bluuebunny/crossref_metadata_2025 huggingface dataset:
Studies of Human Rad51 and Human Rad54 Promoted Strand Exchange / Branch Migration Reactions by an Innovative FRET System
Authors: Urena Damian E. , Zhang Zhaoquing , Chen Junghuei
Published: June 2021

Human Rad54 (hRad54) and Human Rad51 (hRad51) proteins play crucial roles during homologous recombination. Human Rad54 belongs to the Swi2/Snf2 family of DNA dependent ATPases. Human Rad51 forms a right‐handed ssDNA filament and promotes homologous pairing and strand exchange. It has been found that hRad54 promotes branch migration in an ATPase dependent manner. Here, we have developed a FRET‐ DNA assay in order to sensitively monitor these two homologous recombination processes in real time. We have focused on the interactions between hRad54 and hRad51 and their effects on hRad51 promoted strand exchange as well as hRad54 promoted branch migration. Our results indicate that hRad54 greatly stimulates the overall efficiency of hRad51 mediated strand exchange probably by enhancing the rate of branch migration after the formation of Holliday junction. We also find that hRad54 can promote branch migration through various degrees of mismatches more efficiently than the same reactions promoted by hRad51. The results imply that the main role of hRad51 in strand exchange is in the homology recognition and initial strand exchange to form a three‐ or four‐stranded Holliday junction. After the formation of the Holliday junction, then hRad54, which can promote more efficient branch migration, could take over to promote long range strand exchanges.
Bacterial SSB interactome: Structural basis of interaction between E. coli Exonuclease I and SSB and exploitation of this interaction to identify novel anti‐bacterial compounds
Authors: Lu Duo , Bernstein Douglas , Keck James
Published: June 2021

Bacterial single‐strand (ss) DNA‐binding proteins (SSBs) play essential protective and organizational roles in genome biology. To shield ssDNA from potential damage, SSBs bind tightly to ssDNA that arises during the course of DNA replication and repair. Far from being inert, ssDNA/SSB complexes are active DNA processing centers where at least a dozen different enzymes gain access to genomic substrates by exploiting direct protein‐protein interactions with SSB. In all cases examined to date, the last 8‐10 residues of SSB's flexible C‐terminus (SSB‐Ct) form a docking site for heterologous proteins. How proteins bind to the SSB‐Ct sequence and how these essential interactions affect the activities of genome maintenance enzymes remains poorly defined. Moreover, given the essential nature of interactions with SSB, inhibitors that block association with SSB could provide novel avenues for antibiotic development. In our poster, we describe two recent advances from our lab that shed light on these aspects of genome biology. The first defines the physical basis underlying interaction between E. coli Exonuclease I and SSB through a crystallographic approach. The second uses a high‐throughput screen to identify small‐molecule inhibitors that disrupt formation of Exonuclease I/SSB complexes. These inhibitors have robust selective anti‐bacterial properties.
Structure and activity of human DNA polymerase kappa bypass of 8‐oxoG
Authors: Eoff Robert Lawton , Irimia Adriana , Egli Martin , Guengerich Frederick Peter
Published: June 2021

Formation of reactive oxygen species and subsequent reactions with biological macromolecules is central to processes that disrupt normal cellular activity and can lead to cellular senescence and tumorigenesis. Here, we present a detailed analysis of the activity and specificity of human DNA polymerase kappa (hpol kappa) bypass opposite the major oxidative adduct 7,8‐dihydro‐8‐oxo‐guanosine (8‐oxoG). Crystal structures of hpol kappa in complex with dATP opposite 8‐oxoG modified DNA reveal a Hoogsteen pairing mode, and complexes with dGTP opposite 8‐oxoG show that hpol kappa does not adopt the "Type II" frameshift mode that is commonly observed in the archaeal DinB homolog Dpo4. Kinetic analysis suggests that hpol kappa ?forms primarily non‐productive complexes when 8‐oxoG is present and greatly favors insertion of dATP opposite the lesion. Mutating the L508 residue in the little finger domain of hpol kappa to lysine results in a shift towards more accurate bypass of 8‐oxoG, reminiscent of results observed with Dpo4. These results provide insight into error‐prone bypass of oxidative damage and reveal the importance of electrostatic interactions between the little finger domain and template DNA during bypass of 8‐oxoG.
Workshop on developing biochemistry concept inventories
Authors: Sears Duane W , Anderson Trevor R
Published: June 2021

The effective education of biochemistry students requires that educators develop rigorous knowledge and assessment standards. The first standard demands clear articulation of the core knowledge students are expected to master in ways that anticipate what they may need to know as practitioners in this field in the future. The second standard demands that effective evaluative tools be developed for monitoring students' working mastery of that knowledge, during and after their formal education. One interwoven approach for achieving these standards is through concept inventories (CIs). CIs effectively help educators deconstruct the "big ideas" of their discipline into underlying fundamental concepts that then serve as the basis for evaluative testing or assessments. CIs are emerging in many biological disciplines but the development of biochemistry CIs is just beginning. Participants at this workshop will be given an opportunity to make brief presentations about their activities and interests in this area, and they will engage in a group activity aimed at exploring avenues for biochemistry CI development and assessment in order to promote greater awareness of the key issues here.
Sequence Dependence of Stability for Group II Single Nucleotide Bulge Loops
Authors: Serra Martin , Lim Geoff , Hanson Ryan , Manni MIchelle , Estes Julie , Kelpec Kelly , Gratton Jess , Frattini Greg
Published: June 2021

