bluuebunny/crossref_metadata_2025 huggingface dataset:
17β‐estradiol exerts protective effects in skeletal muscle cells at the level of the apoptotic ASK1‐MAP kinase pathway
Authors: Zanetti Noelia I , Milanesi Lorena M , Boland Ricardo L
Published: June 2021

17β‐estradiol is a steroid hormone that promotes cell survival and prevents from apoptosis in a great variety of tissues. Previous investigations with the murine skeletal muscle C2C12 cell line demonstrated that the action of 17β‐estradiol involves stimulation of PI3K‐Akt. Studies with other cell types have shown that upon its activation Akt can inhibit apoptosis by alternative mechanisms. In this report, we investigated whether the antiapoptotic effect of estrogen in muscle cells is mediated by Akt through interaction and dephosphorylation at Thr 845 of ASK‐1, a MAPKKK pro‐apoptotic protein. When proliferating C2C12 cells were treated with 17β‐estradiol (10−8 M), phosphorylation of ASK1 (Thr 845) and JNK was decreased. However, no significant changes occurred in ASK1 and JNK 1/2 protein levels. On the other hand, treatment with the apoptic agent H2O2 caused a sustained (30 min ‐ 24 h) increase in ASK1 levels and phosphorylation at Thr 845 (associated to stimulation of kinase activity) and, in addition, of JNK 1/2 phosphorylation. Furthermore, a 17β‐estradiol‐dependent interaction between p‐Akt and ASK1 was detected by coimmunoprecipitation. The association of both proteins was diminished by H2O2. In conclusion, these results suggest that Akt interaction with ASK1 followed by dephosphorylation of the latter is part of the mechanism by which 17β‐estradiol exerts its antiapoptotic action in skeletal muscle cells challenged with stress stimuli.
Searching for binding partners for the phosphorylase kinase variant PhK γ181
Authors: Polireddy Kishore , Rice Nancy A.
Published: June 2021

Phosphorylase kinase (Phk), a key regulatory enzyme in glycogenolysis, is a hexadecameric protein that contains 4 different subunits with a stoichiometry (αβλδ)4. Alpha and βare regulatory subunits, γis catalytic and d is an intrinsic molecule of calmodulin. The gene which encodes for the γ subunit, PHKG1, undergoes alternative processing to produce a truncated form of γcontaining 181 residues (γ181) instead of the normal 387 residues. Although much of the kinase helical domain is absent, the truncated form is still catalytic with its activity being influenced by PKC, and its mRNA is expressed in high levels in brain. A natural substrate for γ181 has not yet been identified. In the present study, we are using the LexA based yeast two‐hybrid system to identify binding partners for γ181. Gamma181 was cloned into the pLexA vector and used as bait to screen a human fetal brain cDNA library. Interactions between γ181 and cDNA library proteins were monitored by reporter genes. Characterization of the bait, LexA‐γ181, shows that it is efficiently localized to the nucleus and does not activate the reporter genes. The cDNA library was amplified, titered, and then transformed into EGY48 yeast cells for screening. Following screening for leucine prototrophy and β‐galactosidase activity, we obtained 36 colonies that show positive interactions for leucine prototrophy although only 11 of these colonies are β‐galactosidase positive. Positive clones are currently being sequenced for identification. The discovery of novel binding partners for γ181 is critical in determining the physiological role of this unique kinase in brain.

The work was supported in part by NIH grant P20RR16481.
Regulation of Cell Growth by VACM‐1/cul5 is Dependent on its Phosphorlyation by PKA and PKC
Authors: Bradley Shirley E , Burnatowska‐Hledin Maria
Published: June 2021

