bluuebunny/crossref_metadata_2025 huggingface dataset:
Binding of the DNA primase of bacteriophage T7 to its recognition sequence in DNA is modulated by the zinc‐binding domain
Authors: Lee Seung‐Joo , Zhu Bin , Akabayov Barak , Richardson Charles
Published: June 2021

The zinc‐binding domains (ZBD) of prokaryotic DNA primases are critical for recognition of specific sequence in ssDNA at which primers are synthesized. ZBDs are structurally conserved in primases but they direct recognition of distinct trinucleotide sequences. In order to determine regions of the ZBD responsible for sequence‐specific recognition, we carried out homolog‐scanning mutagenesis on T7 DNA primase using a bacterial homolog from Bacillus stearothermophilus. Substitution of a five amino acid residue segment in the ZBD eliminates the ability of the primase to synthesize primers from a specific template sequence. The role of residues neighboring two cysteins (Cys17 and Cys20) in the primase was investigated by generating a library comprised of multiple amino acid substitutions at positions of Pro16, Asp18, and Asn19 followed by genetic screening for functional gene 4 protein. Examinations of the altered proteins reveal that substitutions at the three sites do not change the specific sequence recognized. However, the more positively charged residues in the region facilitates the better binding to a recognition site in a short template, leading to more efficient primer synthesis. Results from this study suggest that sophisticated architecture of ZBD determines recognition of specific sequence by DNA primase.

This work was supported by Public Health Service Grant GM 54397.
Function follows form: Linking epithelial polarity, growth control and morphogenesis in Drosophila
Authors: Bilder David
Published: June 2021

How do cells organize themselves internally, in order to take on a specific external form and function? Each of the >200 cell types in the human body has a distinct, sometimes radical architecture, yet how these properties arise from the relatively isotropic newborn cell remains largely unknown. We are exploring the biology of cell architecture in vivo by studying a simple cell type —epithelia— in a highly manipulable model organism ‐‐Drosophila. Using unbiased, large‐scale forward genetic screens as an entry point, we have identified novel molecular pathways that control cell biological features of epithelial organization, including apicobasal polarity. We have further investigated how this form ensures the proper functioning of cells during development, by influencing cell adhesion, signaling, and morphogenesis. A particularly dramatic and surprising case is seen in the Drosophila ‘neoplastic’ tumor suppressor genes, which simultaneously control both cell polarity and cell proliferation; mutation of these genes induces uncontrolled growth of disorganized cells that show several striking similarities to malignant human tumors. I will discuss insights that our work has provided into links between epithelial polarity, proliferation control, and organ morphogenesis.
In vitro selection for a polymerase ribozyme with improved substrate binding
Authors: Moretti Janina , Huang Ta‐You , Muller Ulrich
Published: June 2021

The origin of life went through a stage in which RNAs acted both as genome and as catalyst—the RNA world. Re‐creating a self‐replicating RNA system in the lab would help us to understand what an RNA based world may have looked like and how it evolved into today's protein and DNA based world. A self‐replicating RNA system would require an RNA catalyst (ribozyme) capable of polymerizing RNA, to replicate the system. A ribozyme capable of catalyzing RNA polymerization has been developed, but its efficiency is about 100‐fold below the level required for self‐replication due to weak affinity for its substrate. To improve substrate binding we are using an in vitro selection approach. In this method the sequences of a few functional ribozymes are selected from a large pool (~ 1014) of ribozyme variants. Previous selections covalently linked the ribozyme to its substrate thereby removing selective pressure for substrate binding affinity. A method recently published by the Unrau lab facilitates selection of polymerase ribozyme that are not covalently linked to their substrate by compartmentalizing ribozymes and substrates in emulsions with about 1014 droplets. We have improved many aspects of this recently published method and will soon be able to perform the selection.
In vivo selection of mRNA repair ribozymes
Authors: Olson Karen , Muller Ulrich
Published: June 2021

Group I introns are catalytic RNAs (ribozymes) that can excise themselves from pre‐mRNAs. These ribozymes can be taken out of their pre‐mRNA context and modified to replace the 3’‐terminus of a substrate mRNA with their own 3’‐terminus. To employ these trans‐splicing ribozymes for therapeutic applications, it is necessary to improve the in vivo mRNA repair efficiency. We developed an in vivo selection method that is able to increase the mRNA repair efficiency of the trans‐splicing Tetrahymena thermophila ribozyme by selecting from pools with several million ribozyme variants. These ribozyme variants contained 10 or 20 randomized nucleotides in their external guide sequences (EGSs), a region of the ribozyme known to increase splicing efficiency and fidelity. Ribozymes with beneficial EGSs were able to repair a mutated mRNA encoding the chloramphenicol acetyl transferase, and allow growth of the E. coli on medium containing chloramphenicol. Our selections isolated several efficient mRNA repair ribozymes. The predicted secondary structures of the selected EGSs contain several distinct structural motifs. Three of these motifs were previously known, and two motifs are new. Further in vivo selections could allow the generation of ribozymes that repair mRNA efficiently enough for therapeutic applications.
The elongation factor eEF1A is a binding target for a novel, non‐hormonal male contraceptive
Authors: Hutton Jenna Louise , Gross Stephane , Tash Joseph , Kinzy Terri Goss
Published: June 2021