Thirty RNA duplexes containing group II single nucleotide bulge loops were optically melted in 1M NaCl, and the thermodynamic parameters DH°, DS°, DG°37, and TM for each sequence were determined. Group II bulge loops have two or more consecutive identical nucleotides at the bulge position such that there is ambiguity as to which nucleotide is the bulge. Sequences from this study were combined with sequences from a previous study (Znosko et al., (2002) Biochemistry 41, 10406‐10417), thus examining all possible group II single nucleotide bulge loop sequences. The combined data were used to develop a nearest‐neighbor model to predict the free energy of an RNA duplex containing a single nucleotide bulge. The free energy increments for the introduction of a group II bulge loop range from 0.9 and 7.3 kcal/mol. The introduction of the bulge was found in all cases to destabilize the duplex. As observed previously for Group I bulge loops (Blose et al., (2007) Biochemistry 46, 15123‐15135), neither the identity of the bulge nor its nearest neighbor had an effect on the destabilization of the duplex by the bulge. The influence of the bulge on the duplex stability was primarily affect by non‐nearest‐neighbor interactions. Specifically, there is a direct correlation between the destabilization of the duplex and the stability of the stems adjacent to the bulge.
Bioluminescent assay for quantitation microRNAs.
Authors: Sun Ye , Gregory Kalvin , Golovlev Val
Published: June 2021

A novel bioluminescent enzymatic assay for high‐throughput detection and quantification of microRNA has been developed. The assay is based on the time‐resolved luminescence detection of inorganic pyrophosphate (PPi) released during microRNA initiated polymerase reaction, which utilizes a specially designed long oligonucleotide template with complementary sequence to the microRNA target of interest. The subsequent primer strand extension gives rise to the nucleotide incorporation and the generation of PPi molecules with the amount proportional to the number of miRNA target molecules in sample. The assay does not need the thermal cycling of PCR reaction. Compared to the current RT‐PCR, microarray, and hybridization‐based microRNA detection methods, this assay provides a simpler, faster, and less expensive platform to be used in life science research, drug discovery, and clinical diagnosis.
The ubiquitin‐dependent chaperone Cdc48/p97(Ufd1‐Npl4) regulates Aurora‐B kinase on mitotic chromosomes
Authors: Meyer Hemmo
Published: June 2021

In interphase, the AAA‐ATPase Cdc48/p97 extracts ubiquitylated substrate proteins from ER membranes for subsequent degradation or recycling. In mitosis, Cdc48/p97 regulates spindle dynamics and nuclear envelope formation, but the molecular basis of this activity has remained elusive. We now show that Cdc48/p97 and its ubiquitin‐adapter Ufd1‐Npl4 regulate chromatin‐associated pools of Aurora‐B kinase and thus control progression of mitosis. In Xenopus egg extracts and C. elegans embryos, chromatin‐bound Aurora‐B inhibits chromatin decondensation and reformation of the nuclear envelope during exit from mitosis. Cdc48/p97(Ufd1‐Npl4) binds Aurora‐B kinase after its ubiquitylation and extracts it from chromatin, thus decreasing chromatin‐associated Aurora‐B activity and promoting nucleus formation. Further evidence now suggests that this pathway is conserved in human somatic cells and relevant for faithful chromosome segregation. Together, our data describe a novel pathway that controls mitosis through spatially restricted ubiquitin‐dependent regulation. Furthermore, they reveal ubiquitin‐dependent extraction as a unifying function of Cdc48/p97 in interphase and mitosis.
Ethanol and the ubiquitin‐proteasome pathway of alveolar macrophages
Authors: D'Souza Alain Joshua , Shellito Judd E , Rudner Xiaowen L , Desai Shyamal , Ruan San Bao
Published: June 2021

The ubiquitin‐proteasome pathway is critical in the inflammatory host response to bacterial infections. Alcohol exposure predisposes to infection, particularly pneumonia caused by Klebsiella pneumoniae (Kp). We tested the effect of alcohol on the ubiquitin‐proteasome pathway using exposure to Klebsiella pneumoniae as our model. Murine alveolar macrophage cell line (MH‐S), macrophage/monocyte cell line (RAW264.7) or primary alveolar macrophages from Balb/c mice were exposed to different alcohol concentrations and challenged with 2x106 colony forming units of heat‐killed Klebsiella. At various time points cell lysates were analyzed for 20S proteasome activity by measuring the release of proteasome‐cleaved fluorigenic substrate. We observed that 20S proteasome activity was decreased, in a dose‐dependent manner, by as much as 61% in the groups exposed to ethanol, as compared to the control group (p<0.05). Cell lysates were also assayed, by Western analysis, for levels of total intracellular free protein ubiquitin and of polyubiquitinated proteins. We observed that alcohol alters levels of intracellular polyubiquitinated proteins but does not affect levels of intracellular free ubiquitin. We conclude that pulmonary infection caused by Klebsiella may be enhanced by alcohol exposure through inhibition of the ubiquitin‐proteasome pathway of alveolar macrophages.

Supported by AA09803, HL076100.
Effect of a ketogenic diet on motor performance and amyloid beta accumulation in a mouse model of inclusion body myositis
Authors: Niedowicz Dana , Beckett Tina L. , Weidner Adam M. , Studzinski Christa M. , Murphy M. Paul
Published: June 2021

