regulate Ty1 retrotransposition, including RTT105; however, the host function of this gene is unknown. RTT105 was originally identified as a negative Ty1 regulator by the insertion of the bacterial transposon, Tn3, creating a disruption at the 212bp of the 627bp gene. The resulting mutant allele, rtt105::Tn3‐LacZ/Leu2, increases transposition of an endogenous Ty1 element. However, our lab isolated the RTT105 gene in a screen of genes that when over‐expressed, increased retrotransposition of a galactose inducible Ty1 element on a 2‐micron (high copy) plasmid. We have shown that a complete deletion of RTT105 results in dramatically less galactose‐induced Ty1 retrotransposition. From this, we hypothesize that the Tn3 allele does not nullify gene function but rather enhances it. Real‐time quantitative PCR and northern blots are being used to determine expression levels of the wild type and rtt105::Tn3‐LacZ/Leu2 allele from RNA extracts of the strains. Since the Tn3 insert is over 7kbp in size, disruption mutants at the same 212bp location are being made with auxotrophic markers varying in length. This will help determine whether expression of the N‐terminal region of RTT105 specifically inhibits transposition or whether sequences within Tn3 may be impacting RTT105 expression. The various insertion mutants will be assayed for retrotransposition and RNA extracts will be used to look at expression levels of the mutant rtt105 alleles. Additionally, we are purifying the Rtt105 protein for antibody production by expressing in E. coli. This research is supported by NIH grant GM074658‐01 to JBK.
Presence of Left‐Handed Z‐DNA in the Epidermis of Melanoma Tissue
Authors: Gagna Claude Eugene , Lambert W Clark
Published: June 2021

Melanoma is a malignant tumor of melanocytes. The frequency of melanoma has continued to increase considerably over the past few decades. New classes of drugs need to be produced that can be used to treat and cure this disease. Z‐DNA is an alternative form of right‐handed double‐stranded (ds‐)‐B‐DNA, which plays a role as a transcriptional enhancer, RNA editing and recombination. We have examined the epidermis of normal human skin for the presence of left‐handed ds‐Z‐DNA. Z‐DNA has been found in the epidermis of normal human skin; it undergoes destruction as these cells are displaced from the stratum basale to the stratum corneum, viz., cell death [i.e., apoptosis and terminal differentiation (denucleation)]. The highest and most consistent levels of Z‐DNA are found in the stratum basale of normal human skin. However, in skin with melanoma, Z‐DNA is not reduced in epidermal cells as they are moving upward to form corneocytes. Increased levels of Z‐DNA have been found in the upper regions of the stratum spinosum and stratum granulosum. Being able to locate Z‐DNA in epidermal genes will allow for additional target sites to produce new classes of drugs, viz., based on B‐DNA to Z‐DNA transitions. This research project was supported in part by a NYIT ISRC grant.
Repeat expansion in intron 1 of the Frataxin gene reduces transcription initiation in Friedreich ataxia
Authors: Kumari Daman , Biacsi Rea Erika , Usdin Karen
Published: June 2021

Expansion of a GAA‐repeat tract in the first intron of the frataxin (FXN) gene causes an mRNA deficit that results in Friedreich ataxia (FRDA). The region flanking the repeat on FRDA alleles is more extensively methylated than normal alleles and is enriched for repressive histone modifications. However, whether these epigenetic changes are responsible for the mRNA deficit is controversial. We show here that certain marks of active chromatin are also reduced in the promoter region of the FXN gene in patient cells. Thus, the promoter chromatin may be less permissive for transcription initiation than it is on normal alleles. Furthermore, we show that the initiating form of RNA polymerase II and histone H3 trimethylated on lysine 4, a chromatin mark tightly linked to transcription initiation, are both present at lower levels on FRDA alleles. Our data thus supports the idea that repeat‐induced chromatin modifications reduce transcription initiation in FRDA. Our findings may have implications both for understanding the mechanism responsible for FRDA as well as for therapeutic approaches to reverse the transcription deficit.
H2B K123 ubiquitination and H3 K4 methylation play a positive role in DNA replication
Authors: Faircloth Lindsay Michelle , Dorn Elizabeth , Carlile Candice , Cook Jean Gowan
Published: June 2021

DNA replication is initiated from sites in the genome known as origins of replication. In eukaryotic cells, control over the function of origins is essential to ensure complete genome replication, and its loss results in genome instability. Despite the importance of controlling origin function, little is known about the determinants of origin identity or the effects of chromatin structure on origin function. We hypothesize that unique elements of chromatin structure are responsible for defining functional origins and regulating replication initiation. Our objective is to characterize the effects of histone modifications on origin function using genetic and biochemical analyses. We have conducted a genetic screen to identify chromatin‐modifying enzymes that affect the licensing of origins. We discovered that loss of the Bre1 E3 ubiquitin ligase compromises the growth of a replication mutant. This enzyme, in conjunction with the E2 ubiquitin‐conjugating enzyme, Rad6, monoubiquitinates H2B K123, and this mark is a pre‐requisite for H3 K4 methylation. We are analyzing the mechanism by which these marks affect DNA replication. We will also determine whether the presence/absence of these marks affects recruitment of replication proteins, origin firing, or replication fork progression. These studies will provide a mechanistic view of how these and other histone modifications regulate DNA replication. (AHA 10PRE3740013, NIH 5R01GM083024‐02)
Exploring the role of the MLL1 complex in leukemia by small molecule targeting of an essential protein‐protein interaction
Authors: Townsend Elizabeth , Dou Yali
Published: June 2021