Hormone‐based oral contraceptives were approved for use in women in 1960 and have become a popular form of birth control. But oral contraceptives are not suitable for all women and other methods have a high failure rate when used incorrectly. According to WHO statistics, 42% of pregnancies are unintended, identifying a need for the development of alternative methods of contraception. Gamendazole (GMZ) was identified from a screen of indazole carboxylic acid analogues as a non‐hormonal compound that causes male infertility in rats. H2‐GMZ, a more potent analogue of GMZ, produced 100% infertility after a single 3mg/kg dose, with 100% reversibility within 6 weeks. Eukaryotic elongation factor 1A (eEF1A) was identified as a binding partner of GMZ. To gain insight into the mechanism of GMZ action, we examined the effect of the drug on the canonical and non‐canonical functions of eEF1A. Total translation rates and nucleotide binding were minimally affected, suggesting that GMZ functions via inhibition of eEF1A's actin bundling function. Co‐sedimentation assays using purified yeast actin and yeast or rabbit eEF1A showed inhibition of actin bundling in the presence of 10 uM GMZ. We are currently developing a light scattering assay to determine kinetics of bundling, reversibility of inhibition and effects of adding competing factors. Preliminary data shows 10 uM GMZ inhibits rabbit eEF1A's actin bundling function.
Construction of retroviral vectors to functionally dissect the mir‐17‐92 oncogenic cluster
Authors: Martinez Mariela Raquel , Olive Virginie , Jiang Iris , He Lin
Published: June 2021

MicroRNAs (miRNAs) are small non‐coding ribonucleic acids involved in gene regulation by inhibiting translation through binding with perfect or almost perfect complementarity with the 3’untranslated region of target mRNAs. Investigations by He et.al.describe a miRNA cluster, the mir‐17‐92 polycistron, overexpressed in B‐cell lymphomas and comprised of the six miRNAs: miR‐17, 18, 19a, 20, 19b, and 92. Enforced expression of this cluster acted with c‐myc expression to accelerate tumor development in a mouse B‐cell lymphoma model and prevented the apoptosis of tumors derived from hematopoietic stem cells. However, the individual potential of the seven miRNAs in the cluster to accumulate tumor formation is largely unknown. To access this problem PCR amplified primary forms of individual miRNAs were used to produce retroviral constructs with the vector CMSV Sfi A, B T7 ires thCD4‐1.ab 1(hCD4). Molecular clones were obtained of miR‐17,18, 19a, 20,1792 and mutated miRNA to unexpress 19a and 92(miR‐Δ19 and 1792Δ92). The constructs where produced to isolate cells expressing the miRNAs by magnetic labeling using the MACS Select System. However, to understand the individual role of these miRNAs further screening and expression assays need to be preformed. Due to the novelty and complexity of miRNAs involvement in cancer progression the quest to solve the miRNA puzzle has just begun.
Parallel assessment of silencing potency of conventional siRNAs vs. Dicer substrate interfering RNAs
Authors: Snead Nicholas , Song Min‐sun , Sakurai Kumi , Rossi John J
Published: June 2021

RNA interference (RNAi) is a sequence specific gene silencing mechanism that can decrease the expression of target proteins. RNAi's sequence specificity has made it a powerful tool for cell biologists and is being developed for therapeutics. RNAi is triggered by short interfering RNA (siRNA), which are ~21bp RNA duplexes designed such that one of the strands (the guide strand (GS)) is complementary to the mRNA of the target protein. Biasing strand selection in favor of the guide strand, rather than the non‐guide strand (the passenger strand (PS)) will improve gene silencing potency. Our lab has developed longer, asymmetric, and chemically modified siRNA variants, termed Dicer substrate interfering RNA (dsiRNA). DsiRNA are engineered such that the GS is preferentially selected. Here we present a parallel screening of gene silencing ability of conventional siRNA vs. dsiRNA. Using a psiCHECK assay and calculating the 50% inhibitory concentration (IC50), we found that the PS of conventional siRNA exhibited > 8‐fold lower IC50 value than the GS (IC50(PS) = 9.75 pM vs. IC50(GS) = 71.55 pM). Meanwhile, dsiRNA reduced the silencing potency of the PS (IC50(PS) = 24.40 pM) while simultaneously increasing the silencing potency of the GS (IC50(GS) = 29.90 pM). Thus, relative to conventional siRNA, the biased strand selection of the dsiRNA improves the silencing ability of the GS and reduces the silencing ability of the PS.
Microbial Degradation of the Antidepressant Sertraline in Wastewater Sludge
Authors: Koechli Chantal Nathalie
Published: June 2021

Sertraline, a selective serotonin reuptake inhibitor (SSRI), is an antidepressant used to treat anxiety and depression. Because of its ability to be an effective SSRI, sertraline is one of the most heavily prescribed drugs in the United States. Thus, its disposal and excretion rates have also been high. Sertraline has been found present in the environment, and furthermore, has been found to produce harmful environmental effects on the aquatic and microbial ecosystems. Microbial degradation and specifically optimizing metabolic pathways of sertraline degradation in bacteria present in wastewater treatment provides one viable hypothesis for the removal of sertraline at a point source before its introduction into the environment. To explore this option, we measured degradation of sertraline over time in microcosms mimicking wastewater treatment and also isolated microbial species from the microcosms that were able to degrade sertraline. We found that sertraline concentrations degraded by a factor of 10 over a period of 28 days. We also were able to isolate two bacterial organisms that were able to use sertraline as a sole carbon source after growth on minimal salt plates with sertraline as the only carbon source. Initial ribosomal gene sequencing shows one isolate in Pseudomonas and one in Serratia, with species level identification still in progress.
The Interaction of N‐Acetylglutamate Synthase with Other Mitochondrial Urea Cycle Enzymes
Authors: Haskins Nantaporn , Tuchman Mendel , Morizono Hiroki , Caldovic Ljubica
Published: June 2021

The urea cycle operates in the mammalian liver to protect the central nervous system from the neurotoxic effects of ammonia via the highly efficient conversion of ammonia into urea. Protein:protein interactions between urea cycle enzymes are thought to contribute to the high efficiency of ureagenesis. We examined the interactions of the three mitochondrial urea cycle enzymes: N‐acetylglutamate synthase (NAGS), carbamylphosphate synthetase I (CPSI), and ornithine transcarbamylase (OTC).