?‐Amyloid (A ?) accumulation is a hallmark of Alzheimer's disease (AD) and is thought to induce the onset of disease. Similarly, A ? accumulates in muscle in inclusion body myositis (IBM), an age‐related disease. Our lab is interested in the potentially shared aspects of the disease process, particularly those involving regulation of expression. There is considerable evidence that diet impacts the progression of AD, a theory supported by the correlation between diabetes and AD. A "ketogenic" diet has been used to treat childhood epilepsy and has shown efficacy in AD models. In this study, we fed aged (18‐19 mo.) IBM transgenic mice either a ketogenic (80% fat) or control (10% fat) diet for 2 months, significantly enhancing ketone levels (0.33 ± 0.06 mM vs. 0.68 ± 0.08 mM). Motor performance was measured longitudinally and tissue was collected after euthanasia. Plasma insulin and cholesterol decreased in mice fed the ketogenic diet (0.93 ± 0.3 ng/mL vs. 0.65 ± 0.08 ng/mL and 2.16 ± 0.41 mg/mL vs. 1.55 ± 0.1 mg/mL respectively), while adiponectin, leptin, and resistin were unaffected. The ketogenic diet markedly prolonged wire suspension times (1.33 ± 0.21 sec. vs. 6.6 ± 2.8 sec.) and induced a trend towards improved grip strength (0.55 ± 0.06 N vs. 0.62 ± 0.04 N) and exploratory behavior (17.6 ± 3.2 meters vs. 21.1 ± 4.3 meters). These data suggest that dietary interventions may have some therapeutic value for both AD and IBM. Supported by NIH NS058382.
Epidermal growth factor receptor activation in an in vitro diabetes model
Authors: Quan Krystle , Hudson Laurie G
Published: June 2021

Diabetes is the fifth leading cause of death in the United States and a major cause of morbidity. One of the more troubling complications in diabetes are non‐healing skin wounds, where high glucose levels and poor blood flow contribute to the formation of infected sores and ulcers. Hyperglycemia leads to the accumulation of advanced glycation end products, which can alter receptor functions. Because the epidermal growth factor receptor (EGFR) plays a crucial role in keratinocyte migration and wound reepithelialization, we tested the impact of high glucose on EGFR signaling in a human keratinocyte cell line. Basal EGFR tyrosine phosphorylation was decreased in cells following chronic exposure to high glucose (4.5 g/l) compared to cells maintained in normal glucose (1.0 g/l). Acute exposure of cells with high glucose diminished ligand‐stimulated EGFR tyrosine phosphorylation. This response was evident within 2‐4 hrs of high glucose treatment and became more pronounced with extended exposure. Similarly, downstream EGFR signaling, as detected by ERK phosphorylation, was decreased with time following high glucose treatment. These experiments demonstrate that high glucose interferes with EGF receptor function in keratinocytes with predicted impacts on cell migration and other EGFR‐dependent functions in wound reepithelialization. Funded by NIH award R01 GM079381.
LOCALIZING THE CYTOHESIN 2/ARNO BINDING SITE IN THE HUMAN PROTEIN INTERACTION PROTEIN FOR CYTOHESIN FACTORS (IPCEF).
Authors: Richardson Clarice Leah , Santy Lorraine C
Published: June 2021

The study and application of locating the Cytohesin 2/ARNO binding site in the Human Protein Interaction Protein for Cytohesin Factors (IPCEF) is a very vital skill. Previous studies indicate that Cytohesin 2/ARNO binds to the IPCEF Protein in the last 121 amino acids. Consequently, our research focused on localizing the binding site of Cytohesin 2/ARNO in the IPCEF Protein while clarifying whether there are any other proteins that have the same function (binding ability) in the region where Cytohesin 2/ARNO binds in the IPCEF Protein. Next, IPCEF DNA was transformed; cultured; and Maxi and Mini prep QIAGEN kits were used to purify the IPCEF DNA. Electrophoresis; mutagenesis; digestion; co‐immunoprecipitation; transfection; and Western Blot were performed. Mutant 389 and Mutant 405 were successfully created and tested for their binding ability with Cytohesin 2/ARNO in the IPCEF Protein. Mutant 389 did not retain its binding ability while Mutant 405 retained its binding ability with Cytohesin 2/ARNO. These results are very vital because it lets us know that at least the regions from amino acid 389 (mutant 389) and amino acid 405 (mutant 405) are needed when conducting tests to locate the Cytohesin 2/ARNO binding site in the IPCEP Protein. In the future, our focus will be to further investigate where the binding site of Cytohesin 2/ARNO is located in the Human IPCEF Protein.
Exploring leucine‐rich repeat proteins as molecular scaffolds: design of an oxidation‐resistant ribonuclease inhibitor
Authors: Rowland Elizabeth Fay , Krawisz Anna , Dickson Kimberly Anne
Published: June 2021

Engineered proteins with specific binding activities have evolved as a powerful technology. Repeat proteins, which exhibit structurally conserved modular frameworks and extensive surface areas, are alluring molecular scaffolds; some have yielded molecules with the specificity and robustness of antibodies. Our long‐term goal is to design novel binding surfaces on the human ribonuclease inhibitor (RI), a horseshoe‐shaped leucine‐rich repeat (LRR) protein that binds to pancreatic‐type ribonucleases with extraordinary affinity (KD ? fM). Exploration of RI as a protein scaffold has been limited by its oxidation sensitivity.

We have constructed oxidation‐resistant variants of RI in which some or all of its 32 cysteine residues have been replaced. Six variants of RI have been produced in which groups of 5‐7 cysteines (at conserved positions within the LRR motif) have been mutated. Preliminary results indicate that the RI variants maintain affinity for ribonucleases, but possess decreased oxidation sensitivity. These data contribute to our understanding of the structural stability of LRR proteins and suggest that RI and other LRR proteins are practical scaffolds for the design of novel binding surfaces. This work was supported by a Monticello College Foundation Research Grant to E.F.R., and start‐up funds (Macalester College and Lawrence University) to K.A.D.
Inherently Antibacterial Hydrogels: Altering Activity via Tryptophan/Arginine Interactions
Authors: Larsen Tyler John , Salick Daphne A. , Nagarkar Radhika , Schneider Joel P.
Published: June 2021

