bluuebunny/crossref_metadata_2025 huggingface dataset:
Role of NAD(P)H Oxidase‐ and Mitochondria‐derived ROS in Coronary Collateral Growth
Authors: Rocic Petra , Chilian William M
Published: June 2021

Relative importance of NAD(P)H oxidase‐ vs. mitochondria (mt)‐derived oxidative stress (OxStress) in cardiovascular pathologies, including impaired arteriogenesis, is unresolved. We investigated the contribution both to regulation of coronary collateral growth (CCG). CCG was stimulated in lean Zucker (LZR) and prediabetic obese Zucker (ZOF), where OxStress is elevated, rats by repetitive ischemia (RI). Coronary blood flow was measured in normal (NZ) and collateral‐dependent (CZ) zones. RI increased CZ flow (0.23 to 0.84 ml/min/g; CZ/NZ 0.84 ± 0.03) in LZR, but failed to increase it in ZOF (0.08 to 0.15 ml/min/g; CZ/NZ 0.16 ± 0.03). Treatment with a ubiquitous ROS scavenger, TEMPOL, partially restored CCG in ZOF (CZ/NZ 0.69 ± 0.05). Treatment with mt‐targeted TEMPOL and another mt‐targeted ROS scavenger (Mito‐Quinone) restored RI‐induced CCG in ZOF to a similar extent (CZ/NZ 0.64 ± 0.01 and 0.75 ± 0.04, respectively). Treatment with the NAD(P)H oxidase inhibitor, apocynin also partially rescued CCG in ZOF and in a rat model of the metabolic syndrome (JCR), where OxStress is similarly elevated and CCG compromised (CZ/NZ 0.50 ± 0.02 and 0.65 ± 0.03, respectively). We conclude that both NAD(P)H oxidase‐ and mt‐derived OxStress are important in the regulation of CCG, and lowering of both may be necessary for restoration of CCG in the prediabetic metabolic syndrome. Support: AHA 0630285N, NIH RR018766, NIH 32788.
MicroRNAs in development and cancer
Authors: Slack Frank John
Published: June 2021

MicroRNAs are small non‐coding RNAs that negatively regulate gene expression to control important aspects of development and metabolism such as cell differentiation, apoptosis and lifespan. let‐7 encodes a microRNA implicated in human cancer. Specifically, human let‐7 is poorly expressed or deleted in lung cancer, and over‐expression of let‐7 in lung cancer cells inhibits their growth, demonstrating a role for let‐7 as a tumor suppressor in lung tissue. let‐7 is expressed in the developing mammalian lung and regulates the expression of important oncogenes implicated in lung cancer, suggesting a mechanism for let‐7's involvement in cancer. We are focused on the role of let‐7 and other oncomirs in regulating proto‐oncogene expression during development and cancer, and on using miRNAs to suppress tumorigenesis.
Taking a LEAP: Training the next generation of pharmacologists
Authors: Sikes Suzanne Sessoms , Schwartz‐Bloom Rochelle D
Published: June 2021

Launch into Education About Pharmacology (LEAP), a science enrichment program for high school students, uses the study of drugs and their effects on the human body to teach basic principles in biology and chemistry. LEAP targets 10th/11th graders underrepresented in the sciences. Students complete an intensive three‐week course in pharmacology during June and engage in mentored research during the school year. Teaching approaches include guided‐inquiry exercises, debates, discovery labs, and presentations by career scientists in diverse areas of pharmacology. LEAP students exhibit a 13–32% gain in basic science knowledge as assessed by a 10‐item multiple‐choice posttest administered at the conclusion of the summer course. Overall, the students rate their interest and self‐efficacy in science as significantly higher after the LEAP experience. In a retrospective analysis, 86% of participants report an increased interest in science. LEAP students have earned a combined total of nine awards in local and state science competitions (including one academic scholarship). These data suggest that topic‐relevant study, coupled with exposure to research science, effectively increases understanding of key scientific concepts and enthusiasm for science education among high school students. LEAP is funded by a SSEP grant from the Burroughs Wellcome Fund.
Three‐dimensional reconstruction of the air‐ and blood capillaries of the avian lung
Authors: Maina John N. , Woodward Jeremy D.
Published: June 2021

In the avian lung, gas exchange occurs between the air capillaries (ACs) and the blood capillaries (BCs). The shapes and the spatial arrangement of the components are uncertain. The models used to characterize pulmonary gas exchange have used inaccurate morphological details. Previously, it assumed that the ACs and the BCs are straight tubules that run parallel and in direct contact with each other and that they are exact copies. The actual forms and the organization vitally differ from these assumptions. A suitable model for assessing respiratory efficiency must include exact structural data that include part of the parabronchial lumen, an infundibulum, and the ACs that arise from it. 3‐D computer reconstruction from the exchange tissue of the lung of a duck, Cairina moschata, using a cross‐correlation based alignment strategy, showed that the ACs consist of globular parts that are joined by narrow conduits; the BCs consist of short segments that are as long as they are wide; and that the respiratory units intertwine intimately. A sheet‐flow arrangement of the BCs surrounds the infundibulum. This corresponds to the organization of the BCs in the interalveolar septa of the mammalian lung. In an unexpected structural analogy, the fine details of the design of the avian lung match those of the mammalian lung. Funded by the National Research Foundation.
Development of the heart in ANP−/− mice during early postnatal period
Authors: Leroux Janette Samantha , Angelis Ekaterini , Tse Yat , O'tierney Perrie , Pang Stephen
Published: June 2021