MLL1 is a histone H3 lysine 4 (H3K4) specific methyltransferase that has essential functions in embryonic development, hematopoiesis and leukemia. Dysregulation of MLL1 activity, by mutation, translocation or amplification of one copy of the MLL1 gene, has been found to play an essential role in many cases of acute leukemia with poor prognosis. In most cases of MLL1‐associated leukemia, MLL1 wild‐type functions coordinately with the mutant copy of MLL1 to aberrantly overexpress MLL1 target genes, specifically HoxA9 and Meis1. Overexpression of these genes is essential for leukemogenesis. In the cell, MLL1 activity is upregulated by stable association of MLL1 with the proteins WDR5, RbBP5 and Ash2L. In this stable enzyme complex, WDR5 serves as the structural platform to mediate complex assembly and facilitate the interaction of RbBP5 and Ash2L with MLL1. We hypothesize that small molecule targeting of the MLL1:WDR5 interaction will downregulate MLL1 activity and provide a therapeutic avenue to treat MLL1‐involved leukemias. To this end, we have developed a potent, peptidomimetic inhibitor of the MLL1:WDR5 interaction. We have found that treatment of leukemia cell lines with this inhibitor downregulates expression of MLL1 target genes and has moderate inhibitory effects on cell viability in leukemia‐derived cell lines.
Real‐time Imaging of Transcriptional Elongation
Authors: Shin Ilchung , Gupta Vinayak , Kraus George , Nilsen‐Hamilton Marit
Published: June 2021

Transcription is a key regulatory step in gene expression that involves initiation, elongation, cotranscriptional modifications, termination and posttranscriptional modification. Gene expression has long been known to be regulated at transcription with recruitment of RNA polymerase II (RNAPII) to the transcription initiation complex. But, there is also evidence that the rate of elongation varies between genes. The purpose of this study is to develop a new technology to measure the elongation rate of RNAPII in vivo and in real‐time. For this technology, IMAGEtags (Intracellular MultiAptamer Genetic tags), encoded in the initial and/or terminal regions of a gene that is expressed from a promoter of choice, are utilized as markers. IMAGEtags are strings of RNA aptamers that have high affinity and specificity for their ligands. For imaging, the cells are incubated with aptamer ligands that are labeled with fluorescent dyes. To increase signal/noise, ligands have been separately conjugated with the FRET pairs, Cy3 and Cy5. To measure the elongation rate, IMAGEtags have been inserted in the initial and/or terminal regions of YLR454 gene after the GAL1 promoter. IMAGEtags are then used to monitor RNAPII passage through defined positions in the gene as it transcribes in living cells in culture. This work was supported by the NIH and the Biological Systems Science Division, Office of Energy Science Research of the U. S. DOE.
miR‐16 and HuR co‐regulate cyclin E1 mRNA in breast cancer cells
Authors: Hartley Rebecca S , Guo Xun
Published: June 2021

HuR is an RNA binding protein implicated in tumorigenesis. HuR stabilizes cyclin E1 mRNA in breast cancer cells, contributing to its overexpression. HuR and cyclin E1 promote proliferation, inhibit apoptosis, and are overexpressed in 30% of breast cancers, where they are associated with highly aggressive cancers. miR‐16 is a tumor suppressor microRNA downregulated in breast cancer that functions by targeting multiple oncogenes, including cyclin E1. Our objective was to ask if HuR and miR‐16 coordinately regulate cyclin E1 in breast cancer cells. We overexpressed or knocked down HuR in the presence of miR‐16 precursor or antagomiR and assessed effects on cyclin E1. Introducing miR‐16 precursor into breast cancer cells of luminal vs. basal subtype, decreased cyclin E1 protein while miR‐16 antagomiR increased it. miR‐16 effects were caused by cyclin E1 mRNA destabilization. Knockdown of HuR did not alter the effects of miR‐16 on cyclin E1, while miR‐16 precursor blocked HuR‐mediated upregulation of cyclin E1. These results suggest that miR‐16 keeps cyclin E1 and proliferation in check, with decreased miR‐16 enabling HuR upregulation of cyclin E1. miR‐16 had no effect on HuR, despite a miR‐16 target site in its mRNA. We are currently testing if miR‐16 RISC interferes with HuR binding to cyclin E1 mRNA. Support: NIH R01 CA095898, UNM Cancer Center Inflammatory Breast Cancer Grant and Rauth Family Basic Scientist Award.
Novel transcript variants arise from alternate promoter usage and alternative splicing in 5′ regions of cAMP‐dependent Protein kinase A subunit genes in mouse
Authors: Banday Abdul Rouf , Azim Shafquat , Tabish Mohammad
Published: June 2021

Protein kinase A (PKA) is composed of four membered structure with two regulatory (R) and two catalytic (C) subunits. There are four different R subunits (Iα and Iβ, IIα and IIβ) and two different C subunits (Cα and Cβ) in mouse, each encoded by separate gene. Earlier, few alternative spliced variants have been identified with tissue specific function. The objective of this study was to analyze splicing events in 5′ region of R and C subunit genes of PKA in mouse.