Hydrogels are heavily hydrated materials that show considerable promise as artificial extracellular matrices for use in tissue regenerative therapies. The development antibacterial hydrogels has been of great interest to the hydrogel research community as a means to combat the threat of infection during material implantation. We have developed MAX1, a self‐assembling β‐hairpin peptide hydrogel whose surface exhibits inherent antibacterial activity against several pathogens prevalent in hospital settings. Under physiological conditions, MAX1 self‐assembles into a highly crosslinked, mechanically rigid hydrogel whose solvent‐exposed fibrils display positive charge, which is thought to be important for antibacterial activity. This study aims to investigate the contributions of a cation‐π interaction to the antibacterial activity of a newly designed peptide hydrogel. Cation‐π interactions are a common feature of many antibacterial peptides, where they assist in the disruption of bacterial membranes. Thus, a new β‐hairpin peptide (RWMAX1) was designed, incorporating a cross‐strand Tryptophan/Arginine pair, in the hope of creating a more potent antibacterial hydrogel. The folding and self‐assembly properties were assessed using circular dichroism and rheology and the antibacterial activity was investigated against E. coli and S. aureus. Supported by the Beckman Foundation
An investigation into the relationship between calmodulin protein conformation and binding specificity
Authors: Dunlap Tori , Bradley Luke H.
Published: June 2021

In the presence of calcium, calmodulin (CaM) undergoes large conformational changes throughout the protein to present a structurally dynamic surface that can recognize and bind numerous target proteins to transduce the cell's calcium signal. Most of these structural changes occur in the protein's unique central linker region, which serves as a molecular hinge between the N‐ and C‐terminal dual EF‐hand calcium binding domains. Numerous studies have led to the hypothesis that the interactions provided by this region are primarily responsible for CaM‐target binding affinity and thus, CaM's binding specificity. To further examine the relationship between protein sequence, conformation, and CaM's pluripotent binding specificity, we constructed linker‐region protein libraries designed to differentially restrict the conformational freedom of the molecule. Individual, non‐selected library members were characterized by circular dichroism spectroscopy to investigate alterations in conformational change upon calcium binding, and fluorescence binding studies with CaM‐peptide targets to investigate changes in binding specificity. We present the initial structural and binding characterization of these randomly selected library proteins.
Advanced Glycation End Products in Histone H1 In Vivo
Authors: Cervantes Laurean Daniel
Published: June 2021

Oxidative stress leads to protein and DNA damage, which is involved in aging and cancer. The role of protein damage in the alteration of genomic integrity is not well defined. Protein damage by glycation, non‐enzymatic reaction of hydroxy aldehydes with amino groups in proteins, correlates with complications of diabetes, actinic elastosis and the aging process. Glycation is a reversible, non‐enzymatic reaction between reducing sugars and amino groups of proteins that undergo rearrangements to stable ketoamines, which lead to the formation of advanced glycation end products (AGEs) such as fluorescent (argpyrimidine) and non‐fluorescent (Nε−carboxymethyllysine, CML) protein adducts and protein crosslinks. We look for in vivo AGEs adduct formation in nuclear proteins, specifically histone H1, using CML and argpyrimidine as biomarkers. Histone H1 isolated from calf thymus was examined due to its relevance in genomic integrity homeostasis. It was determined the presence of CML and argpyrimidine in histone H1, purified by HPLC, using antibodies against CML and argpyrimidine. Furthermore, CML was quantified using a sensitive chemical method. MS/MS mass spectroscopy analysis of histone H1 revealed the presence of two lysine residues being modified with CML adducts. Our results indicate that glycation of important nuclear targets accumulates in histone H1 under in vivo conditions and this should open the field into exploring AGE protein damage ant its role in the pathophysiology of aging and diabetes.
Ypt/Rab GTPases and Traffic Coordination
Authors: Segev Nava , Chen Shu , Sundaram Geetanjali , Suresh Veena , Taussig David , Tokarev Andrei , Lipatova Zhanna
Published: June 2021

Ypt/Rab GTPases are key regulators of intracellular trafficking in all eukaryotes. These GTPases are activated by guanine‐nucleotide exchange factors (GEFs). When in the GTP‐bound state, Ypt/Rabs interact with effectors that mediate individual steps of the trafficking pathways. An attractive possibility is that Ypt/Rabs and their activators not only regulate separate steps of transport pathways, but also coordinate them. We uncovered a number of novel mechanisms for such coordination in the yeast exocytic pathway. One such mechanism involves a dual‐specificity GEF. The conserved modular‐complex TRAPP is a GEF for the Golgi gatekeepers, Ypt1 and Ypt31/32. However, it is not known how Golgi entry and exit are coordinated. TRAPP exists in two configurations: the seven‐subunit TRAPPI is required for ER‐to‐Golgi transport, whereas the ten‐subunit TRAPPII functions in the late Golgi. We showed that in vitro, the two essential TRAPPII‐specific subunits, Trs120 and Trs130, are required for switching the GEF specificity of TRAPP from Ypt1 to Ypt31. In vivo, mutations in the TRAPPII‐specific subunits confer opposite effects on the intracellular localization of these GTPases. We suggest that the Trs120/Trs130 sub‐complex joins TRAPP in the late Golgi to switch its GEF specificity from Ypt1 to Ypt31/32. Such a switch‐able GEF could ensure sequential activation of these Ypts, thereby coordinating Golgi entry and exit.
Establishment of a timeline of morphological changes in kidney tissue during progression of diabetes
Authors: Tomcho Kayce A , Gay Shanna B , Hahn Timothy F , Birckbichler Paul J
Published: June 2021