Disruption of the proANP gene results in marked cardiac hypertrophy, chronic and salt‐sensitive hypertension. It is unclear when cardiac hypertrophy in ANP−/− mice appears during development. The present study was designed to examine cardiac hypertrophy in ANP−/− mice during early postnatal development and correlate it with expression of the renin‐angiotensin (RAS) and the extracellular matrix (ECM) systems. Hearts were excised from 1–5 week old neonatal and 12 week adult male ANP+/+ and ANP−/− mice. Total mRNA was extracted from whole neonatal hearts and adult left ventricles, and evaluated using Northern blot analysis and quantitative RT‐PCR. Cardiac hypertrophy as indicated by an increase in heart/body weight ratio was evident in ANP−/− mice at all age groups. The most significant molecular changes were an increase in the expression of cardiac BNP mRNA level and a decrease in metalloproteinase 2 (MMP‐2) mRNA of ANP−/− as compared to the ANP+/+ mice. These data support the idea that the renin‐angiotensin system plays a minor role in the development of cardiac hypertrophy in the proANP gene disrupted mice and suggest the essential role of ANP and ECM in the development and maintenance of cardiac hypertrophy in the ANP−/− mice. Supported by the Heart and Stroke Foundation of Ontario.
Tbx3 is required for normal cardiac conduction system development
Authors: Frank Deborah U , Moon Anne M
Published: June 2021

TBX3 is mutated in human Ulnar‐Mammary Syndrome. Tbx3 null mice die in mid‐gestation with mammary gland, limb, and yolk sac defects. Tbx3 is also expressed in the conduction system and valves of the developing heart.

We generated mutants from a series of null, and severe and mild hypomorphic Tbx3 alleles to examine the dosage‐dependent requirements for Tbx3 during mouse cardiac development. Echocardiography, EKGs, and histology were used for cardiac phenotyping. Severe hypomorphs die by e13.5; and limb defects are indistinguishable from null homozygotes. Mild hypomorphs survive to e13.5 with mildly affected limbs. Real time rt‐PCR levels of Tbx3 correlate with the severity of phenotype.

Serial echocardiography of embryos reveals a range of embryonic death in severe hypomorphs which correlates with arrhythmias. Embryos that die from e10.5‐12.5 have marked bradycardia prior to death, and those dying from e12.5–13.5 have atrioventricular (AV) block. Some mutants display poorly coordinated ventricular contraction. Mild hypomorphs develop AV block a day later. The developing sinoatrial and AV nodes are smaller in Tbx3 mutants. There are morphologic abnormalities of the developing valves and ventricles.

Our data support a dosage‐dependent requirement for Tbx3 during cardiac development. Supported by R01HD046767‐01 to AMM. DF is supported by T32‐HL07576‐22 and UofU CHRC.
The Transverse Vesical Fold is a Nontransient and Vascularized Structure
Authors: Boaz Noel T , Martin Alexander H. , Ferreira Curvin , Forest Lynette
Published: June 2021

Laparoscopy provides a dynamic perspective that can alter conceptions of anatomical structures in the peritoneal cavity. The transverse vesical fold (plica vesicalis transversa) named by Waldeyer is a salient peritoneal laparoscopic feature of the anterior pelvic cavity and stretches between the lateral side of the urinary bladder to the pelvic wall, subdividing the paravesical fossa. However, it is rarely clearly visualized in the embalmed cadaver and is not figured in most current anatomical atlases. Early editions of Gray's Anatomy state that the transverse vesical fold disappears when the bladder is full. We report here that this is not the case. Although showing individual variability, the transverse vesical fold in the living patient is a nontransient structure and in fact becomes more prominent as the bladder is stretched during filling. Cadaveric dissection of the transverse vesical fold demonstrates that this structure is a vascularized peritoneal fold usually containing one or more branches of the superior vesical artery. We suggest that Gray's error refers to the shallow groove on the posterosuperior border of the bladder connecting the medial ends of the two transverse vesical folds. Waldeyer figured this area but clearly labelled it as the peritoneal “roof” (“obtecta”) of the bladder. We propose that this peritoneal groove be termed the “obtectal groove of the bladder,” and suggest that it, not the transverse vesical ligament, tends to disappear with a filled bladder.
Characterization of a Novel Cardiac Isoform of the Cell Cycle‐related Kinase
Authors: Qiu Hongyu , Shen You‐Tang , Vatner Dorothy E. , Vatner Stephen F. , Depre Christophe
Published: June 2021

We examined a genomic profile in monkey hearts subjected to myocardial infarction (MI) followed by pacing‐induced heart failure (HF), to identify novel mechanisms involved in the pathogenesis of HF. One candidate was the transcript encoding the cell cycle‐related kinase (CCRK), which decreased by 50% in HF (P<0.05). The CCRK sequence cloned from the heart showed the same N‐terminal kinase domain as the “generic” isoform cloned previously, but a different C‐terminal half due to alternative splicing with frame shift. Expression of the protein, confirmed by monoclonal antibody, was limited to heart, liver and kidney. Upon overexpression in cardiac myocytes, both the generic and cardiac isoforms promoted cell growth and decreased apoptosis (P<0.05 vs control). The “generic” but not the cardiac CCRK interacted with cyclin H and casein kinase 2 in a yeast two‐hybrid, phosphorylated the cyclin‐dependent kinase 2, and increased the number of myocytes in the S and G2 phases of the cell cycle (P<0.05 vs control). Therefore, the heart expresses a variant of CCRK, which promotes cardiac cell growth and survival similarly to the “generic” form, but both isoforms differ in terms of protein‐protein interactions, substrate specificity and re‐entry in the cell cycle. Antagonizing the down‐regulation of CCRK expression in the transition to HF could protect the heart and enhance regeneration of failing myocardium.
IDENTIFICATION OF BASE EXCISION REPAIR ACTIVITIES ASSOCIATED WITH THE HERPES SIMPLEX VIRUS TYPE‐1 REPLISOME
Authors: Boehmer Paul E , Bogani Federica , Corrediera Ilsa , Fernandez Virneliz
Published: June 2021