Analysis was done with the help of combinatorial approach of bioinformatics tools and molecular biology techniques like gene and exon prediction programmes, ORF finders, EST searches, alignment tools, 5′ RACE, RT‐PCR, sub‐cloning, sequencing etc. We were able to sequence two types of cDNA clones from mouse tissues, one type having alternative 5′ untranslated exon (s) and other type having alternative 5′ first coding exon. In former the variants arise either from alternate promoter usage or splicing of 5′ untranslated exons and in later new exon replaces the canonical first coding exon to exhibit different N‐terminal to translated protein. However, alternate promoter usage is mostly linked to regulatory subunit genes.

The numbers of new transcript variants sequenced were four in Cα, two in Cβ, three in R Iα and four in R Iβ. Their expression profiles showed variation across the tissues together at different development stages. The research was funded by DBT, India.
Thermodynamic Stability and Divalent Ion Interactions of RNA Containing Purine‐Rich Bulges
Authors: Strom Shane
Published: June 2021

Bulges are a common non‐helical motif found in RNA that play an important role in RNA recognition. Small asymmetrical bulges occur in many different RNA including HIV‐RNA and various micro RNA. In the P5abc region of group I intron, an X nucleotide adenine rich bulge coordinates two Mg2+ ions in order to allow for the formation of a specific structure necessary for ribozyme catalysis. Previous studies in our lab have shown that TAR RNA containing cytosine‐rich trinucleotide bulges are ~2 kcal/mol more stable than those containing uracil‐rich bulges in 1 M KCl. Magnesium ions stabilize TAR RNA containing uracil‐rich bulges by ~1–2 kcal/mol and cytosine‐rich bulges by ~ 1 kcal/mol, relative to 1 M KCl conditions. In this study, we are examining the differences in RNA stability as the size of a purine bulge is varied between one and five nucleotides. Thermal denaturation experiments are being performed on varying concentrations of RNA in buffers containing either 1 M KCl or varying concentrations of divalent ions. Altering the nucleotide sequence of the bulge alters RNA stability as well as its ion binding properties. A comparison of thermodynamic properties of purine and pyrimidine‐rich bulges will be presented.
miR‐17‐3p Regulates Metabolism of Prostate Cancer Cell
Authors: Velez Enid , Xu Yong
Published: June 2021

Micro RNA (miRNA), ~ 22 nucleotide RNA molecules that generally repress the translation of target messenger RNAs, is involved in various aspects of physiogenesis and pathogenesis. Our previous studies have demonstrated that miR‐17‐3p is able to suppress tumorigenicity of prostate cancer through inhibition of mitochondrial antioxidant proteins, such as manganese superoxide dismutase (MnSOD), glutathione peroxidase‐2 (GPX2) and thioredoxin reductase‐2 (TrxR2). In the present study, we test whether or not miR‐17‐3p enhances glycolysis pathway based on the inhibition of mitochondrial antioxidants. The miR‐17‐3p expressed in prostate cancer PC‐3 cell was cultured for isolation of RNA and proteins. Since lactate dehydrogenase (LDH) is an important marker for glycolysis pathway, the effect of miR‐17‐3p on the expression of LDH was examined. The RNA was reversely transcripted into cDNA and then quantified by real‐time PCR (RT‐PCR). The cellular proteins were quantified by western blots. Finally, concentration of lactate in the culture media was measured. The miR‐17‐3p increases expression of LDH‐A at both mRNA and protein levels. Consistently, the lactate concentration in media is also increased in miR‐17‐3p expressed PC‐3 cell culture. These results support the hypothesis of that miR17‐3p may enhance glycolysis pathway by inhibiting mitochondrial metabolism.
ERAD and autophagy cooperatively facilitate clearance of aggregation‐prone mutant Surfactant Protein C
Authors: Maguire Jean Ann , MacNabb C. Marshall , Guttentag Susan H. , Beers Michael F.
Published: June 2021

We have previously shown that mutations within the BRICHOS domain of Surfactant Protein C (SP‐C), a conserved C‐terminal region of ~100 amino acids found in a number of degenerative disease‐related proteins, lead to UPR activation, cytosolic aggregation, and activation of caspase‐mediated apoptotic pathways. We hypothesized that in addition to upregulation of quality control mechanisms, SP‐C BRICHOS mutants would induce autophagy as a means of cytoprotection. To study this, A549 and HEK293 cells were transfected with human SP‐CΔexon4 mutant. Expression induced autophagosome formation, assessed by detection of LC3 isoform conversion, and by alteration of the intracellular localization of GFP‐tagged LC3. These results were also observed in primary human alveolar type II cells. Inhibition of either the 26S proteasome with MG‐132, or autophagy by 3‐methyladenine resulted in a significant increase in expression levels of mutant SP‐C, suggesting both pathways play a prominent role in mutant clearance. Inhibition of autophagy in cells expressing SP‐CΔexon4 also led to pronounced Caspase 3 activation, while augmentation of autophagy through rapamycin treatment enhanced the clearance of mutant protein. These results indicate that macroautophagy is important for lung epithelial cell homeostasis via its participation in quality control responses activated by aggregation‐prone BRICHOS mutants.