The effects and progression of diabetes mellitus can be studied through an examination of the glomerulus. When diabetes is present, glomerular filtration rate is lowered, filtration selectivity is altered and membrane proteins accumulate. Our hypothesis is that biochemical changes precede morphological changes in the kidney. If the hypothesis is correct, ultimately, these changes might be utilized to predict individuals at risk for development of kidney complications. In this study, we are interested in establishing a timeline of measurable changes in kidney morphology during progression of diabetes. Kidney sections were prepared from both diabetic and non‐diabetic BBZDR/Wor rats, animals that mimic type 2 human diabetes, between 26‐395 days of age. Damage analyses were performed independently using a visual semi‐quantitative estimation of overall sclerotic damage and glomerular damage, and a quantitative measurement of glomerular diameters. Diabetic rats showed retraction within the glomerulus, increased collagen expression and increased interstitial space when compared to non‐diabetic rats. Glomerular diameters increased in diabetic rats as early as 34 days of age. Glomeruli analyses showed a positive correlation between the glomerular diameter and the glomerular damage in both groups, but more so in the diabetic group.
MitoNEET dimerization uncouples mitochondria and reduces oxidative stress
Authors: Guerin Theresa , Smart Eric J
Published: June 2021

MitoNEET is a novel mitochondrial membrane protein that contains a 2Fe‐2S center and may be involved in regulating redox reactions. The goal of these studies was to determine if mitoNEET dimerization uncouples mitochondrial respiration and thereby reduces oxidative stress (ROS). A cell line, Swiss 3T3 cells, that does not express mitoNEET was transfected with mitoNEET cDNA and clonal lines established. In the absence of oxidative stress mitoNEET is a monomer, mitochondrial ROSs were low, and oxidative capacity was low. In the presence of exogenous oxidative stress such as hydrogen peroxide and peroxynitrate, mitoNEET dimerized, mitochondrial ROSs were high, and oxidative capacity was increased. Endogenously induced oxidative stress was generated by incubating the cells in high glucose (20 mM) or high fatty acids (0.5 mM). The cells responded similarly to endogenous oxidative stress as they did with exogenous oxidative stress. In contrast, cells that were not expressing mitoNEET underwent apoptosis when identical levels of oxidative stress were employed. Time course studies demonstrated that mitoNEET dimerized as ROS levels increased. This correlated with an increase in oxidative capacity and mitochondrial uncoupling. MitoNEET returned to a monomer as ROS levels decreased. These data suggest that the dimerization of mitoNEET plays a role in limiting mitochondrial ROS levels and cell apoptosis.
Regulation of Caveolin‐1 Trafficking by the Na/K‐ATPase/Src Interaction
Authors: Cai Ting , Xie Jeffrey , Ye Qiqi , Harris Tanoya L
Published: June 2021

We have shown that graded knockdown of the Na/K‐ATPase increases endocytosis and mobility of caveolin‐1(Cav1), resulting in decreases in the plasma membrane pool of Cav1 (Cai, et al, J Cell Biol. 2008, 182:1153‐69). Moreover, the Na/K‐ATPase interacts and inhibits Src and knockdown of the Na/K‐ATPase increases cellular Src activity. Interestingly, inhibition of Src by PP2 reduced the endocytosis of Cav1 and partially restored the plasma membrane pool of Cav1, suggesting that Src‐mediated phosphorylation of Cav1 may play an important role in regulation of Cav1 trafficking. To further test this hypothesis, we mutated two well‐characterized phosphorylation sites in Cav1, namely Y14 and S80. Cells were transiently transfected with YFP‐tagged wild type Cav1 or Cav1 mutants. Confocal imaging analyses indicate that most Cav1S80A‐YFP, like Cav1wt‐YFP, resided in the cytosol in the Na/K‐ATPase knockdown cells. In contrast, most of the Cav1Y14F‐YFP mutant was detected in the plasma membrane. Consistently, particle tracking analyses demonstrated that Y14F mutation significantly reduced the mobility of YFP‐Cav1 vesicles. These findings suggest that the Na/K‐ATPase may regulate tyrosine phosphorylation of Cav1 Y14 and thus Cav1 trafficking by interacting and modulating cellular Src activity.
Two basic patches in the flexible region of Huntingtin‐interacting protein 1 (HIP1) drive the binding of clathrin
Authors: Ybe Joel Alcasid , Clegg Mary , Illingworth Melissa , Gonzalez Claire , Alphonse Ignatius Arun
Published: June 2021

Huntingtin‐interacting protein 1 (HIP1) is a dumbbell‐shaped molecule that carries out multiple functions in animals. We published two crystal structures of contiguous segments of HIP1 that give us new insights into how HIP proteins participate in clathrin‐mediated endocytosis and Huntington's disease (HD). The objective of the study is to understand if charged patches in the flexible region of HIP1 are involved in mediating the binding of clathrin light chain. A basic patch centered on K494 interrupts the S3 path and acidic residues mark the end of the path. A second basic patch centered on K474 (~50 ? away from K494) is critical for the binding of clathrin light chain b (LCb). These electrostatic features that are also conserved in HIP12/R (related to HIP1, connects clathrin‐coated vesicles to the cytoskeleton) are in a flexible part of the HIP1 coiled‐coil. To assess the impact of replacing K474 and K494 with alanine or glutamic acid, we performed GST‐pulldowns. Circular dichroism (CD) and NMR experiments show the opened region of HIP is stable at low temperatures, but highly labile at 37° C. NMR data indicate that the opened region has "hard wired" structure that is stable even at 83° C. We conclude both K474 and K494 are necessary for the productive binding of clathrin LCb and that this region involved in binding htt, HIPPI and clathrin is flexible.
Possible Biochemical Mechanism of Action of Petroleum‐Ether Extract of Calotropis procera leaves (Asclepiadaceae) in Gravid Pregnant Sprague Dawley Rats
Authors: James Ayorinde Babatunde , Magbagbeola Olubunmi Adeola , Saibu Gbemisola Morounke , Oloyo Ahmed
Published: June 2021

Calotropis procera (Asclepiadaceae) is one of the traditionally used antifertility plants in Nigeria. Previous studies have shown that this antifertility plant has abortifacient property but none of them has reported its possible biochemical mechanism of action.