There are two known isoforms of NAGS protein, NAGS‐M and NAGS‐C, which differ only in the presence (NAGS‐M) or absence (NAGS‐C) of a ~ 50 amino acid N‐terminal variable segment. The distribution of NAGS‐M and NAGS‐C within the mitochondria was examined using ultracentrifugation and western blotting. NAGS‐M was loosely associated with the inner mitochondrial membrane, while NAGS‐C interacted strongly with the inner mitochondrial membrane. Co‐immunoprecipitation (CO‐IP) was used to determine if NAGS, CPSI and OTC interact. The NAGS‐M, but not NAGS‐C co‐immunoprecipitated with CPSI and OTC. In addition, CO‐IP was used to examine if the variable segment of mouse NAGS could compete with NAGS for interaction with CPSI. Results indicate the variable segment is sufficient for the binding of NAGS to CPSI. These finding suggest localization and interaction of mitochondrial urea cycle enzymes are important for regulation of ureagenesis.
Biochemical and Histological Changes in the Kidney of Streptozotocin‐Induced Diabetic Rats: Effects of Garlic and Ginger Compared to Aspirin
Authors: Alhouli Mouna J , Thomson Martha , Al‐Qattan Khaled , Muslim Ali Divya J.S.
Published: June 2021

This study was conducted to determine the biochemical and histological efficacies of garlic and ginger in comparison to aspirin in streptozotocin‐induced diabetic rats. One week diabetic rats (blood glucose > 300 mg/dl) were divided into four groups of which three groups were treated for 8 weeks with daily IP doses of fresh extracts of garlic and ginger (500 mg/kg each) or aspirin (10 mg/kg). Kidney malondialdehyde (MDA), lactate dehydrogenase (LDH), catalase (CAT), protein and total antioxidants were measured. The results showed that kidney MDA and LDH levels of diabetic non‐treated rats were significantly increased as compared to both the normal controls and the garlic‐, ginger‐ and aspirin‐treated animals. Total antioxidants levels of untreated diabetic rats were significantly lower than those of both the normal controls and the three treated groups. CAT and protein levels of untreated diabetic rats were significantly decreased as compared to the normal controls and the garlic‐ and ginger‐treated groups. Aspirin‐treated rats were not statistically different from the diabetic non‐treated rats in regard to CAT and protein and from the garlic‐ and ginger‐treated rats with respect to MDA and total antioxidants. Diabetic renal corpuscles showed general distortion, in particular deterioration in capsular membrane and reduction in capsular space. All treatments attenuated the renal corpuscle modulation with more potency exerted by garlic and ginger. Results suggest that the hypoglycaemic effects of garlic and ginger can help in ameliorating diabetic nephrology, and it is concluded that these two herbs can be administered as a supplementary precaution against diabetes.

This work was partially supported by the College of Graduate Studies, Kuwait University.
Effect of hyperketonemia on chemokine secretion, adhesion molecule expression, and monocyte adhesion to endothelial cells
Authors: Rains Justin Lee , Jain Sushil K
Published: June 2021

Frequent episodes of hyperketonemia are associated with higher incidences of vascular disease. We examined the hypothesis that hyperketonemia increases monocyte adhesion to endothelial cells (EC). Human U937 monocytes or THP‐1 monocytes and HUVECs were cultured with AA (0–10 mM) or BHB (0–10 mM) for 24 hours. Results ( ± SE) show that there was significant increase in IL‐8 and MCP‐1 secretion in AA treated THP‐1 and U937 monocytes and HUVECs, however, BHB had no effect. There was a 10.5% ± 0.04 (ns) increase in protein levels of LFA‐1 in 4 mM AA treated U937 monocytes compared to control and a 13% ± 4.3 (p<0.04) increase in surface expression. There was a 21% ± 3.5 (p<0.01) increase in CD14 surface expression on U937 and a 3.9% ± 1.8 (ns) in THP‐1 treated with 4 mM AA compared to control whereas BHB had no effect. Surface ICAM‐1 increased by 23.8% ± 0.02 (p<0.05) and total protein expression by 40% in HUVECs treated with 4 mM AA compared to the control. Results showed a 33.7% ± 2.3 (p<0.01) increase in U937 adhesion and a 23.5% ± 3.8 (p<0.05) increase in THP‐1 adhesion to HUVEC monolayer's treated with 4 mM AA compared to the control. Monocyte adhesion could be blocked by 14% ± 3.6 (p<0.04) when HUVEC's were pre‐incubated with ICAM‐1 antibody. Thus, hyperketonemia increases monocyte adhesion to EC's by upregulation of LFA‐1, CD‐14 and ICAM‐1, which may contribute to the excess vascular disease in diabetes.
Differences among white adipose tissue depots in mice
Authors: Sala Lucila Sackmann , Munn Rachel , Lubbers Ellen , Berryman Darlene E , Kopchick John J
Published: June 2021