Funding: HL19737
A novel ATP dependent conformational change of p97 ND1 fragment revealed by crystal structures of disease related mutants
Authors: Tang Wai Kwan , Li Chou‐Chi , Xia Di
Published: June 2021

Mutations in human p97, a type II AAA+ protein, have been implicated in IBMPFD (Inclusion Body Myopathy associated with Paget's disease of the bone and Frontotemporal Dementia). Previous crystallographic analyses showed that wild type p97 are invariably bound with ADP in the D1‐domain. Here, we show the crystal structures of ND1 fragment of the disease‐related p97 mutants, for the first time, with ATPγS bound in the D1‐domain. In the present structure, the N‐domain undergoes rotational and translational movement. This conformational change has been confirmed to be nucleotide‐driven as the movement is reversible by ADP. We found that the amount of prebound ADP in D1‐domain is consistently lower in mutants. Interestingly, titration with ATPγS revealed two types of binding sites in the mutants but not in wild type p97, suggesting the ADP‐prebound D1 sites can be occupied by ATPγS in the case of mutants but not in wild type. We believe that the non‐uniform nucleotide‐binding states of D1 in various subunits of wild type p97 hexamer are essential for its function. External stimulus such as ATP hydrolysis in D2‐domain or interaction with adaptor proteins may be needed to trigger the nucleotide exchange at the ADP‐prebound sites of wild type p97. The present work also shed light into how the mutations might affect the function of p97 leading to the disease.
Folding of individual subunits of AcrB before trimerization
Authors: Zhong Meng , Lu Wei , Wei Yinan
Published: June 2021

AcrB and its homologous are the inner membrane components of the major multidrug transporter in Gram‐negative bacteria. In Escherichia coli, AcrB recognizes and exports a broad range of functionally and structurally unrelated substrates. The pumps confer elevated drug tolerance to bacteria, thereby increasing the difficulty to cure infections caused by these bacteria. AcrB exists and operates as a homotrimer. To investigate to what extent each individual subunit folds before they oligomerize, we created a reporter platform to detect subtle conformational changes in AcrB in vivo. We expect this thiol‐trapping based method to be generally useful in the study of many other inner membrane proteins that contain periplasmic domain. Taking advantage of this reporter platform, we created an AcrB mutant that existd as a monomer in the cell membrane. The mutant AcrB was not functional, but folded into a conformation highly resembling that of the wild type AcrB. The success in the creation of a well‐folded AcrB monomer suggests that after translation, each individual subunit folds to a conformation very similar to its final state before trimerization occurs.

The work was supported by faculty start‐up fund to Wei Y. and RCTF fellowship from University of Kentucky.
Enhancing glutaminolysis in glutamine dependent breast cancer cells with TRO provides a therapeutic window for GDH inhibition by EGCG inducing massive cell death
Authors: Friday Ellen , Oliver Robert , Turturro Francesco , Welbourne Tomas
Published: June 2021

We previously demonstrated that breast cancer cells depend upon glutaminolysis and derived glutamate metabolized via GDH (NH4+) rather than ALT (ALA production). One strategy for limiting tumor growth is to enhance glutaminolysis and then block GDH. To effect this, MCF‐7 cells were incubated for 24h in DMEM containing 5mM Glc + 2mM Gln or 2mM Gln resulting in increased NH4+and decreased ALA; NH4+/ Gln utilized increased (1.1 to 1.4) indicating increased GDH flux. Lactate production (Lac) in Glc/Gln reversed to utilization consistent with reentry of pyruvate into mitochondria subserving anaplerosis. Cell protein content had increased incorporation of 14C‐U‐glutamine and mitochondrial membrane potential (Ψm) increased. Adding 25 uM TRO to Gln media enhanced NH4+ and the NH4+/ Gln ratio (1.4 to 1.8) consistent with accelerated GDH flux while Ψm increased; Lac uptake reversed to production while ALA plummeted (↓85% vs Gln alone) consistent with limiting mitochondrial pyruvate uptake. Addition of 100 uM EGCG to Gln media reduced the NH4+/gln ratio(1.4 to 1.0) with GDH inhibition; cell protein content decreased 32%. EGCG added to Gln media + TRO reduced NH4+ and NH4+/gln ratio(1.8 to 1.0) and prevented Lac‐ formation. Cell protein content dropped 55% while Ψm decreased 17% associated with an 8 fold increase in cell death.

Research support: Southern Arizona Research Foundation (TW) and Feist‐Weiller Cancer Center (FT)
Retinol supplementation had no significant effect on worm burden in Balb/c mice
Authors: Eneh Peace N , Bath Jennifer
Published: June 2021

Soil transmitted helminths have attracted interest in clinical research due their ability to parasitize human hosts. These parasitic species such as H. polygyrus infest host cell and suppress the immune system by secreting a fatty acid and retinol binding protein called FAR protein. Previous research has indicated that these parasites secrete proteins (Hp‐FAR‐1) that down‐regulate Th2 response by mopping up the amount of retinol needed to trigger the above mentioned immune response thereby compromising the immunity of the host. Thus the goal of this experiment was to investigate the effect of high level retinol in the diet on the resulting levels of H. polygyrus in an effort to see if these retinol doses could assist the host in improving humoral immunity. To carry out this study, retinol was administered to our experimental group while the control group received no retinol. In the 3 studies conducted, there was no decrease in the average worm burden for the retinol group as expected. Therefore more research is needed to better understand the role of retinol and other biomolecules in the Th2 immune response pathway against helminthes.
The anticancer of thioflavanone in human breast cancer cells via antiproliferative activity and apoptosis induction
Authors: Kim Gun‐Hee , Choi Eun Jeong , Lee Jae In
Published: June 2021