We are interested in studying events at the HSV‐1 replication fork, processes that are dependent on six core viral gene products: the heterodimeric DNA polymerase‐clamp assembly (UL30/UL42), the DNA helicase‐primase (UL5/UL8/UL52) and the single‐strand DNA binding protein (ICP8/UL29). In an effort to identify additional factors that are active at the viral DNA replication fork, we searched for polymerase interacting proteins. We identified the viral uracil DNA glycosylase (UL2) as a protein that co‐purifies with the polymerase through numerous chromatographic steps, an interaction that was verified by co‐immunoprecipitation. We found that the interaction with UL2 is mediated through the UL30 subunit. Moreover, UL2 co‐localizes with UL30 to nuclear viral replication compartments. In addition, we have observed that UL30 specifically cleaves abasic DNA in a manner independent of divalent cation, resembling the activity of a lyase. This property would make the HSV‐1 DNA polymerase functionally analogous to the mitochondrial DNA polymerase gamma in as much as it possesses DNA synthesis, proofreading and lyase activities. These findings raise the interesting notion that the viral replisome promotes replication‐coupled base excision repair.
Plasmid Involvement In Biodegradation Of Polycyclic Aromatic Hydrocarbons By Bacteria Isolated From Nigeria
Authors: Igwo‐Ezikpe Miriam Nwanna , Gbenle George Olabode , Okpuzor Joy , Ilori Joy Matthew , Idoko Joy Ejiofor , Amosa Joy Adebola , Soyoye David Olasukanmi
Published: June 2021

Six Gram‐negative bacteria isolates designated PYR 1, FLU 1, FLU 2, FLU 3, FLU 4 and FLU 5 capable of utilizing fluoranthene, pyrene and phenanthrene as sole source of carbon and energy were isolated from diesel‐oil contaminated soil in Nigeria and evaluated for the role of plasmid in biodegradation of polycyclic aromatic hydrocarbons (PAHs). 97% degradation of fluoranthene and pyrene (100mg/L) were observed for all the isolates whereas phenanthrene (100mg/L) was degraded to undetectable level by HPLC analysis within 8days. Co‐metabolizing of fluoranthene and pyrene in the presence of phenanthrene showed statistical significant difference (p<0.05) in degradation and total viable cell count compared to degradation of fluoranthene and pyrene. Large plasmids (>500bp) were detected in isolates; PYR 1 (880.24bp), FLU 2 (1521.42bp and 880.24bp), FLU 3 (1521.42bp and 880.24bp), FLU 4 (1521.42bp and 880.24bp) and FLU 5 (34693.65bp, 1521.42bp and 880.24bp) while FLU 1 had no plasmid under the condition of assay. SDS‐curing of the isolates lead to complete loss of plasmid and PAHs degradation activity. This study suggests that these mega plasmids could be responsible for the isolates degradation of PAHs.
Proteasome malfunction increases autophagosomes in cardiomyocytes and mouse hearts
Authors: Zheng Qingwen , Mizushima Noboru , Wang Xuejun
Published: June 2021

Background:

Autophagy and the ubiquitin‐proteasome system (UPS) are two main routes for intracellular protein degradation. The effect of proteasome malfunction on autophagy in cardiomyocytes and the heart remains to be demonstrated.

Methods & results:

Proteasomal function in cultured neonatal ventricular myocytes (NRVMs) or mouse hearts is manipulated using pharmacological and/or genetic means. The resultant effects on autophagy are assessed by two methods: LC3‐II/LC‐I ratio measured by western blot analysis; autophagy index measured via confocal microscopic analysis of GFP‐LC3 distribution. Cardiac‐restricted expression of a 7‐amino‐acid deletion mutation of the desmin gene (D7‐des) has been linked to human desminopathy which develops aberrant desmin aggregation and severe proteasome malfunction in heart muscle cells. D7‐des increases LC3‐II/LC3‐I ratio and punctate GFP‐LC3 in mouse heart as early as 2 month. The increased autophagosomes is also detected in mouse heart 12 hours after an intravenous injection of proteasome inhibitor MG262 (5umol/kg). In NRVMs, proteasome inhibition by MG132 (1uM) increases the LC3‐II/LC3‐I ratio and autophagic flux measured as the difference of autophagosomes in the absence and presence of lysosomal protease inhibitors.

Conclusions:

Pharmacological and/or genetic proteasome inhibition increase autophagosomes in both NRVMs and mouse hearts.
Biotin Synthase: A role for the FeS cluster assembly chaperone HscA in regenerating the [2Fe‐2S] cluster substrate
Authors: Jarrett Joseph T , Reyda Michael R
Published: June 2021

Biotin synthase is an is an S‐adenosylmethionine (AdoMet) radical enzyme that catalyzes the addition of sulfur to dethiobiotin, generating the thiophane ring of biotin. Biotin synthase contains two FeS clusters: a [4Fe‐4S]2+ cluster is involved in radical generation via reductive cleavage of the AdoMet sulfonium, and a [2Fe‐2S]2+ cluster provides the sulfur for the biotin thiophane ring. During a single turnover in vitro, the [2Fe‐2S]2+ cluster is destroyed and the enzyme rendered inactive, although in vivo data suggest that each enzyme molecule undergoes multiple turnovers. Using equilibrium binding methods, we report that the FeS cluster chaperone HscA forms a tight complex with biotin synthase. Further, a 1:1 complex of biotin synthase and HscA also binds the FeS cluster scaffold protein IscU, generating a 3‐way complex. Using mass spectrometry methods, we have identified 3 loop regions near the biotin synthase active site that bind to 3 separate sites on HscA. We suggest a specific role for HscA in targeting IscU to low‐abundance iron‐sulfur proteins such as biotin synthase, facilitating regeneration of the active FeS clusters.
Catalytic metal ion switching in zinc‐dependent deacetylases
Authors: Gattis Samuel Guy , Hernick Marcy , Gantt Stephanie L. , Fierke Carol A.
Published: June 2021