Organ bath experiments using cumulative doses of the extract on rat uterine rings in Dejalon's solution aerated with 95% O2 + 5% CO2 produced a non significant (P>0.05) increase in tension (% response). An EC50 value of 0.1059 mg/ml was obtained and the intrinsic activity was found to be above 1 in comparison with oxytocin.

Blocking muscarinic receptors with 35µg/ml atropine caused the dose‐response curve to shift to the right with a non‐significant (P>0.05) decrease in maximal tension (% Response). However, the ?2‐adrenergic blocker (1µM propanolol) caused a significant (P<0.05) shift in the dose‐response curve.

The extract also exhibited its effect on cAMP modulation of β2‐adrenergic receptors by causing a significant (P<0.05) decrease in cAMP level after treatment of cultured uterine cells with the extract.

In conclusion, this study suggests that pet‐ether extract of Calotropis procera (Asclepiadaceae) exerts its abortifacient effect by inducing myometrial contractions of the uterus by binding to β2‐adrenergic receptors thereby causing a decrease in the level of cAMP. cAMP reduction may reduce the activation of protein kinase A (PKA) preventing PKA from inactivating myosin light chain kinase (MLCK).
Investigation of human follicle stimulating hormone residency in membrane microdomains
Authors: McKenzie Kevin Andrew , Dias James A , Cohen Brian D
Published: June 2021

The human follicle stimulating hormone receptor (hFSHR) is a G protein‐coupled receptor involved in reproduction in both males and females. hFSHR is involved in complex networks of signaling molecules beyond the G proteins and understanding the ability of the receptor to participate in many signaling pathways is of significant interest for those looking to modulate receptor function. One possibility is that microdomains in the plasma membrane, such as lipid rafts, play an important role in signal transduction. The aim of this study was to determine the role of plasma membrane microdomains in hFSHR function. To analyze this, lipid rafts were disrupted using the cholesterol withdrawing agent methyl‐β ‐cylcodextrin (MβCD) and hFSH activation of MAP kinase pathways was investigated. Removal of membrane cholesterol caused the loss of hFSH induced p38‐MAP kinase activation but did not alter p42/44 MAP kinase activation. To investigate further the residency of hFSHR in lipid rafts, co‐immunoprecipitation experiments were performed to determine if caveolin‐1 interacted with hFSHR. Caveolin‐1 was overexpressed in cells stably transfected with hFSHR. A monoclonal antibody against hFSHR was used to immunoprecipitate the receptor and caveolin‐1 was shown to co‐immunoprecipitate. Although the physiological role of this interaction has not been determined, it presents a new opportunity to study hFSHR function.
Cyclooxygenase‐2 and RhoA Signaling Disrupts Cell‐Cell Adhesion by the Regulation of the Transcriptional Repressor, ZEB‐1
Authors: Bean Ronald R. , Jakobi Rolf , Marlin Jerry , Chang Yu‐Wen E.
Published: June 2021

Cyclooxygenase 2 (COX‐2) plays a critical role in colon cancer and loss of the adhesion molecule E‐cadherin is one of the hallmarks that indicate a tumor cell has become invasive. The aim for this study is to investigate if the transcription repressor ZEB1 is a crucial component linking COX‐2 and RhoA/ROCK signaling to down‐regulation of E‐cadherin. In the cells lines that expressed COX‐2 , activity of RhoA was elevated, and formation adherens junctions were disrupted, which was concomitant with down‐regulation of E‐cadherin. In contrast, inhibition of COX‐2, inactivation of RhoA by expressing dominant negative RhoA or inhibition of ROCK, restored E‐cadherin levels in the same COX‐2 expressing cells. Quantitative real‐time RT‐PCR showed that E‐cadherin was down‐regulated at transcriptional level in cells expressing COX‐2. Furthermore, both protein and mRNA levels of ZEB1 were highly elevated in cells expressing COX‐2. We also found that expression of constitutively active RhoA resulted in increased ZEB1 and decreased E‐cadherin expression, which were prevented when ROCK was inhibited. Importantly, silencing ZEB1 expression in cells expressing COX‐2 markedly increased E‐cadherin mRNA and protein levels. Thus, our results showed for the first time that the RhoA/ROCK pathway mediates COX‐2 signaling to increase ZEB1 expression, which is required to modulate E‐cadherin and formation of adherens junctions.
Hypertrophy and Postischemic Contractile Dysfunction in Mice Overexpressing the Active Form of Zea maize RacD in the Heart
Authors: Awad Mohamed M , Talukder Hassan , Kulkarni Aditi , abouelnaga zeinb , Hessein‐Hassona Mohamed , Mostafa Adel , Abdelhamid Mostafa , Alhaj Mazin , Zweier Jay , Kuppusamy Periannan , Bergese Sergio , Hassanain Hamdy
Published: June 2021