Excess white adipose tissue (WAT) is widely associated with insulin resistance, type 2 diabetes (T2D) and cardiovascular disease (CVD). In fact, intra‐abdominal fat is better correlated with insulin resistance than subcutaneous fat. However, the physiological impact of adipose depot location is not well understood. Our goal was to investigate cellular and molecular differences between WAT depots. Cell sizes and proteomic profiles from subcutaneous (inguinal) and intra‐abdominal (mesenteric, epididymal and retroperitoneal) fat pads from C57BL6/J mice were analyzed. Proteomes resolved by two‐dimensional gel electrophoresis followed by mass spectrometry showed significant differences in proteins involved in glycolysis, energy production, lipid transport, stress resistance, cytoskeleton structure, and others. Some of these proteins showed significant correlations to plasma insulin, leptin and/or adiponectin levels. Mean adipocyte cross‐sectional area was measured by digital image analysis of paraffin embedded tissue and was highest in epididymal and lowest in inguinal and mesenteric fat. Together, these results show marked differences among WAT depots, even within intra‐abdominal fat. This and further studies will help clarify the depot‐specific characteristics and their influence on insulin resistance in an effort to establish new diagnostic and/or therapeutic targets for T2D and CVD.

Supported in part by the State of Ohio's Eminent Scholar Program that includes a gift from Milton and Lawrence Goll, by NIH Grants DK075436‐01, AG019899‐06, 1P01AG031736‐01A1, by the Diabetes Research Initiative and the BioMolecular Innovation and Technology Partnership at Ohio University, and by AMVETS.
Absence of intestinal gluconeogenesis in type 1 diabetic rats: carbon 13 NMR and arteriovenous concentration difference measurements
Authors: Baverel Gabriel , Gadet Rudy , Nazaret Rémi , Martin Mireille , Boghossian Michelle , Martin Guy , Ferrier Bernard , Conjard‐Duplany Agnès
Published: June 2021

Recent reports have indicated that 48–72h of fasting, type 1 diabetes and high‐protein feeding induce gluconeogenesis in the small intestine of adult rats in vivo. Since this would (i) represent a dramatic revision of the prevailing view that only the liver and the kidneys are gluconeogenic, and (ii) have major consequences in the metabolism, nutrition and diabetes fields, we have thoroughly re‐examined this question in the situation reported to induce the highest rate of gluconeogenesis. For this, metabolically viable small intestinal segments from 72h‐fasted adult rats were incubated with [3‐13C]glutamine as substrate. After incubation, substrate utilization and product accumulation were measured by enzymatic and NMR methods. Although the segments utilized 13C‐glutamine at high rates and accumulated 13C‐labeled products linearly for 30 min in vitro, no substantial glucose synthesis could be detected. This was not due to the re‐utilization of 13C‐glucose initially synthesized. Arteriovenous metabolite concentration difference measurements across the portal vein‐drained viscera of 72h‐fasted Wistar and Sprague‐Dawley rats clearly indicated that glutamine, the main if not the only gluconeogenic precursor taken up, could not give rise to detectable glucose production in vivo. Therefore, we challenge the view that the small intestine of the adult rat is a gluconeogenic organ.
Contribution of MAP Kinases ERK1/2 in Beta Cell Function
Authors: Dioum Elhadji Mamadou , Schneider Jay W. , Cobb Melanie H
Published: June 2021

Pancreatic islet development and beta‐cell function are dependent on the coordinated activity of multiple islet‐enriched transcription factors, including PDX‐1, NKX6.1, MafA and BETA2. Somatic mutations in these factors have been implicated in maturity‐onset diabetes of the young (MODY). Understanding of the molecular mechanisms of pancreatic beta‐cell function and integrity is necessary for the development of anti‐diabetic drugs. Activation of the mitogen activated protein kinases (MAPKs) Extracellular signal‐related kinases 1 and 2 (ERK1/2) in beta cells is required for insulin gene transcription and secretion. Using in vitro and cell culture approaches, we showed that ERK1/2 phosphorylate BETA2, PDX‐1 and MafA and modulate their DNA binding activity and ability to form ternary complexes required for insulin gene transcription and beta‐cell function.

Isoxazole, a compound identified in a small molecule screen, shows improved beta‐cell function through chromatin remodeling and transcription factor activation in a pleiotropic pathway involving ERK1/2. We are exploring the molecular mechanisms involved in this signal transduction pathway.

Source of funding:

NIH R37 DK34128

NIH RO1 DIC55310
THE HEXOSAMINE BIOSYNTHESIS PATHWAY IS ESSENTIAL FOR PANCREATIC BETA CELL DEVELOPMENT
Authors: Guillemain Ghislaine , Filhoulaud Gaelle , Scharfmann Raphael
Published: June 2021

Pancreatic exocrine and endocrine cells develop during embryonic life from endodermal progenitors. This process depends on activation of a hierarchy of transcription factors. While information is available regarding the mesodermal signals controlling pancreas development, little is known about the role of environmental factors such as nutrients— including glucose—that also may impact development. Previously, we showed that glucose plays an important and specific role in beta cell development by activating the transition of Neurogenin3‐positive endocrine progenitors into beta cells. Here, we examined the implication of glucose metabolism and more precisely the role of the hexosamine biosynthesis pathway (HBP) to understand the mechanisms by which glucose regulates beta cell development. We have established an in vitro model of endocrine and exocrine cells development from E13.5 rat pancreases in a manner that replicates in vivo pancreas development perfectly. Using this model, we tested the effect of selective inhibitors and activators of the HBP. If the HBP has a modest effect on cell proliferation and exocrine cell differentiation, beta cell development is tightly controlled by the HBP. Specifically, HBP activators increase beta cell development, whereas inhibitors repress such development. Importantly, both the HBP and glucose control the same steps in beta cell development.
Mapping and measuring molecular networks in cells
Authors: Aebersold Ruedi
Published: June 2021

At a molecular level biological systems such as cell signaling or metabolism can be represented as dynamic networks of interacting molecules. In the living cell multiple such networks are concurrently present and active. These include the gene regulatory (transcriptional) networks, protein interaction networks, networks of enzymes and their substrates (e.g. protein kinase substrate networks), networks of microRNA and their substrates etc. Notably, most of these types of networks consist of or involve proteins. Therefore, the systematic analysis or the proteins that constitute a biological system, their interactions and the networks they constitute is a central theme of the emerging field of systems biology.