The metallohydrolase UDP‐3‐O‐((R)‐3‐hydroxymyristoyl)‐N‐acetylglucosamine deacetylase (LpxC) catalyzes the first committed step in Lipid A biosynthesis, a critical component of the outer leaflet of gram‐negative bacteria. LpxC activity has previously been shown to be activated by a divalent transition metal ion (Zn(II), Co(II), or Ni(II)) and has been described as a mononuclear zinc enzyme. Fe(II) has not yet been considered as the in vivo catalytic metal ion. However, LpxC reconstituted with Fe(II) exhibits increased catalytic activity vs. Zn(II) and the activity of LpxC in E. coli cell extracts is oxygen‐sensitive. When LpxC is purified anaerobically from E. coli grown in media with defined metal concentrations, the ratio of iron/zinc bound to LpxC varies linearly with the iron/zinc ratio in the cell lysate, suggesting that the catalytic metal ion of LpxC depends on the cellular conditions. Binding of Fe2+ vs. Zn2+ has been measured and compared to free cellular metal concentrations. Two possibilities arise from these results – either LpxC lacks specificity for its active site metal, or the enzyme is subject to a regulatory mechanism that responds to intracellular metal concentrations. Preliminary results indicate another Zn‐dependent deacetylase, human histone deacetylase 8 (HDAC8), may also be able to use Fe(II) in vivo, suggesting this may be a broader theme in “zinc”‐dependent deacetylases.
Zinc metallation of yeast alkaline phosphatase and its protein level response to zinc
Authors: Qiao Wei , Ellis Charissa D. , Wu Chang‐yi , Eide David J.
Published: June 2021

Alkaline phosphatase of Saccharomyces cerevisiae (ScALP/Pho8) is a zinc‐dependent vacuolar enzyme. ScALP moves through the secretory pathway as a zymogen before being activated by the Pep4 vacuolar protease. Interestingly, we found that both ScALP activity and protein level are affected by the availability of zinc ions; both decrease dramatically when cells are Zn limited and this effect is very specific to ScALP. Decreased ScALP level in low zinc was dependent on Pep4 suggesting that the apoprotein is degraded. By studying ScALP activity in wild type and various Zn transporter mutants, we found that ALP accumulation and activity is mainly dependent on the major vacuolar Zn transporters Zrc1 and Cot1, rather than zinc transporters acting upstream in the secretory pathway. Our experiments show that this transporter dependency is not due to direct channeling of zinc from these transporters to the enzyme nor is proteolytic activation in the vacuole required for metallation. Our current hypothesis is that the vacuolar environment is specifically favorable for ScALP metallation for two reasons. First, the level of zinc in the vacuole may be higher than in upstream compartments of the secretory pathway. Second, in vitro studies indicate that the low pH of the vacuole (5.5) promotes metal binding relative to more alkaline conditions. Failure to metallate ScALP in the vacuole results in its degradation by vacuolar proteases.
VPO1 and VPO2: identification and characterization of two new heme‐containing peroxidases
Authors: Cheng Guangjie , Salerno John C , Cao Zehong , Pagano Patrick J , Lambeth J David
Published: June 2021

Heme‐containing peroxidases play diversified roles in innate immunity, synthesis of thyroid hormone and pathogenesis of inflammatory diseases. Based on the recent discovery of new ROS‐generating Nox enzymes, and the known relationship between classical Nox enzymes and peroxidases, we hypothesized the existence of novel peroxidases. Using Duox1 peroxidase domain, we carried out homology searching of the NCBI databases. Two novel heme‐containing peroxidases were identified in humans and mice. One shows highest tissue expression in heart and vascular wall, and is termed VPO1 (vascular peroxidase 1). A second, VPO2, present in humans but not in mice, is 63% identical to VPO1, and is most highly expressed in heart. VPO1 and VPO2 show 42% and 38% identity to myeloperoxidase (MPO), respectively. A molecular model of the peroxidase region of VPO1 shows a structure that is highly similar to other peroxidases, including a conserved heme‐binding cavity, critical catalytic residues, and a calcium‐binding site. Absorbance spectra of VPO1 are similar to lactoperoxidase. VPO1 purified from heart or expressed in HEK cells is catalytically active. When co‐expressed in cells, VPO1 can utilize H2O2 produced by Nox enzymes. VPO1 may carry out peroxidative reactions in the vascular system previously attributed exclusively to MPO. This work was supported by the AHA grant 0635122N and NIH grant CA105116.
Identification of a novel ketogenic enzyme
Authors: Montgomery Christa L. , Miziorko Henry M.
Published: June 2021

Ketogenic metabolism has been associated with mitochondria, which contain HMG‐CoA synthase, HMG‐CoA lyase, and 3‐hydroxybutyrate dehydrogenase. A bioinformatic approach has identified several mammalian proteins, designated as HMGCLL1 (HMG‐CoA lyase like), as putative members of the HMG‐CoA lyase family. These proteins are characterized by a unique 37 residue N‐terminus that contains a potential myristoylation site but not a mitochondrial leader sequence. In order to test the function of these proteins, full length human HMGCLL1 coding sequence has been isolated from a MGC repository clone and introduced into expression vectors. Expression in Pichia pastoris of the 340 residue enzyme with a C‐terminal his‐tag has facilitated protein purification and demonstration of substantial HMG‐CoA cleavage activity (>140 U/mg). Substrate Km (28 ± 3 μM) and the divalent cation requirement (for Mg2+, Km = 47 ± 9 μM) have been evaluated. The G2A mutant protein, for which myristoylation is precluded, has been expressed and shown to retain activity; the mutation does not affect substrate Km (24 ± 2 μM). Results suggest that HMGCLL1 could function as an extramitochondrial HMG‐CoA lyase. NIH DK21491
The binding and activation of Nitric Oxide Synthase by modified central linker and “phosphomimetic” Calmodulin proteins
Authors: Israel Odi K , Spratt Donald E , Taiakina Valentina , Guillemette J. Guy
Published: June 2021