The present study was designed to investigate the antiproliferative and pro‐apoptotic activity of thioflavanone in vitro. The antiproliferative activity of thioflavanone was investigated using MTT assays with several human breast cancer cell lines (MCF‐7, MDA‐MB‐231 and MDA‐MB‐453). MCF‐7 cell line was the most responsive in terms of cell proliferation. Significant antiproliferative effects of thioflavanone were first observed from at 1ƒÝM of MCF‐7 (p<0.05), and this inhibition showed in a dose‐dependent manner. The IC50 of MCF‐7, MDA‐MB‐231 and MDA‐MB‐453 were 63.8, 88.9 and 62.2 ƒÝM, respectively. Moreover, these cells exposed to thioflavanone at each IC50 underwent apoptosis, as determined by DAPI staining and caspase‐3 activity assay. Compared to respective control level, exposure to the thioflavanone at each IC50 concentration resulted in a remarkable increase in caspase‐3 activities (32.0, 38.3, and 42.0% in MCF‐7, MDA‐MB‐231 and MDA‐MB‐453, respectively, p<0.05). Based on theses results, the present results show that the antiproliferative effect of thioflavanone resulted in apoptosis induction, which are consistent with the notion that thioflavanone has been used for the useful anticancer drug candidate.
Digestive physiological outcomes related to polydextrose and soluble corn fiber consumption by healthy adult men
Authors: Boler Brittany Vester , Serao Mariana Rossoni , Bauer Laura , Boileau Thomas , Swanson Kelly , Fahey George
Published: June 2021

The objective of this study was to evaluate fermentation characteristics of novel fibers by healthy adults. Twenty‐one healthy adult males were utilized in a crossover design. Each subject received each treatment of 21 g of polydextrose (PDX) or soluble corn fiber (SCF), or a no‐fiber control (NFC) in a snack bar. Each period lasted 21 d and feces were collected during the last 5 d of each period. Food intake, including fiber intake (average 14.7 g/d) did not differ among treatments. Cramping, flatulence, and distention were greatest (P<0.01) when subjects consumed PDX. Reflux, flatulence, and distension were greater (P<0.01) when subjects consumed SCF compared to NFC. All tolerance scores were very low (<2) indicating only small decreases in comfort. Fecal ammonia, 4‐methylphenol, indole, and branched‐chain fatty acid concentrations were decreased (P<0.01) when subjects consumed the novel fiber sources compared to NFC. Fecal acetate, propionate, and butyrate concentrations were lowest (P<0.05) when subjects consumed PDX compared to SCF and NFC. Fecal pH was lower (P=0.01) when subjects consumed SCF compared to NFC, while PDX was intermediate. Fecal wet weight was greatest (P=0.03) when subjects consumed SCF compared to NFC. Fecal dry weight tended to be greater (P=0.07) when subjects consumed PDX compared to NFC. The novel fibers led to 1.4 and 0.9 (PDX and SCF, respectively) gram increases in fecal dry mass per gram of supplemental fiber intake. These novel fibers appear to be beneficial to gut health while leading to minimal gastrointestinal upset in healthy adult males.
Cuomarin‐6‐sulfonamides May be Useful Photoaffinity‐labeling Reagents for Analyzing the Kinetics of Drug Transport and ATP Hydrolysis in ABC Multidrug Transporters
Authors: Spierenberg Sarah , Basanagouda Mahantesha , Kulkarni Manohar V. , Golin John
Published: June 2021

The photoaffinity reagent [125]‐iodoarylazidoprazosin (IAAP) is used to study both the ATPase catalytic cycle and the behavior of drug‐binding sites in a number of ABC multidrug transporter proteins including those found in fungi and mammalian cells. This reagent has several limitations, however. It is synthesized with a radioactive label and the lack of a cold counterpart makes measuring the Kd at a drug binding site onerous. Furthermore, multidrug transporters have multiple drug binding sites and IAAP labels at best only a minority of these. Both of these limitations are potentially addressed with a set of cuomarin‐6‐sulfonamides that contain a free C4‐azidomethyl group that should be suitable for photoaffinity labeling. We have begun to test some of these reagents using the yeast multidrug transporter Pdr5. We demonstrate that these derivatives are strong Pdr5 drug substrates that don't interfere with the protein's ATPase activity. Using whole cell transport assays, we show that several of these compounds are transported from at least one site that is distinct from the one photoaffinity labeled with IAAP.
Discovery of a Human Extramitochondrial Hydroxymethylglutary‐CoA Lyase
Authors: Montgomery Christa , Miziorko Henry M , Pei Zhengtong , Watkins Paul A
Published: June 2021