Mass spectrometry is the most widely used method for the analysis of proteins and proteomes. In this presentation we will discuss new mass spectrometry based technologies for the analysis of different types of cellular networks and their dynamic change as a function of cellular state.
Reduced mitochondrial respiration and improved coupling in infarcted heart following stem cell therapy
Authors: Hughey Curtis , Ma Lianli , James Freyja , Young Pampee Paul , Wasserman David , Rottman Jeff , Shearer Jane
Published: June 2021

Aim

To determine the changes in mitochondria following stem cell therapy in the infarcted heart.

Methods

C57BL/6J mice were randomly divided into three treatment groups, SHAM (n=8), MI (myocardial infarction)(n=9) and MI‐STEM (n=8) receiving direct myocardial injection of mesenchymal stem cells. Echocardiography was performed at baseline, 1 and 3 wk post‐infarct. Mitochondrial respiration (pmol/s*mgww/IUCS) in permeabilized fibers bordering the infarct was determined at 4wk following treatment. State 2 (absence of ADP) respiration, state 3 (ADP‐stimulated) respiration and state 4 respiration were examined. Citrate synthase activity (CS) was determined.

Results

MI‐STEM state 2 was lower than the MI and SHAM groups (20.98 vs. 29.46 and 32.45). State 3 was lower in the MI and MI‐STEM groups compared to the SHAM (77.01 and 68.46 vs. 97.18). Respiratory Control Ratio (RCR) was higher in the MI‐STEM compared to MI group (3.37 vs. 2.71) and state 4 was lower in the MI‐STEM compared to the SHAM group (67.93 vs. 96.23).

Conclusion

Cell therapy does not prevent declines in state 3 respiration, however, the higher RCR and lower state 4 respiration suggests stem cell treatment reduces futile respiration and increases respiration efficiency. These findings show cell therapy to improve cardiac function by reducing the energetic mismatch between supply and demand following a MI. Research supported provided by CIHR.
Up‐regulation and inhibition of human peptide deformylase in cancer cells and tissues
Authors: Sorensen Karly , Reindl Katie
Published: June 2021

The human isoform of peptide deformylase (PDF) has been recently discovered and found to localize to mitochondria; however, its role in the deformylation of newly‐synthesized mitochondrial polypeptides is uncertain. Our hypothesis is that human mitochondrial PDF (hsPDF) is functional in human cells. The objectives of this study were to quantify hsPDF in normal and cancerous cells and tissues and to determine the effects of hsPDF inhibition on cell proliferation. Quantitative real time PCR was used to measure hsPDF mRNA expression in cancerous and normal cell lines and tissues. Additionally, various cancer and normal cell lines were treated with increasing concentrations of a known PDF inhibitor, actinonin, and cell proliferation was assessed. Increases in hsPDF mRNA levels were seen in most cancer cell lines when compared to the normal. hsPDF was upregulated in most cancer tissues compared to the normal, with the greatest differences observed in breast, colon and lung tissues. The antiproliferative effects of actinonin were greater in prostate, colon, and breast cancer cell lines than in the normal cells. These results suggest that hsPDF is up‐regulated in certain cancers and that it may play an active role in proliferation of human cells. This work is supported by the NDSU Center for Protease Research (NIH 2P20 RR015566) and start‐up funds from NDSU to KR.
The MAP kinase Pmk1 is required for rotenone resistance in the fission yeast, Schizosaccharomyces pombe
Authors: Wang Yiwei , Gulis Galina , Marcus Stevan
Published: June 2021

The plant‐derived isoflavonoid, rotenone, induces Parkinson's disease (PD)‐like symptoms when chronically administered to rats and death of dopaminergic neurons in culture. Although rotenone is a potent inhibitor of complex I of the electron transport chain characteristic of mitochondria in animal cells, recent evidence suggests that it can induce death of dopaminergic neurons independently of complex I inhibition. We have investigated the effects of rotenone in the fission yeast, Schizosaccharomyces pombe, which like some other fungi lacks complex I and carries out rotenone‐insensitive cellular respiration. Microscopic analyses using fluorescent probes revealed that rotenone induces disruption of mitochondrial organization and generation of reactive oxygen species in treated S. pombe cells, two previously described effects of the toxin in mammalian cells. While rotenone only modestly inhibits growth of wild type S. pombe cells, it is profoundly inhibitory to growth of mutants lacking the stress responsive ERK‐type MAP kinase, Pmk1, but not mutants lacking the p38 MAP kinase homolog, Spc1. Indeed, cells lacking Spc1 are more resistant to rotenone than wild type S. pombe cells. Our findings demonstrate the usefulness of S. pombe as a model organism for elucidating complex I‐independent mechanisms of rotenone‐induced cytotoxicity and resistance, which might be insightful with regard to the pathogenesis of PD.
Emblica officinalis (Amla) improves glucose regulation by modulating hepatic nuclear transcription factor, peroxisome proliferator‐activated receptor (PPAR)
Authors: Buesa Lance Martinez , Nerurkar Pratibha Vivek
Published: June 2021