Calmodulin (CaM) is a ubiquitous calcium‐binding protein that is known to interact and regulate hundreds of intercellular functional proteins. The promiscuity of CaM is partially due to its highly flexible central linker region that separates its two terminal domains which interact with its target protein. Modification of the central linker region of CaM can result in changes in the proximity of the two terminal domains, their relative orientation, binding affinity and activation of the CaM target proteins. The present study was designed to investigate the role of the central linker of CaM by introducing select mutations, including phosphomimetic residues, into the CaM central linker region. The effects of these modifications on the binding and activation of the three mammalian nitric oxide synthase (NOS) isozymes have been investigated using biophysical techniques including circular dichroism, FTIR and gel shift mobility assays. Assays have been performed to assess the effect that modified CaM linker residues have on NOS enzyme activation and electron transfer. This research is supported by NSERC (J.G.G.)
Novel dithiolethione (DTT) compounds abrogate S6K1‐dependent insulin resistance
Authors: Kim Young Mi , Bae Eun Ju , Yang Yoon Mee , Kim Sang Geon
Published: June 2021

Current therapeutic interventions of insulin resistance leading to several metabolic disorders are limited and therefore expected to be advanced. This study investigated the effects of oltipraz, a prototype dithiolethione, and new DTTs, on hepatic and systemic insulin resistance and the molecular basis. DTTs were tested on TNFα‐induced insulin resistance and glucose homeostasis in vitro and in vivo by immunoblot, plasmids transfection and kinase assays. The effects of DTTs were also tested in the cellular model of insulin resistance induced by hyperosmotic stress. An experiment using dominant negative S6K1 supports the essential role of S6K1 in the hyperosmolarity‐stimulated IRS1 phosphorylation. DTTs inhibited the S6K1 activity, a downstream of mTOR leading to the protection of insulin signals. Moreover, DTTs activated AMPK. S6K1 inhibition and protection of insulin signaling by DTT (oltipraz) treatment was abolished by either dominant negative mutant of AMPK or constitutively active S6K1 transfection. DTT (oltipraz) prevented hepatic insulin resistance in C57BL/6 mice challenged with endotoxin (or TNFα) and lowered blood glucose level in lepob/ob mice and in mice fed a high‐fat diet. In conclusion, we demonstrated the potential therapeutic role of intervention of S6K1 signaling with the identification of a new class of DTT compounds potentially effective for the treatment of insulin resistance.
MICE WITH A TISSUE SPECIFIC DELETION IN THE GENE FOR PHOSPHOENOLPYRUVATE CARBOXYKINASE (PEPCK) ARE INSULIN RESISTANT
Authors: Milllward Carrie Ann , Heaney Jason D. , Gornicka Agnieszka , Croniger Colleen M.
Published: June 2021

Obesity has become a world‐wide health problem that is associated with metabolic syndrome, including insulin resistance and the development of type 2 diabetes. The acute elevation of free fatty acid (FFA) levels that occurs in obesity is controlled by the triglyceride/FFA cycle. We have investigated the role of the triglyceride/free fatty acid cycle in the development of insulin resistance using a mouse model with a deletion of the peroxisome proliferator activator response element (PPARE−/−) in the cytosolic form of the phosphoenolpyruvate carboxykinase (PEPCK‐C) gene promoter. This mutation results in ablation of PEPCK‐C gene expression in white adipose tissue leading to insulin resistance and glucose intolerance. We measured the degree of insulin resistance through a hyperinsulinemic‐euglycemic clamp and portal vein injections of insulin. The insulin signaling cascade was severely blunted in the adipose tissue. In addition, the metabolic parameters including triglyceride, free fatty acid, β‐hydroxybutyrate, and cholesterol levels are significantly higher in the PPARE−/− mice compared to the wild type mice. Newborn pups with the deletion of the PPARE site demonstrated altered glucose homeostasis as early as day 14 of birth. The data indicates that the PPARE site in the promoter of the PEPCK‐C gene plays an important role in the regulation of glucose homeostasis and the triglyceride/FFA cycle.
Hyperglycemic concentrations of glucose directly affect PPARα
Authors: Hostetler Heather A. , Huang Huan , McIntosh Avery L. , Atshaves Barbara P. , Payne H. Ross , Kier Ann B. , Schroeder Friedhelm
Published: June 2021