Expression of hydroxymethylglutaryl‐CoA lyase like protein (HMGCLL1) in Pichia pastoris produces the 340 residue human protein. Isolated protein catalyzes ketogenic HMG‐CoA cleavage with efficiency (144 U/mg) comparable to human mitochondrial HMGCL (159 U/mg). HMGCLL1 contains a unique N‐terminus that includes a potential myristoylation target (MGNVPS) rather than a mitochondrial leader sequence. Metabolic labeling with exogeneous myristate of wt‐HMGCLL1 protein, but not of the G2A mutant, supports this prediction. Highly specific antibodies detect HMGCLL1 on blots of rat organ homogenates with a pattern that contrasts with controls using Ab to mitochondrial HMGCL. Plasmids encoding wt or G2A HMGCLL1 proteins support exogenous overexpression in COS cells. Immunofluorescent detection of overexpressed wt‐HMGCLL1 indicates golgi localization with additional punctate staining of another organelle. For G2A, myristoylation is precluded and the observation of diffuse G2A distribution suggests that localization is myristoylation dependent. Strong endogenous expression in mouse neuroblastoma cells also indicates a punctate pattern and HMGCLL1 association with a nonmitochondrial organelle.
Yeast coq null mutants harboring multi‐copy COQ8 accumulate novel intermediates in coenzyme Q biosynthesis
Authors: Xie Letian X , Chen Jia Yan , He Cuiwen , Watanabe Shota , Pierrel Fabien , Clarke Catherine F.
Published: June 2021

Coenzyme Q (ubiquinone or Q) is a lipid electron carrier in the electron transport chain. In yeast Saccharomyces cerevisiae nine genes, designated COQ1 through COQ9, have been identified as being required for Q biosynthesis. Deletion of any one of the nine COQ genes leads to decreased steady state levels of other Coq polypeptides, including Coq4, Coq6, Coq7, and Coq9 (Hsieh et al., 2007). In this study, we find overexpression of Coq8p in certain coq null mutants restores steady state level of Coq4p, Coq7p and Coq9p; suggesting Coq8p facilitates the stabilization of the high molecular weight Coq polypeptide complex. Six of the coq null mutants (coq3 – coq9) accumulate early Q‐intermediates such as 3‐hexaprenyl‐4‐amino‐benzoic acid (prenyl‐PABA) and 3‐hexaprenyl‐4‐hydroxy‐benzoic acid (HHB) (Marbois et al., 2010). Here, we utilize liquid chromatography and mass spectrometry (LC‐MS/MS), and show that certain coq null mutants harboring multi‐copy COQ8 now accumulate late stage Q‐intermediates. For example, demethyl‐demethoxy‐Q6 was found in a yeast coq5 null mutant harboring multi‐copy COQ8. We suggest that over‐expression of Coq8 facilitates the assembly of and/or stabilizes the Coq polypeptide complex, allowing the synthesis of Q‐intermediates that can be diagnostic of the blocked step in the corresponding coq null) mutant.
Molecular Architecture of Viral Assembly and Bud Site Formation
Authors: Stahelin Robert V , Adu‐Gyamfi Emmanuel , Cossell Christina M , Soni Smita
Published: June 2021

Viruses cause an array of diseases, some fatal and others a minor nuisance, some transmitted through the air and others through bodily fluids. Strides have been made in eradicating some viruses from the planet through vaccination but others such as HIV, Ebola or the Human T‐cell Leukemia virus (HTLV) lack significant treatment options or preventive vaccination. The pathogenesis of these viruses depends on infection, replication and generation of new virions. This process requires concerted action of newly synthesized viral proteins and hijacking of mammalian machinery. The generation of new virions requires budding from the host cell membrane into a new particle with the ability to infect more cells. Over the last several years the matrix proteins (MA) and capsid proteins (CA) have been implicated in association with lipid membranes and budding from the host cell to create new virions but mechanistic insight is lacking. To address basic questions underlying virion formation, we have used biochemical, biophysical, structural, cellular, and single molecule analysis to investigate how MAs bind lipid membranes and change their shape. We then map these interactions in human cells and in the formation of new virions.
A cafeteria diet modifies the response to chronic variable stress in rats
Authors: Zeeni Nadine , Daher Costantine , Darcel Nicolas , Fromentin Gilles , Tome Daniel
Published: June 2021

Stress is known to lead to metabolic and behavioral changes. The aim of this study was to investigate the effect of long‐term nutrient intake on the response to chronic variable stress (CVS). Adult Sprague‐Dawley rats were fed one of three diets for 6 weeks: high carbohydrate (HC), high fat (HF), or “Cafeteria” (CAF) (Standard HC and a choice of highly palatable cafeteria foods: chocolate, biscuits and peanut butter). After the first 3 weeks, half of the animals from each group were stressed daily using a CVS paradigm while the others (controls) were kept undisturbed. Rats were sacrificed at the end of the 6‐week period. Abdominal fat, serum lipids and corticosterone were measured. Results showed higher total cholesterol and LDL, and lower HDL associated with both chronic stress and the CAF diet; as well as an increase in body weight (P =0.03), food intake (P <0.001) and intra‐abdominal fat (P <0.001) in the CAF group compared to the other dietary groups. In addition, there was a significant interaction between stress and diet on serum corticosterone levels (P=0.012). More precisely, there was an increase in corticosterone levels of stressed rats compared to non‐stressed rats in the HC and HF groups but not in the CAF group. These results show that a highly palatable diet offering a choice of food items is associated with a reduction in the response to CVS and could validate a stressor‐induced preference for comfort food.
Consumption of higher dairy and dietary protein during diet‐ and exercise‐induced weight loss promotes a metabolically favourable body composition change in overweight and obese young women
Authors: Josse Andrea R , Atkinson Stephanie A , Tarnopolsky Mark A , Phillips Stuart M
Published: June 2021