We investigated the effect of increased reactive oxygen species (ROS) in the heart on myocardial injury. Our hypothesis is overexpressing Rac 1 in the heart will have increased ROS generation, hypertrophy and poor contractile recovery during ischemia reperfusion (I/R). We have generated a TG mouse model that expresses the cDNA of a constitutively active mutant of Zea maize Rac‐D gene in the heart of FVB/N mice using a mouse ?‐myosin heavy chain promoter. IHC analysis confirmed the expression of Rac‐D protein in the heart of TG mice. Echocardiographic analysis showed significantly low ejection fraction in TG mice compared to control mice [20±4 (TG) vs. 62±5 (control), P<0.05]. Magnetic Resonance Imaging (MRI) study revealed a significant left ventricular hypertrophy in TG mice.Western blotting confirmed the activation of the hypertrophic signaling cascades in the heart of TG mice, both phospho‐ERK1/2 and phospho‐JNK were significantly higher in TG mice.I/R studies performed on isolated hearts demonstrated markedly poor post ischemic contractile recovery with frequent abnormal ventricular rhythms. This study shows the importance of Rac 1 in NADPH/ROS‐mediated myocardial injury during I/R. In conclusion, the Rac‐D transgenic model provides an advantage to investigate the signaling mechanisms regulating ischemic heart diseases and helps to develop therapeutic approaches for ischemic heart diseases.
Analysis of exposed cysteines in cortactin by sulfhydryl modification
Authors: Jenness Christopher , Kruchten Anne E
Published: June 2021

The actin binding protein cortactin is involved in cytoskeleton restructuring and many instances of cancer cell motility. We examined the structure of cortactin and a mutant mimicking tyrosine phosphorylation using 5,5′‐Dithiobis(2‐nitrobenzoic acid) (DTNB). DTNB will form a covalent bond with exposed cysteines, releasing an TNB anion that can be tracked spectrophotometrically at 412 nm. Cysteines, being hydrophobic amino acids, are typically embedded within a protein, which inhibits this binding. We found that wildtype cortactin has approximately two (of three) cysteines exposed, while a cortactin mutant mimicking tyrosine phosphorylation had only one of the cysteines exposed. This difference suggests that cortactin is an intrinsically unstructured protein that oligomerizes or folds when phosphorylated. Further work will mutate individual cortactin cysteines to alanines to determine which are exposed and which regions are folded. This work was supported by a Faculty Start Up Grant to A.E. Kruchten by The Murdock Charitable Trust and by a Linfield College Faculty Student Collaborative Research Award to A.E. Kruchten and C. Jenness.
Nitric oxide dependent increase in free radical generation mediates release of extracellular traps from human neutrophils
Authors: Jyoti Anupam , Keshari Ravi Shankar , Kumar Sachin , Patel Satyananda , Srinag Bangalore Suresh , Verma Anupam , Barthwal Manoj Kumar , Krishnamurthy Hanumanthappa , Bajpai Virendra K , Dikshit Madhu
Published: June 2021

High availability of NO at the inflammatory/infection site is noticed often with oxidative stress and neutrophil extracellular traps (NETs) contents, but role of NO remains unexplored in NETs formation. In the present study incubation of adhered human neutrophils with DETA‐NONOate led to NETs release in a time and concentration dependent manner, as assessed by confocal microscopy and by measuring extracellular DNA and NET‐bound elastase, which was blocked by N‐acetyl cysteine, suggesting role of free radicals. A time and concentration dependent augmentation in free radical formation by NO donors was measured by using DCF‐DA, NBT, and by p47 phox migration to the neutrophils membrane. NO mediated formation of NETs and free radicals was significantly attenuated by pretreatment of neutrophils with diphenyleneiodonium, a dual inhibitor of NADPH‐oxidase/NO synthase (NOS), 4‐aminobenzoic acid hydrazide, a myeloperoxidase inhibitor and 7‐nitroindazole, a NOS inhibitor, suggesting enzymatic free radical generation. We thus provide first experimental evidence that NO by augmenting free radical formation in human neutrophils mediates NETs release.
Saturated fatty acids increase peroxynitrite formation via a SR‐BI/caveolin/AMP kinase signaling pathway
Authors: White James , Smart Eric J
Published: June 2021

The goal of these studies was to determine the mechanism whereby saturated fatty acids increased peroxynitrite formation. Mouse aortic endothelial cells were treated with 10 μM of saturated or unsaturated fatty acids. Saturated fatty acids but not unsaturated fatty acids caused an increase in peroxynitrite. In contrast, saturated fatty acids did not increase peroxynitrite in aortic endothelial cells isolated from SR‐BI or caveolin‐1 null mice. To further explore the requirement of SR‐BI and caveolin‐1, aortic endothelial cells lacking these proteins were treated with adenovirus encoding either mouse SR‐BI or caveolin‐1. Expression of SR‐BI and caveolin‐1 restored the ability of saturated fatty acids to induce peroxynitrite formation. Bovine aortic endothelial cell lines that do not express AMP kinase or express AMP kinase from an adenoviral construct demonstrated that AMP kinase is also required for saturated fatty acids to induce peroxynitrite formation. Further studies demonstrated that saturated fatty acids induce the phosphorylation, and thus, activation of AMP kinase and nitric oxide synthase. Competition and binding assays demonstrated that saturated fatty acids directly bind to SR‐BI. Taken together these data suggest that binding of saturated fatty acids to SR‐BI initiates a signaling cascade that involves caveolin and AMP kinase which results in the production of peroxynitrite.
[13C]‐Carotenoid Production from hp‐1 Tomato Cell Cultures for Human Metabolic Tracing
Authors: Engelmann Nancy J. , Rogers Randy B. , Rupassara S. Indu , Garlick Peter J. , Lila Mary Ann , Erdman John W.
Published: June 2021