The pathophysiology of diabetes exhibits elevated glucose and long chain fatty acids (LCFA). Since LCFA are high affinity ligands for the peroxisome proliferator‐activated receptor alpha (PPARα), which regulates transcription of multiple genes in fatty acid and glucose metabolism, it was hypothesized that glucose might also serve in this role. Indeed, PPARα also functions as a high affinity glucose sensor, exhibiting high glucose sensitivity prior to any physical manifestation, such as autoglycation, seen much later in chronic exposure to high glucose. Circular dichroism, co‐immunoprecipitation, binding assays, and transactivation assays were utilized to examine the effects of hyperglycemic concentrations of glucose on PPARα. High glucose altered: (i) conformational of PPARα; (ii) lipid binding to PPARα; (iii) interaction of co‐factors, such as the liver fatty acid binding protein (L‐FABP), with PPARα; (iv) altered the nuclear distribution of a fluorescent LCFA—ligands bound and transported by L‐FABP; (v) increased PPARα transactivation, and (vi) hyperactivated PPARα in the presence of peroxisome proliferators. These data suggest that PPARα is an endogenous sensor not only of LCFA, but also glucose, and may be a drug target for glucose as well as LCFA regulated complications in diabetes. Supported in part by the NIH DK41402 (FS, ABK); DK066732 & DK77573 (HAH).
Leptin is essential for normal development in zebrafish
Authors: Londraville Richard Lyle , Liu Qin
Published: June 2021

We have cloned and sequenced leptin from zebrafish (Danio rerio). The coding region consists of 594 bp (with a 20bp signal peptide), and the predicted amino acid sequence is ~20% identical to mammalian leptins, 12% to Fugu leptin, and 60% to carp leptins. Leptin message was amplified from brain, heart, ovary, and liver cDNAs. Zebrafish leptin appears to be expressed early in development, starting at 9–10 hours post fertilization (which is earlier than other ectotherms and mammals). We injected morpholino antisense oligonucleotides directed against the ATG start site of zebrafish leptin into 1–2 cell stage embryos and compared their development to controls. Leptin morphants expressed dramatically altered (and generally reduced) development of brain, eye, heart, otic vesicle, pectoral limb buds, caudal fin, and pigmentation. Leptin morphants showed little yolk absorption, even at 70 hpf, and had significantly lower heart rate. It is unlikely that the effects of leptin knock‐down are simply due to slow development, as these effects persisted even to 7 days post‐fertilization. These data indicate that leptin has pleiotropic effects in zebrafish, and that it is essential for normal development. Supported by a Univ. Akron Faculty Research Grant and Integrative Bioscience grants to RLL and NIH R15 Ey13879‐03 to QL and RLL.
Alpha‐helix formation studied with pressure perturbation calorimetry
Authors: Sharaf Naima Gabriela , Fuchs Kristin M , Tripathy Ashutosh , Pielak Gary J
Published: June 2021

Pressure perturbation calorimetry (PPC) is a relatively unexplored technique that quantifies the temperature dependence of a solute's thermal expansion coefficient. PPC data provides information about solute‐solvent interactions. To investigate the formation of an alpha‐helix in solution, PPC was used to characterize helix‐solvent and random coil‐solvent interactions. Helix and random coil models were obtained by using two peptides, Ac‐DKDGYISAAE‐NH2 (P12) and Ac‐DKDGDGYISAAEAAAQ‐NH2 (P16) which contain the same sequence for the first twelve amino acids. Circular dichroism spectropolarimetry was used to determine the helix content. In the presence of La3+ the last four residues of P16 form an alpha‐helix. When La3+ is absent, both peptides are random coils, with a small amount of residual helix, which decreases with increasing temperature. The temperature dependence of the corrected thermal expansion coefficient suggests that the helix‐solvent interactions are more hydrophobic, while random coil‐solvent interactions are more hydrophilic. Therefore, alpha‐helix formation is marked by a change from hydrophilic to hydrophobic solvent interaction. This result is consistent with the accepted view that helix formation results in a net loss of hydrophilic surface area.
Tracing studies in rat provide sensitive gene expression data for neurons innervating the bladder and urethra
Authors: Brinkmann Hannah , Miner Wesley Dennis , McMurray Gordon , Walsh Roddy , Gardner Clare Evelyn , Ward Sari Annelie , Christy Clare
Published: June 2021

The aim of the study was to explore novel tissue based techniques to generate gene expression profiles of both afferent (sensory) and efferent (motor) neurons that specifically innervate the rat bladder and urethra.

Retrograde neuronal tracing studies in combination with laser capture microdissection (LCM) and microarray analysis were used to generate gene expression profiles of both afferent and efferent neurons that specifically innervate the rat bladder and urethra. A complete rat study (n=12) was performed in which fluorescently labelled cholera toxin B was injected into the bladder and urethra. Traced neurons were isolated by LCM, RNA was extracted, amplified and analysed by microarray.

Extensive analysis of all the data derived from this study give very sensitive and powerful gene expression data from only 50–100 microdissected neurons. Expression of sensory neuron specific markers could be confirmed in dorsal root ganglion neurons whereas expression of genes involved in motor function of the lower urinary tract could be confirmed in efferent neurons e.g. Onuf's Nucleus.

The resulting gene expression profile will increase our understanding of signalling pathways involved in the control of the lower urinary tract. Data derived from this study will not only be relevant for safety and efficacy but will also enable us to identify targets for the treatment of urinary frequency and urge incontinence (UFUI).
How to build a signalling organelle: A molecular pathway for biogenesis of the ciliary membrane
Authors: Nachury Maxence
Published: June 2021

The primary cilium is a hair‐like projection present at the surface of nearly every vertebrate cell. How such a complex organelle is assembled remains an open question that has become all the more relevant with the emergence of primary cilium dysfunction as the central pathomechanism in a range of inherited disorders characterized by obesity, brain malformations, retinal degeneration, kidney cysts and skeletal abnormalities.

Strikingly, all these symptoms coincide in Bardet‐Biedl Syndrome (BBS). However, despite the identification of twelve BBS genes, the molecular basis of BBS had remained elusive. Last year, the discovery of a stable complex of seven BBS proteins (the BBSome) considerably simplified the apparent molecular complexity of BBS. Most tellingly, the BBSome associates the ciliary membrane and with Rabin8, the guanosyl exchange factor for the small GTPase Rab8 and Rab8‐GTP enters the primary cilium to promote extension of the ciliary membrane.