While diet and exercise induced body weight loss has significant health benefits, the ratio of fat:lean tissue loss may be more important. We aimed to determine how daily exercise and different hypoenergetic diets, varying in protein and calcium, affected changes in the tissue composition of weight lost in otherwise healthy young, overweight and obese women over 16wk. Ninety subjects were randomized to 3 groups (n=30 ea): HiDairyPro (HDP), DairyPro (DP) and Control (CON). They differed in the type of protein consumed (high, moderate or low dairy, respectively) and amount (30%, 15% and 15% of energy, respectively). Body composition (DXA) was measured at 0, 8 and 16wk, and a subset of subjects (n=39) underwent MRI to assess visceral adipose tissue (VAT) volume at 0 and 16wk. All groups lost body weight and fat (P<0.05, for all), however, the net loss of fat during 8–16wk was greater in HDP versus DP and CON. HDP gained muscle with a greater increase during 8–16wk than the other groups while DP maintained muscle and CON lost muscle. HDP also lost more VAT versus CON. We conclude that diet and exercise induced weight loss regimens with higher dairy, calcium and protein versus those without promoted weight loss in women with a more metabolically favourable body composition change characterised by greater total and visceral fat loss and muscle mass gain.

Grant Funding Source: The Dairy Farmers of Canada, CIHR, and The US Dairy Research Institute
Structural basis of Ncb5or, a multi‐domain redox enzyme implicated in diabetes and lipid metabolism
Authors: Deng Bin , Parthasarathy Sudharsan , Wang WenFang , Sturms Ryan , Hargrove Mark , Gibney Brian , Battaile Kevin , Lovell Scott , Benson David , Zhu Hao
Published: June 2021

NADH cytochrome b5 oxidoreductase (Ncb5or) is a multi‐domain enzyme implicated in diabetes and lipid metabolism. It contains two redox domains that are similar to cytochrome b5 (b5) and its reductase (b5R), a bridging domain with the CS motif, and a novel N‐terminal (N) domain. The heme center has a relatively low redox potential (E0 = −108 mV). To elucidate the structural basis of inter‐domain electron transfer and properties of the heme redox center, we generated individual domains or various combinations of domains, namely, b5, N‐terminus + b5, b5R and CS+b5R for X‐ray diffraction, kinetic and spectroscopic measurements. A 1.25 Å crystal structure of the b5 domain reveals an orthogonal orientation of the heme‐ligating histidines, consistent with a highly anisotropic low spin EPR signal, the first example of such a heme environment in the b5 superfamily. Kinetic studies of inter‐domain electron transfer suggest an electrostatic “b5‐b5R” interaction which is weaker than in microsomal “Cyb5A‐Cyb5R3” complex, in agreement with fewer charged residues on the surface of b5 than of Cyb5A. This interaction is facilitated by the CS domain and by the N‐terminus, which is shown to be unstructured by CD spectroscopy. Stopped‐flow kinetics suggested a limited role of invariant b5 surface residue Trp114 in b5R→b5 electron transfer. Thus, Ncb5or employs a unique mechanism for inter‐domain electron transfer. (Supported by NIH)
Transglutaminase response during differentiation and apoptosis in myelogenous leukemia cells
Authors: Miller Logan Tyler
Published: June 2021

Tissue transglutaminase (tTg) covalently modifies proteins by the formation between an ε‐amino group of a lysine residue and a γ‐carboxamide group of a glutamine residue. The expression of tTG is a widely used marker for apoptosis and cellular differentiation. The K562 cells, a myelogenous leukemia cell line, are differentiation‐incompetent, programmed cell death‐competent cancer cells. The objective of the study was to examine tTg response following exposure to chemicals known to induce differentiation and/or apoptosis.

The K562 cells were treated with various chemicals including sodium butyrate and heteroarontinoids (HETS), retinoids with one aromatic ring and at least one heteroatom. The chemicals were introduced to cellular suspensions of K562 cells and McCoy's Modified 5a media. After 48 hours of exposure the cells were processed using trypan blue exclusion, Hoechst/propidium iodide assay, and a morphology evaluation from May Grunwald‐Giemsa stained cytospin slide preparations. The expression of tTg was evaluated using SDS‐PAGE Electrophoresis/Western Blotting.

Preliminary results show that tissue transglutaminase is unresponsive to retinoic acid treatments. The heteroarotinoids showed varying levels of tissue transglutaminase expression as well as differing levels of apoptosis. Sodium butyrate effected the growth of K562 cells by decreasing the growth rate. Support was provided by SRU.
Uncovering novel therapeutic targets and pathways against neurodegeneration elicited by oxidative stress
Authors: Cho Kyoungin , Yi Haiqing , Ferreira Paulo A
Published: June 2021

Oxidative stress is linked to a plethora of diseases and aging manifestations. Identification of factors modulating oxidative stress may uncover therapeutic targets to unmet clinical needs. We report that haploinsufficiency of RAN‐binding protein‐2 (RANBP2+/−) protects photoreceptors and the retinal pigment epithelium (RPE) from degeneration by light damage, whereas it increases and decreases the susceptibility of dopaminergic neurons of the brain and retina to MPTP toxicity, respectively. RANBP2+/− mice upon light‐elicited oxidative stress suppresses membrane dysgenesis and modulates the levels of a subset of functionally diverse partners of RANBP2 (e.g. RAN GTPase, ubc9, S1 subunit of the 26S proteasome), ubiquitylated proteins and a set of orphan nuclear receptors. RANBP2+/− suppresses also the oxidative stress‐induced formation of lipid deposits in the RPE. On the other hand, MPTP in RANBP2+/− decreases the locomotor activity and leads to damage of TH+‐neurons of SNpc, whereas it promotes an increase of GFAP+‐glial cells and TH+‐amacrine neurons of the retina. We found also ten brain metabolites significantly modulated by MPTP between RANBP2+/+ and RANBP2+/−. Hence, insufficiency of RANBP2 leads to cell‐type‐dependent regulation of proteostasis and lipid homeostasis acting on functionally interconnected pathways in response to oxidative stress.