Ayurvedic medicine Emblica officinalis (amla, EO) has been traditionally used as rejuvenative and anti‐inflammatory therapy. The objective of our study was to investigate the anti‐diabetic effects of EO in mice fed 58% kcal high‐fat‐diet (HFD) for 16 weeks. Male C57BL/6J mice were randomized into four groups: 1) control, 2) EO (10% w/w), 3) HFD and 4) HFD + EO. After 16 weeks, EO had no significant effect on daily food intake but improved liver weights, plasma and hepatic glutathione, fasting glucose as well as glucose and insulin tolerance in mice fed HFD. Mechanistic studies indicated that EO normalized hepatic mRNA expression of nuclear transcription factor, peroxisome proliferator‐associated receptor alpha (PPARα) and improved hepatic insulin signaling as measured by phosphorylation of Akt proteins by Western blot. Future studies are warranted to identify the active components and physiologically effective doses of EO. [Grants: NCCAM (R21AT003719), USDA‐CREES (2004‐34135‐15182)].
In isolated rabbit renal proximal tubules, the antibiotic cephaloridine does not inhibit the mitochondrial uptake of succinate, a biomarker of its nephrotoxicity: A cellular metabolomic study with carbon 13 NMR
Authors: Baverel Gabriel , Cadi‐Soussi Sophia , Martin Guy
Published: June 2021

Cephaloridine is a model nephrotoxicant. In isolated rabbit renal mitochondria, cephaloridine (i) inhibits succinate uptake and (ii) reduces respiration. We have re‐examined this problem using isolated proximal tubules. Tubules were incubated in Krebs‐Henseleit buffer containing either 1,4‐13C‐succinate, or 2,3‐13C‐succinate or succinate + 13C‐bicarbonate without or with cephaloridine. Substrate removal and product formation were measured by enzymatic and carbon 13 NMR methods. Enzymatic fluxes were calculated by combining these results with an original mathematical model of renal succinate metabolism. Succinate was converted mainly into fumarate, malate, glucose and CO2. Cephaloridine induced an increase in the accumulation of fumarate, malate and a decrease in the production of glucose, CO2 and the level of ATP. Surprisingly, it did not change the removal of succinate. Fluxes through malate dehydrogenase, glucose 6‐phosphatase and citrate synthase were also diminished by cephaloridine.

In conclusion, the absence of inhibition of mitochondrial uptake of succinate in intact renal proximal tubules strongly suggests that results obtained with isolated mitochondria do not necessarily apply to mitochondria in intact cells and, therefore, should be interpreted with great caution. These results also suggest that mitochondrial functions should be studied in intact cells.
Variable toxicological response to loss of OXPHOS through 1‐methyl‐4‐phenylpyridinium‐induced mitochondrial damage or anoxia in diverse immortal cell lines
Authors: Soliman Karam F , Mazzio Elizabeth A
Published: June 2021

Many cell lines are often of malignant origin having unusually high glycolytic activity to acclimatize to hypoxic conditions. The current study investigates variation of cell survival during loss of mitochondrial OXPHOS. The data show a significant difference between the metabolic rate amongst mouse‐N2A, rat‐PC‐12 and human SH‐S5Y5 neuroblastoma, which accounted for speed of glucose use and vulnerability to 1‐methyl‐4‐phenylpyridinium (MPP+) toxicity. After MPP+ exposure in N‐2A cells have shown an anaerobic shift occurring as early as 2 hr evidenced by a rise in lactate, followed by a drop in glucose at (4hr) and exhaustion of glucose supplies at (22 hr), corresponding to loss of viability. MPP+ did not induce a rise in reactive oxygen species and was not afforded protection by catalase or SOD. In contrast, toxicity was attenuated by glucose, glycolytic fuels or pyruvate ± mitochondrial monocarboxylate transporter blocker (a‐cyano‐4‐HCA) or a pyruvate dehydrogenase inhibitor (octanoyl‐CoA). The cells also survived a complete loss of OXPHOS during O2 removal– anoxia (Na+2 dithionite) in high glucose media. In summary, immortal cell lines have robust anaerobic capability to perpetuate survival during loss of mitochondrial function or anoxia, whereby vulnerability is a function of metabolic rate, cell density and glucose concentrations within the experimental media. (Supported by a NIH grant RR 03020)
Role of Ethanol Metabolism in Intravital Hepatic Mitochondrial Depolarization
Authors: Zhong Zhi , Ramshesh Venkat K , Rehman Hasibur , Lemasters John J
Published: June 2021

Mitochondrial dysfunction plays an important role in alcoholic liver disease. This study investigated the relation between alcohol metabolism and mitochondrial depolarization in vivo. Mitochondrial polarization and cell death were assessed in living mice by intravital multiphoton microscopy of rhodamine 123 and propidium iodide, respectively. Intravital mitochondrial depolarization occurred as early as 1 h after acute ethanol treatment (6 g/kg, i.g.). After 6 h, 94% hepatocytes contained depolarized mitochondria, but cell death was barely detectable. Similar intravital depolarization occurred in alcohol dehydrogenase (ADH)‐positive deer mice. By contrast in ADH‐negative deer mice, mitochondrial depolarization only occurred in 26% of hepatocytes. Ethanol‐induced mitochondrial depolarization was blunted by ~25% in livers of CYP2E1 deficient mice and after treatment of wildtype mice with a cytochrome P450 inhibitor aminobenzotriazole. Disulfiram, an aldehyde dehydrogenase inhibitor, accelerated ethanol‐induced mitochondrial depolarization, and acetaldehyde infusion into the liver alone caused mitochondrial depolarization. Together, these data suggest that acetaldehyde formation mediates, at least in part, ethanol‐induced mitochondrial depolarization in vivo and that ethanol metabolism by ADH plays a larger role than CYP2E1 in producing this effect (NIAAA).
The Multifaceted Human Transcriptome
Authors: Kapranov Philipp
Published: June 2021