[13C]‐carotenoids (CAR), while most appropriate for human metabolic research, are not commercially available. The goal of this research is to use tomato suspension cultures to efficiently produce [13C]‐lycopene (LYC), phytoene (PE), and phytofluene (PF). Two sources of [13C] for cell cultures are labeled glucose (glc) or sucrose (suc). The effect of unlabeled glc and suc on total CAR yield was first evaluated by HPLC in the standard tomato cell line, VFNT Cherry. Glc treatment resulted in greater total CAR (2147 ± 466 ug/L culture) than providing suc (975 ± 102 ug/L). CAR and harvest mass from high LYC hp‐1 tomato suspensions were optimized using response surface methodology (RSM). Plant growth regulators (GR), indole‐3‐acetic acid and all‐trans zeatin, were varied by two factors: ratio (0.71‐15) and additive amount (6‐8 mg/L), with treatment points determined by D‐optimality to model the two‐dimensional design space. A significant linear relationship for the overall model was observed for harvest mass and growth regulator ratio (p = 0.03). The models for PE, PF, and LYC concentration were significant when they included both GR ratios and amounts (p= 0.04, 0.02, and 0.01, respectively). The optimal conditions predicted by RSM will be confirmed by evaluating harvest mass, CAR concentration, and [13C] enrichment from [13C]‐glucose treated cultures by LC‐MS/MS and GC/MS methodologies. (NIH/NCI CA 112649‐01A1)
Effects of Obesity, short‐term soy vs casein protein diet and DMBA on Liver CYP 1A1 and CYP 1B1 Expression in Ovariectomized Obese Zucker Rats
Authors: Hakkak Reza , Shaaf Saeid , MacLeod Stewart , Korourian Soheila
Published: June 2021

Obesity is a risk factor for post‐menopausal breast cancer development. We have shown that obesity increases DMBA‐induced mammary tumor development in intact and ovariectomized Zucker rats. Several data suggest that DMBA requires metabolic activation to exert its carcinogenic effects. The objective of this study was to investigate the effect of obesity and high‐isoflavone soy (high‐isof soy) vs casein diet on hepatic expression of CYP1A1 and CYP1B1 following DMBA treatment in obese and lean ovariectomized Zucker rats. Forty day‐old, ovariectomized obese (n=40) and lean (n=40), Zucker rats were administered a diet containing either casein or high‐isof soy protein isolate and 10 days later were orally gavaged with either with sesame oil (control) or with 65 mg/kg DMBA in sesame oil. All rats were sacrificed 24 hrs post‐DMBA treatment. Liver microsomes were prepared and CYP1A1 and CYP 1B1 expression was measured by Western blotting using goat anti‐mouse CYP1A1 and CYP1B1 antibodies. DMBA treatment significantly (p<0.001) increased expression of CYP1A1 in lean and obese ovariectomized rats in both casein and high‐isof soy‐fed group compared to the oil control groups. High‐isof soy diet decreased (p<0.001) CYP1A1 expression in DMBA treated lean rats compared to lean casein‐fed rats but CYP1A1 expression was increased (p<0.001) by DMBA in obese soy‐fed rats compared to high‐isof soy‐fed lean rats. CYP1B1 expression was not affected by diet, obesity or DMBA. These data suggest that obesity can increase the expression of enzymes that are responsible for DMBA metabolism in the ovariectomized Zucker rat model. (Supported by Susan G. Komen Foundation and ABI to RH).
A quantitative assessment and systematic review of prospective cohort studies of animal fat intake and breast cancer
Authors: Lowe Kimberly Anne , Alexander Dominik , Morimoto Libby
Published: June 2021

The association between intake of dietary animal fat and female breast cancer is equivocal. Thus, we conducted a meta‐analysis and systematic review of epidemiologic cohort studies that reported animal fat intake as a specific nutrient or as a percentage of total energy. Our analysis included data reported in the Pooling Project publication of prospective cohorts (n = 8) and data from subsequent publications of cohort studies (n = 3). Random and fixed effects models were utilized to generate summary relative risk estimates (SRREs), and sensitivity and influence analyses were conducted to discern any potential sources of heterogeneity. No significant association was observed comparing the highest category of animal fat intake with the lowest (SRRE = 1.03; 95% CI: 0.76‐1.40). In addition, no significant association between a 5% increment of energy from animal fat intake and breast cancer was observed (SRRE = 1.02; 95% CI: 0.97‐1.07). The available epidemiologic data are not supportive of a positive association between consumption of animal fat and breast cancer. Epidemiologic investigations of other factors, such as diet early in life or effects by hormone receptor status are limited, and additional evaluations of these factors may provide valuable insight to potential patterns of associations between animal fat intake and breast cancer. This work was partially funded by the Cattlemen's Beef Board and NPB.
Meta‐analysis of animal fat intake and colorectal cancer
Authors: Alexander Dominik D , Cushing Colleen A , Lowe Kimberly A
Published: June 2021

The association between animal fat and colorectal cancer is unclear. Therefore, we conducted a meta‐analysis of six prospective cohort studies published through 2007 that reported data for animal fat as a nutrient variable. These studies contributed approximately 1,070 cases of colorectal cancer and 1.5 million person‐years of follow‐up. We used random effects meta‐analyses to model data across studies. Statistical analyses consisted of high vs. low intake comparisons, categorical dose‐response evaluations, and assessments of heterogeneity. In addition, data from three case‐control studies were combined with the cohort data. The summary relative risk estimate (SRRE) for these studies was 1.04 (95% CI: 0.83‐1.31, p‐value for heterogeneity = 0.221), based on high vs. low intake. When data from case‐control studies were combined with the cohort data, the resulting SRRE was 1.15 (95% CI: 0.93‐1.42) with increased variability (p‐value for heterogeneity = 0.015). In our dose‐response analysis of the cohort studies, no association between a 20g/day increment of animal fat intake and colorectal cancer was observed (SRRE = 1.01; 95% CI: 0.94‐1.08). Based on the results of this quantitative assessment, the available epidemiologic evidence does not support a causal association between consumption of animal fat and colorectal cancer. Partially funded by the Cattlemen's Beef Board and the NPB.