Expression of VACM‐1/cul‐5 gene in endothelial and in cancer cell lines in vitro inhibits cellular proliferation and decreases phosphorylation of mitogen‐activated protein kinase (MAPK). Structure‐function analysis of VACM‐1 protein sequence identified consensus sites specific for phosphorylation by protein kinases PKA and PKC. Mutations at the PKA specific site in VACM‐1 (S730AVACM‐1) sequence resulted in increased cellular growth of rat endothelial cells, RAMEC. The aim of our study was to examine if PKC activity also regulates VACM‐1 dependent cell growth. To induce PKC activity, cells were treated with Phorbol 12‐myristate 13‐acetate (PMA) for 16 hours and growth was monitored. PKC specific phosphorylation of VACM‐1 was examined using anti‐PKC substrate specific antibody. Our results show that cells transfected with S730AVACM‐1 cDNA and treated with PMA for 16 hours grew significantly faster than control cells treated with PMA. Further, Western blot analysis of cell lysates from both groups probed with anti‐PKC substrate specific antibody revealed a higher level of VACM‐1 phosphorylation in S730AVACM‐1 cDNA transfected cells when compared to controls. These results suggest that the antiproliferative effect of VACM‐1/cul5 is dependent on its posttranslational phosphorylation by PKA and PKC.
Scanning mutagenesis of Gα12 reveals a role for the C‐terminal region in RhoGEF interaction
Authors: Ritchie Benjamin Joseph , Foster Lori A , Smith Benjamin R , Meigs Thomas E
Published: June 2021

The alpha subunit of the heterotrimeric G protein, G12, has been shown to modulate many cellular processes including cell‐to‐cell adhesion and oncogenic transformation. Among downstream effectors of G12 signaling, the Rho‐specific guanine nucleotide exchange factors (RhoGEFs) p115RhoGEF and leukemia‐associated RhoGEF (LARG) are of keen interest due to their ability to activate the monomeric G protein Rho, leading to an array of responses that include cell migration and transcriptional activation through serum response factor (SRF). The functional interaction between G12 proteins and RhoGEFs has been studied extensively; however, these studies have required the use of chimeric Gα proteins. In the present study, we have examined the interaction between a panel of engineered Gα12 mutants and immobilized forms of p115RhoGEF and LARG. We have identified several Gα12 mutants that are impaired in binding both RhoGEF proteins. Surprisingly, several mutations near the C‐terminus of Gα12 disrupt RhoGEF interaction and demonstrate a reduced ability to stimulate SRF mediated transcription. In conclusion, our findings implicate a previously unexamined region of Gα12 as critical for its interaction with RhoGEFs.
Polyketide Synthase Engineering Toward Improved Antibiotics
Authors: Schnarr Nathan A. , Borketey Lawrence S. , Dulaney Caroline A.
Published: June 2021

The steady rise of drug‐resistant bacteria coupled with decreasing numbers of viable therapeutics in the pharmaceutical pipeline underscores the need for novel strategies focused on rational exploration of chemical space to design and identify the next generation of antibiotics. Polyketides and non‐ribosomal peptides make up an enormous class of natural products whose unique biosynthetic assemblies provide great potential for engineered product variation in well‐defined chemical scaffolds. Unfortunately, most attempts to generate new biologically active analogs of these molecules have suffered from poor yields largely due to inherent specificity within the enzymes responsible for their production. Using an array of chemical and biological tools, our research focuses on understanding recognition motifs leading to this selectivity to potentially improve engineering efforts at both the genetic and molecular levels. If successful, this work will permit access to a wide‐variety of important compounds based on well‐studied, natural frameworks.
Protein PEGylation prevents benzyl‐alcohol induced aggregation
Authors: Rodríguez‐Martínez José Arcadio , Rivera‐Rivera Izarys , Griebenow Kai
Published: June 2021

Benzyl alcohol is currently the most commonly employed antimicrobial preservative added to protein and peptide pharmaceutical formulations. However, it has been reported that benzyl alcohol induces protein aggregation thus presenting an obstacle to safe protein formulation. Specifically, upon incubation of the model protein chymotrypsinogen at 45°C for 24 h, benzyl alcohol (0.9%) increased the formation of insoluble aggregates from 2% to 13%. We tested the effectiveness of the covalent modification of the protein with poly(ethylene glycol) (PEG) on preventing benzyl alcohol induced protein aggregation. PEGs with molecular weights (MW) of 700 and 5000 were employed and between 2 and 5 molecules were bound to chymotrypsinogen. It was found that PEG5000 reduced aggregation to 0% while PEG 700 was largely ineffective. Since we found that there was only a marginal difference in thermodynamic stability for the various PEG‐protein conjugates, this protective effect was likely afforded by steric shielding of hydrophobic protein surface area patches. In conclusion, PEG‐modification of proteins with large PEG molecules is an effective strategy for preventing benzyl alcohol induced protein aggregation.
Combining Label‐free and Flourometric Functional Assays with Biophysical Binding Assays in Drug Discovery: A Comparison of Positives from an HTS Campaign
Authors: Olson Matthew Wayne , Kervinen Jukka , Schubert Carsten , Kirkpatrick Jennifer , Kranz James , Ma Hongchung , Sun Wenfeng , Lawson Ed , DeCorte Bart , Jordan Alfonzo , Hlasta Dennis , Kuo Lawrence , Todd Matthew
Published: June 2021