A large amount of experimental data collected over the last decade has shown that genomic organization is very complex and has highlighted the fact that the current set of gene annotations does not fully capture this complexity. Furthermore, the transcriptome complexity has expanded in several directions. First, there is a significant amount of RNA that can be detected in a cell that originates from outside the exons of annotated genes, representing a multiplicity of novel exons of protein coding genes and stand alone transcripts. Second, exons of annotated and unannotated transcripts separated by large genomic distances can be joined together in chimeric transcripts. Third, individual compartments within the cell differ in the amount of un‐annotated transcription with nucleus and polyA‐ transcriptomes being highly enriched in un‐annotated transcripts. Fourth, the short RNA component of the transcriptome is vast and underappreciated. These results suggest that the functional space of the human genome is not limited to the annotated exons of protein coding genes and is likely to produce a vast amount of non‐coding RNAs. What are the implications of such genomic architecture in terms of increased information content, transcriptional complexity, evolution and disease states?
Mathematical modeling of elasticity changes in the cortical bone of HIP/RPL29‐ deficient mice
Authors: Sloofman Laura Gail , Pelesko John A. , Schleiniger Gilberto F. , Kirn‐Safran Catherine B.
Published: June 2021

Mathematical modeling, or using equations to describe a system, is a powerful technique in transgenic research. Our laboratory created the first viable mutant mouse model lacking an individual ribosomal protein (HIP/RPL29). A short stature phenotype associated with increased bone fragility is observed in HIP/RPL29 null mice. We recently noted that a decrease in collagen cross‐linking during the growth of HIP/RPL29 null bone precedes an overall enhancement in the mineral‐to‐matrix ratio in adult bone. We hypothesize that we can build on existing mathematical models to describe the changes in bone structure and composition resulting from HIP/RPL29 deficiency. Hierarchical multi‐scale models of wild type and HIP/RPL29 null cortical bone will be used to simulate adult control and mutant bone. We aim to relate the changes in null bone samples to elasticity measurements collected via three‐point bending tests. Since the equations describing mutant and control bone are interrelated, we can extrapolate relationships among the mentioned properties. We will also use this model to predict the elasticity in control and mutant bones at three months of age. All calculations will be compared with accepted experimental values. This model will be altered as the three‐month mutant bone phenotype is refined. Funding was provided by the Howard Hughes Medical Institute to LGS and NIH P20‐RR016458 to CBKS.
The plasma microparticle proteome updated
Authors: Little Kristina Maria , Smalley David M , Harthun Nancy L , Ley Klaus
Published: June 2021

All cell types shed ectosomes and exosomes, collectively known as microparticles (MPs) (0.1–1.0 μm in diameter) when activated or stressed, or during apoptosis. Normal human plasma contains 2μg MP protein/ml, most of which are derived from platelets and other vascular cells. The number and composition of plasma MPs are altered in many diseases, including acute coronary syndrome, diabetes mellitus, sepsis, and sickle cell disease; the protein composition of MPs reflects a subset of the parent cell's proteome. Here, we analyzed plasma microparticles from 42 patients (mean (±SD) age in years = 68 (±12)), most with cardiovascular diseases such coronary artery disease and hypertension. Using label‐free tandem mass spectrometry (MS/MS), we detected 483 proteins with high confidence (SEQUEST XCor ≥ 2.0, 2.2, and 3.3 for charge states +1, +2, and +3, and at least 2 unique peptides per protein). Of these, 93 were detected in at least 90% of patients, representing a core set of plasma MP proteins. This core is primarily composed of cytoskeletal proteins, integrin complex proteins fundamental to the plasma membrane, and proteins involved in coagulation, confirming several detections from previous MP studies. The protein composition of plasma MPs is influenced by age, gender, race, and several comorbidities, suggesting that the MP proteome may be a useful source of biologically relevant biomarkers.
Tellurite Resistance Transformation for Simplified X‐Ray Crystallographic Protein Structure Analysis
Authors: Mancuso Matthew Cian , Boles Jeffrey O
Published: June 2021

One of the most common techniques for determining protein structure is X‐ray crystallography using selenium‐substituted amino acids. The disadvantage of this method, is the high cost and long wait times for a synchrotron x‐ray source, the only device capable of MAD (Multi‐wavelength Anomalous Diffraction), which is necessary for resolution of a structure with selenium. Telluromethionine‐containing proteins, on the other hand, can be imaged using more common x‐ray crystallographic devices. In previous work, tellurium has shown to be toxic to bacterial cells which causes poor integration and low cell growth rates. This is being overcome via the introduction of a tellurium resistance gene into a methionine auxotroph to mitigate toxicity.
Painting the Cysteine Chapel: New Tools to Probe Oxidation Biology
Authors: Carroll Kate S
Published: June 2021