Thus, BBS is likely caused by defects in vesicular transport to the primary cilium. With this realization comes a flurry of questions that will be addressed in my presentation: What is the precise molecular activity of the BBSome and Rab8 in this process? What are the effectors of Rab8 involved in ciliogenesis? Does the ARF‐like GTPase ARL6/BBS3 also function in vesicular transport to the cilium?
Effects on Epidermal Actin Composition in Wounded Drosophila Grainy head Zygotic Mutants
Authors: Sandoval Efren G. , Juarez Michelle T. , McGinnis William
Published: June 2021

Epidermal wound healing is a complex process whose genetic controls are poorly understood, despite decades of study. Within the fruit fly Drosophila, a single epithelial cell layer secretes the hexapod cuticle armor whose primary function is to support and protect the organism from external threat. Several genes linked to the regulation of cuticle formation and repair of epidermal wounds have been found. The transcription factor Grainy head plays an important role in epidermal development and barrier function in a wide range of model organisms, including mammals. Mutations in Drosophila Grainy head disrupt cuticular integrity and response to epidermal wounds. In our quest to study the effects of cellular migration in Grainy head mutants, we have utilized actin‐GFP reporter genes that label cell borders in living Drosophila embryos. After mechanical or laser induced wounds in wild‐type and Grainy head mutant embryos, we compare the two genotypes. Powerful confocal microscopy and live imaging can illuminate potentially different cell migratory behaviors in the Grainy head mutants, as well as sub‐cellular differences in actin distribution. We hypothesize the Grainy head mutation will compromise actin microfilament integrity at the wound site to cause inappropriate cellular migration during epidermal closure.
Positive cooperativity in catalysis of pyrene 1‐hydroxylation by cytochrome P450 1A2
Authors: Sohl Christal Dyane , Isin Emre M. , Eoff Robert L. , Stec Donald F. , Guengerich F. Peter
Published: June 2021

Cytochrome P450s (P450s) are a class of heme‐containing enzymes that oxidize endogenous and exogenous compounds such as steroids and drugs. P450s generally show Michaelis‐Menten kinetics, but several P450s display non‐hyperbolic behavior for some substrates. Prior work in this laboratory identified cooperativity in oxidation of 1‐alkoxy‐4‐nitrobenzenes by rabbit P450 1A2. We analyzed the kinetics associated with the oxidation of P450 1A2 substrates α‐naphthoflavone (αNF) and pyrene. While rates of formation of the 5,6‐epoxide from αNF generate hyperbolic plots, rates of pyrene 1‐hydroxylation yield sigmoidal behavior. Homology modeling of rabbit P450 1A2 using a human P450 1A2 crystal structure shows that only one αNF molecule fits but two pyrenes may be stacked in the P450 1A2 active site, possibly explaining the positive cooperativity. Binding rates measured using stopped‐flow kinetics show that interactions with the ligand and P450 1A2, as indicated by ligand fluorescence quenching, precede changes associated with the heme spectrum. Circular dichroism studies suggest rapid conformational changes upon ligand binding. Kinetic fitting of these data suggests a possible three‐step mechanism for pyrene oxidation in which pyrene monomer binds followed by dimer formation and oxidation. (Supported by USPHS R37 CA090426, T32 ES007028).
Potential lipid microdomains of the endoplasmic reticulum: how does this affect the P450 system
Authors: Brignac‐Huber Lauren Michelle , Reed James R , Backes Wayne L
Published: June 2021

Little is known about the organization of NADPH‐cytochrome P450 reductase (CPR) and multiple P450 enzymes in the endoplasmic reticulum (ER). Although these proteins are known to form a 1:1 molar complex in the ER, in most tissues the P450 concentration exceeds that of CPR by about a 20:1. The goal of this study is to examine how components of the P450 system are organized, and to determine if they are heterogeneously distributed in lipid microdomains of the ER. Liver microsomes from untreated rabbits and those pretreated with P450 inducing agents were partially solubilized with 1% Brij 98 at 4°C, and centrifuged, separating the detergent‐resistant (pellet) and ‐soluble (supernatant) membranes. The supernatants and pellets were then examined by immunoblotting to determine the regional distribution of CPR and P450s in the membrane. Both CPR (∼90%) and CYP1A2 (∼50%) remained in a region of the membrane that is resistant to solubilization, whereas CYP2E1 and CYP2B4 were located in a more detergent‐sensitive region. Lipid analysis of these samples illustrated that the pellet was enriched with saturated and mono‐unsaturated fatty acids of phosphatidylcholine and sphingomyelin while the supernatant was enriched with polyunsaturated fatty acids. These results suggest that the proteins of the ER are not randomly distributed in the membrane, but appear to exist in specific lipid microdomains. (supported by NIEHS ES00434)
Post‐Translational Regulation of Mitochondrial Steroidogenic Cytochrome P450s by NAD+‐Dependent SIRT Deacetylases
Authors: Li Donghui , Rogers Sharon , Dammer Eric B
Published: June 2021

Cortisol is synthesized in the adrenal cortex in response to adrenocorticotropin (ACTH) signaling from cholesterol via a series of reactions that are catalyzed by members of the cytochrome P450 superfamily and hydroxysteroid dehydrogenases. Both the first and last steps of cortisol biosynthesis occur in mitochondria. Based on our previous findings demonstrating that activation of the ACTH signaling cascade changes the ratio of NAD(P)+:NAD(P)H in adrenocortical cells, we hypothesized that these changes in pyridine nucleotide concentrations may regulate the activity of the mitochondrial NAD+‐dependent SIRT deacetylases. We show that both P450scc (side chain cleavage enzyme) and P450 11β hydroxylase are acetylated in mitochondria of H295R human adrenocortical cells. Stimulation with ACTH or dibutyryl cAMP increased the levels of acetylated P450scc and P450 11β, whereas activating SIRT activity using resveratrol decreased P450 acetylation. Resveratrol also increased P450scc and P450 11β protein expression, indicating that acetylation may modulate protein stability. These studies identify a role for NAD+‐dependent SIRT deacetylases in regulating the expression of mitochondrial steroidogenic P450s.
Inhibitor Complexed Structures of the Cytochrome bc1 from the Photosynthetic Bacterium Rhodobacter Sphaeroides
Authors: Xia Di , Esser Lothar , Yu Chang‐An
Published: June 2021