HTS campaigns push functional assays to their limits for multiple reasons. As such, labeled substrates and secondary detection methods that minimize interference issues are often employed. Fluorescent labeling of a substrate results in the generation of a substrate that no longer resembles the physiological target. Secondary or tertiary detection techniques often result in an assay where initial velocity requirements are lost. In an ideal world, one would the physiological substrates requiring the implementation of label‐free system. The reality of label free‐detection are the amplification of interference issues and/or use substrate concentrations well above the KM‐value. The coupling of high‐throughput mass spectroscopy (HTMS) in a functional assay eliminates these issues and as such one can use substrates that more closely reflect the in vivo condition. HTMS assays are sensitive and they eliminate complications from secondary detection methods. HTMS allows for the examination of the substrates and products from multi‐functional enzymes reactions independently. In the present study, functional assays are coupled with a label‐free biophysical binding assay (ThermofluorTM). The overlap between these orthogonal in vitro technologies enables rapid lead generation and lead optimization of compounds that can progress into full development.
Peer to peer mentoring for pre‐tenure faculty in the chemical biology field.
Authors: Chambers James J. , Knapp Michael J.
Published: June 2021

We have developed a Chemical Biology Mutual Mentoring Group as a team‐building initiative to bridge the gap between the physical and life sciences. While many of the current advances in the natural sciences are being made in interdisciplinary areas, faculty are often still hired by traditional departments leading to professional isolation. In the absence of mentoring, pre‐tenure faculty can be pushed away from interdisciplinary research and teaching. We have begun to build a network of mentors to support the development of research and teaching in this chemical biology interface.
Chitosan nanoparticles for siRNA‐mediated gene knockdown to promote neuronal regeneration
Authors: Mittnacht Ursula , Hartmann Hanna , Andersen Morten , Hein San , Howard Ken , Kjems Jorgen , Schlosshauer Burkhard
Published: June 2021

Axons in the adult vertebrate central nervous system (CNS) exhibit almost no regeneration after injury, partly because of the presence of neurite outgrowth inhibitors in the CNS. The aim of this study is to enhance functional recovery and axon regrowth using siRNA. Thereby, we want to transiently inhibit the intracellular signalling pathway of central components into which various repulsive inputs converge. An effort concerning this objective is to find an appropriate carrier matrix for binding, stabilization, and promoting cellular uptake of siRNA.

Chitosan is a biocompatible and biodegradable material which enables a strong electrostatic binding of oligonucleotides. In this study we present the effective binding of siRNA to chitosan under formation of nanoparticles. Analysis of different kinds of chitosan‐siRNA nanoparticles using gel‐retardation assays demonstrated high stability of those complexes abrogated only after addition of competing agents. Cell transfection experiments with fluorescently labelled siRNA showed efficient uptake of chitosan‐siRNA nanoparticles into different cells which play an important role in the nervous system.

Our results therefore highlight the potential application of chitosan in siRNA‐mediated gene knockdown to promote neuronal regeneration after spinal cord injury.

This project is supported by the Landesstiftung Baden‐Württemberg.
Selective isolation of vein graft endothelial and smooth muscle cells using laser capture microdissection for microarray gene analysis
Authors: Huang Zhen Shao , Malek Junaid Yusuf , Bhasin Manoj , Contreras Mauricio , LoGerfo Frank W
Published: June 2021