Oxidants are mediators of signaling pathways, where they modify proteins via chemoselective oxidation of redox sensitive cysteine residues. We have developed a chemical approach for selective detection of sulfenic acid‐modified proteins in living cells [Mol Biosyst 2008]. Application of this new method to the discovery of protein targets of oxidation in mammalian cells has yielded identification of almost 200 proteins [ACS Chem Biol 2009]. A small subset of these proteins is known to be oxidized in cells, but the majority of the proteins have not been previously characterized as oxidation targets. In addition, we have used this method to dissect the mechanism of peroxide sensing in yeast [Chem Biol 2009] and to discover a periplasmic reducing system that protects single cysteines from oxidation [Science 2009]. To further expand our capacity to investigate the dynamic sulfenome, we have also developed new immunochemical [PNAS 2009] and ratiometric approaches. The ability to monitor cysteine oxidation in living cells opens new avenues for the rapidly expanding field of oxidation biology and we continue to develop these reagents for further study and application.
Pretreatment with N‐Acetyl‐L‐Cysteine Increases Viability of C6 Glioma cells Following DNB Exposure
Authors: Rivera Berenise , Miller R. Timothy
Published: June 2021

In previous studies, N‐acetyl‐L‐cysteine (NAC), which increases intracellular concentrations of GSH was shown to protect oligodendrocytes in culture against exposure to toxic stimuli such as organo‐ mercury. The aim of the present in vitro study was to determine if pretreatment with NAC would protect C6 Glioma cells from exposure to the three individual DNB isomers. Cytotoxicity was determined using MTT reduction for mitochondrial integrity and neutral red (NR) uptake for lysosomal integrity. C6 Glioma cells were pretreated with NAC (2μM) for 24 hours, and then exposed to one of the DNB isomers (10–250 μM) for up to 6 hours. The protective effect of NAC (2μM) on C6 Glioma cells exposed to DNB isomers was in the following order 1,2‐<1,3‐<1,4‐DNB using the MTT assay. On the other hand, using the NR assay, an equal protective effect by NAC was observed when C6 Glioma cells were exposed to the DNB isomers. Based on the results obtained with these two assays, MTT was the most sensitive in detecting changes in cell viability in this model. Our results indicate that NAC pretreatment has a concentration‐ and isomer‐dependent effect on cell viability. Studies are underway to explore the cellular mechanism(s) and DNB intermediates involved in NAC mediated protection of C6 Glioma cells from DNB toxicity with regard to, and also separate from, the glutathione detoxification process. This project was supported in‐part by 5G12‐RR008124 from NCRR to UTEP and by NIEHS/NIH grant ES011982 to RTM.
Paraquat‐Induced Protein Expression Changes in Rat Liver Mitochondria
Authors: Venkatakrishnan Priya , Nakayasu Ernesto S , Almeida Igor C , Miller R T
Published: June 2021

Paraquat (PQ) is a widely used synthetic herbicide and redox‐cycling agent. Upon one‐electron reduction of PQ, the paraquat radical (PQ.) is formed. PQ. preferentially accumulate in the lung via transport by polyamine transporters and cause pulmonary edema. Case reports of PQ toxicity indicate that death is due to multisystem organ failure, including hepatotoxicity. Earlier studies identified that PQ causes reactive nitrogen species‐mediated toxicity by affecting the activity of mitochondrial complex I. Therefore, we investigated mitochondrial proteome in order to identify the proteins that are modulated during early stage (3 hrs) of acute PQ exposure (40 mg/kg). Proteomic analyses were performed with purified mitochondria and whole liver homogenate of PQ‐treated rats using nanospray‐coupled linear ion trap mass spectrometry in conjunction with 18O labeling. Data from technical replicates, obtained under stringent conditions, indicated levels of antioxidant and detoxification proteins such as peroxyredoxin 6, thioredoxin‐1 and glutathione‐s‐ transferase p to be modulated. Immunochemical analyses confirmed that peroxyredoxin 6 levels were downregulated in mitochondria. Further studies are underway in order to better understand the role of effected proteins in mitochondria and tissue homogenate during PQ toxicity (Supported by ES011982 from NIEHS/NIH to RTM and 5G12RR008124 from NCRR to UTEP).
Transcriptional mediator subunit Med1 is required for the expression of metastasis‐associated gene, The death associated protein kinase‐1 (DAPK1)
Authors: Gade Padmaja , Kalvakolanu Dhananjaya V
Published: June 2021

The death associated protein kinase‐1 (DAPK1) is a ca+2/calmodulin regulated serine/threonine kinase and is a major tumor suppressor gene. Its expression is lost in multiple tumor types although the mechanisms are unclear. Recently we showed that both the basal and IFN‐γ induced dapk1 expression levels were critically dependent on transcription factor C/EBP‐β (P.Gade et al (2008) Mol. Cell. Biol.), which plays major role in regulating energy metabolism, immune response and cell differentiation. C/EBP‐β associates with various cellular factors to exert its activity in gene context and signal specific manner. We have recently reported that Med1 subunit of the transcriptional mediator complex specifically interacts with C/EBP‐β for stimulating dapk1 expression in an IFN‐induced manner. We investigated if the mechanism responsible for the loss of dapk1 expression in human cancers involves a failure to recruit C/EBP‐β and Med1 to the dapk1 promoter. We found a significantly lower occupancy of Med1 and C/EBP‐β at dapk1 promoter in human cancer cell lines compared to the normal control cells. More importantly, the loss of Med1 expression correlated with a corresponding loss of dapk1 expression in a number of primary human lung carcinomas. Restoration of Med1 into cancer cells not only induced the expression of dapk1 but also suppressed their metastatic potential in vivo. Thus, our studies identified Med1 as one critical parameter that limits dapk1 expression in human cancers.