NIH support GM083165 & EY019492
THE ROUTES OF ERK ACTIVATION IN PROLACTIN‐STIMULATED BREAST CANCER CELLS
Authors: Aksamitiene Edita , Achanta Sirisha , Kiyatkin Anatoly , Hoek Jan B
Published: June 2021

Understanding the interactions that occur among the distinct signal transduction pathways, triggered by activation of prolactin receptor (PRL‐R), is essential in studying pathogenesis of metastatic breast cancer. Using quantitative Multi‐strip Western blotting of immunoprecipitates or total cell lysates, we show that PRL induces tyrosine phosphorylation of adaptor proteins of IRS, Grb2 and Shc families and concurrently activates Src family kinases (SFKs), Janus kinase/signal transducer and activator of transcription (JAK/STAT), Ras/mitogen activated protein kinase (MAPK) and phosphoinositide‐3 (PI3)‐kinase/Akt signaling pathways in T47D and MCF‐7 human breast cancer cells, derived from the patients with infiltrative ductal carcinoma and expressing different amounts of PRL‐R. Employing inhibitors against JAK2, STAT5, SFKs, PI3K, PDK1, Akt, PKC, Rac, PAK and Ras, we found that (1) PI3K/Akt and MAPK pathways cross‐talk upon PRL stimulation; (2) PI3K‐mediated ERK1/2 activation via c‐Raf occurs regardless of STAT5, Akt and PKC, but depends on activities of JAK2 and SFKs; (3) activated PRL‐R largely utilizes PI3K/PDK1‐dependent Rac/PAK pathway rather than canonical Shc/Grb2/SOS/Ras route to initiate, augment and sustain MAPK signaling. These findings suggest that by interconnecting such diverse pathways PLR may enhance breast cancer cell proliferation, survival, migration and invasiveness.
Extracellular signal‐regulated kinase modulates sleep and sleep homeostasis in Drosophila melanogaster
Authors: Vanderheyden William M , Shaw Paul J
Published: June 2021

Although humans spend a third of life asleep, the function this sleep serves is still unknown. Using the model system Drosophila melanogaster, we have begun to identify genes that alter either sleep or sleep homeostasis, the increase in sleep observed after sleep loss. One of the genes we have identified, extra‐cellular signal regulated kinase (ERK), is of particular interest given its role in synaptic plasticity. Thus, we expressed an activated form of ERK (UAS‐ERKact) in adult flies using the pan neuronal Geneswitch driver Gsw‐elav. We have found that RU486 (RU) fed GSw‐elav/+>UAS‐ERKact/+ display a significant increase in sleep compared to vehicle (ETOH) fed controls. Importantly, the pan neuronal expression of ERKact in the adult fly also results in a significantly larger sleep rebound than controls. Consistent with these results, we have observed a reduction in sleep time and an attenuated sleep rebound in two loss of function lines for the Drosophila homologue of ERK, rolled (rl1) and GSw‐elav/+>rlRNAi. Surprisingly, no sleep rebound is observed when ERKact is expressed in the adult Mushroom Body (MB) indicating that ERKact plays a circuit dependent role in sleep regulation. Given the relationship between sleep and synaptic plasticity, it will be interesting to further elucidate the common signaling cascades linking ERK, sleep and plasticity.

Research funded by 5 R01 NS051305‐03.
Covalent binding of retinoic acid to cytokeratins 16 and 10 accompanies its action in mouse skin tissues
Authors: Takahashi Noriko , Ohba Toshihiro , Imai Masahiko , Sasaki Yuichi , Kumaoka Takaya , Takahashi Katsuhiko , Fujiu Yasunori
Published: June 2021

Retinoic acid (RA) has various biological effects in mammalian cells and tissues. In epidermal cells, RA is an inhibitor of differentiation to the squamous phenotype. The molecular mechanisms underlying the primary effects of RA are mediated by RA nuclear receptors as well as by retinoylation (acylation by RA), a protein post‐translational modification. To understand those components arising from the actions of RA via RA nuclear receptors versus retinoylation on epidermal cells, we examined the effects of topical RA treatment on the skin of hairless mice. RA binding proteins were determined by immunoblotting using anti‐RA antibodies and identified by MALDI‐TOF‐MS. RA increased epidermal thickness and decreased matrix metalloprotease and hyaluronidase activities in mouse skin tissues. We identified eight RA‐binding proteins that were increased by topical RA treatment. Three of these proteins were identified as cytokeratin 10, cytokeratin 16 and serum albumin. These results raise the possibility that RA binding to cytokeratins in vivo may be involved in effects of RA on keratinocytes of mouse skins. This investigation was supported in part by the Ministry of Education, Culture, Sports, Science and Technology, Japan and the Open Research Center Project.