The cytochrome bc1 complex (bc1) is a major contributor to the proton motive force across the membrane by coupling electron transfer to proton translocation. The crystal structures of wild type and mutant bc1 complexes from the photosynthetic purple bacterium Rhodobacter sphaeroides (Rsbc1), stabilized with the quinol oxidation (QP) site inhibitor stigmatellin alone or in combination of with the quinone reduction (QN) site inhibitor antimycin, were determined. The high quality electron density permitted assignments of a new metal‐binding site to the cytochrome c1 subunit and a number of lipid and detergent molecules. Structural differences between Rsbc1 and its mitochondrial counterparts are mostly extra membranous and provide a basis for understanding the function of the predominantly longer sequences in the bacterial subunits. Functional implications for the bc1 complex are derived from analyses of 10 independent molecules in various crystal forms and from comparisons with mitochondrial complexes.
Identification of G1‐Regulated Genes in Normally Cycling Human Cells
Authors: Beyrouthy Maroun J. , Alexander Karen E. , Baldwin Amy , Whitfield Michael L. , Bass Hank W. , McGee Dan , Hurt Myra M.
Published: June 2021

Obtaining synchronous cell populations is essential for studies of the cell cycle. Methods such as serum withdrawal or use of drugs which block cells at specific points in the cell cycle almost certainly alter cellular events upon re‐entry into the cell cycle. Here we use a robotic mitotic shake‐off apparatus to select cells in late telophase of mitosis. Using two separate microarray experiments we examined gene activity in early G1: one isolated RNA hourly and the second isolated RNA every 15 minutes. We have also included data obtained by a variety of experimental methods to verify synchrony of these cell populations, including BrdU uptake, FACS, and microarray analyses of histone gene activity as well as activity of stress response genes. Our approach enabled us to identify a unique set of genes whose functions are currently unknown, in cells progressing normally through the cell division cycle. This group of genes may contain future targets for drug development and treatment of human disease.
Heat stress prevents overload‐induced hypertrophy of myosin heavy chain type I fiber area, but does not alter structural damage or localized patterns of cellular replication
Authors: Frier Bruce Charles , Locke Marius
Published: June 2021

Heat stress (HS) inhibits compensatory hypertrophy in the rat plantaris muscle by preventing the accumulation of myosin heavy chain type I (MHC‐I) protein. However, it is unknown whether HS also affects MHC‐I fiber area, structural changes, nuclear accumulation and localized satellite cell activation in overloaded plantaris. Thirty Sprague‐Dawley rats underwent synergistic ablation and were divided into non‐heat stressed (NHS) and heat stressed (15‐min, 42°C) groups. The contralateral limb was used as a control. Muscles were assessed after 1, 3 and 7 days of functional overload. MHC‐I fiber area was not significantly different between NHS and HS plantaris muscles after 1 day of overload (0.228 mm2vs. 0.172 mm2,respectively); however, MHC‐I fiber area was significantly greater in the NHS plantaris compared to HS plantaris after 3‐ and 7‐days (0.364 mm2vs. 0.193 mm2 and 0.442 mm2vs. 0.237 mm2, respectively). Localized Myf‐5, PCNA and MyoD suggested a similar pattern of satellite cell activation in both NHS and HS plantaris muscles on all days. Overload induced progressive structural damage and massive accumulation of nuclei in the deep portion of the plantaris regardless of a prior HS. These results suggest that HS may prevent an overload‐induced increase in MHC‐I fiber area by restricting protein expression in existing fibers, rather than via altered replication signals, tissue strain or nuclear accumulation.
Visualization of an interaction between Ras and Ras‐binding domain in living cells by bimolecular fluorescence complementation.
Authors: Tsutsumi Kaori , Tsuda Masumi , Kawaguchi Hideaki , Ohba Yusuke
Published: June 2021

Recent accumulating evidences converge on the importance of spatiotemporal regulation to determine the specificity and diversity of intracellular signalings. Here, we constructed a system to analyze the protein‐protein interaction between Ras and its effectors in living cells by using bimolecular fluorescence complementation (BiFC) system, in which the dynamic interactions could be observed as an increase in fluorescent intensity. We used H‐Ras and Ras‐binding domains (RBDs) of Raf, PI3K, and RalGDS, and found that the binding of Ras to the effector molecules was specific in a manner dependent on subcellular localization. Ras and Raf bound predominantly at the plasma membrane followed by at Golgi apparatus by epidermal growth factor (EGF) stimulation, as reported previously. On the other hand, the interaction between H‐Ras and PI3K‐RBD upon EGF stimulation was observed at the endosome‐like vesicles, on which early endosome autoantigen 1 (EEA1), a marker for endosome, were localized. This result indicates that PI3K activated by Ras may play an important role in the production of phosphatidylinositol triphosphate (PIP3) at the endosome, wherein PIP3 is highly enriched. Since we used only RBD but not functional domains of Raf, PI3K and RalGDS in the present study, it will be thereby necessary to clarify the mechanism how RBDs dictate the selectivity of localization of H‐Ras‐PI3K binding.