Our study employed laser capture microdissection to selectively collect endothelial (EC) and smooth muscle cells (SMC) from patent canine femoral artery bypass cephalic vein grafts (n=3) and normal contralateral cephalic vein controls (harvested after 24 hours). Total RNA was purified, converted to cDNA, and prepared for Affymetrix Canine 2.0 GeneChip hybridization (43,036 probes, 16,984 genes). Sample purities were measured via qRT‐PCR (EC: CD31, eNOS, vWF; SMC: α‐actin 2, SMC‐MHCII). Chip normalization and analysis were executed with BioConductor and dChip. Significantly up‐ (UR) or downregulated (DR) graft genes had =2.0 fold change versus control and absolute lower confidence bound of =2.0. Human genes were matched using BLAST PERL. Average isolate purities were 43‐78% for EC and 79‐100% for SMC. In grafts versus controls, 2101 genes were differentially expressed in EC and 1753 in SMC. UR genes: 1112 in EC, 729 in SMC, 322 in common. DR genes: 989 in EC, 1024 in SMC, 444 in common. Clustering analysis showed unique clustering of graft versus control EC and SMC. Gene ontology analysis exhibited predominant gene functions in protein binding (EC & SMC), cellular processes involved in inflammation (EC) and cell motility (SMC) and localizations to the plasma membrane (EC) and ECM (SMC). LCM is effective in separating gene responses in the intima from the media of vein grafts. NIH 5 T32 HL007734 & 5 R01 HL086741 to FWL
Inhibition of platelet aggregation by capsaicin is not mediated by the TRPV1 receptor
Authors: Nelson Richard A. , Daanen Jerome F. , King Andrew J. , Kort Michaele E. , Kym Philip R. , Lubbers Nathan L. , Lynch James J. , Cox Bryan F. , Mittelstadt Scott W.
Published: June 2021

Capsaicin is an agonist of the TRPV1 receptor which appears to play a role in the integration of the nociceptive response to a variety of noxious stimuli. In addition to neuronal effects, capsaicin can affect non‐neuronal tissue. Previous in vitro studies have demonstrated that capsaicin can inhibit the platelet aggregation response induced by collagen. However, these studies have not investigated if this occurs through the TRPV1 receptor. The purpose of the present study was to investigate if capsaicin induced platelet aggregation could be inhibited with A‐993610, a selective TRPV1 antagonist.

Methods

Venous blood obtained from dogs was incubated with vehicle, capsaicin, and/or A‐993610 for 10 minutes followed by the application of collagen (2 µg/ml). An aggregometer was used to quantify the platelet response.

Results

Capsaicin caused a significant inhibition of the collagen‐induced platelet aggregation at concentrations of 10 & 30 µg/ml. A‐993610 alone had no effect on platelet aggregation. In addition, A‐993610 did not have an effect on the inhibition of platelet aggregation by capsaicin.

Discussion

The current results agree with previous findings demonstrating that capsaicin inhibits platelet aggregation. However, the effects of capsaicin on platelet aggregation are not mediated via the TRPV1 receptor since the inhibition could not be blocked by a specific TRPV1 antagonist.
Expression of Matrix Metalloproteinase 7 and Fibronectin in Papillary Thyroid Cancer: Gene Expression Profiling using real time PCR
Authors: ABEDALTHAGAFI MALAK , GUO HUIMIN , HARTMANN DAN‐PAUL , MARSHALL BLAIR , AZUMI NORIO
Published: June 2021

Papillary thyroid cancer (PTC) is usually indolent with a high frequency of lymph node metastasis. Rarely, PTC behaves in a more aggressive fashion; however, the mechanisms of invasion and metastasis in thyroid cancer remain poorly understood. We studied 4 cases of PTC in which fresh frozen tissue were available. Two had a distant metastasis to the lung and bone and the other two had only local disease. Benign thyroid with nodular hyperplasia was used as a normal control. Frozen sections were prepared and the tumor and normal tissues were microdissected. We used the Human Tumor Metastasis RT2 profiler PCR Array (SA Bioscience, Frederick, MD) and a high performance SYBR green real‐time PCR (LightCycler, Roche, Indianapolis, IN). The array represents 84 genes known to be involved in metastasis. After processing, statistical analysis was performed using the PCR Array Data Analysis Web Portal.The analysis of 2 PTCs without hematogenous metastasis showed down‐regulation of matrix metalloproteinases (MMPs) and fibronectin (FN). Significant over‐expression of MMPs, especially MMP7, and FN were found in 2 PTC with hematogenous metastasis .Compared to normal control and PTC without hematogenous metastasis, ratios of FN and MMP7 expression were 612.6 and 833.62, respectively. Our findings of significant up‐regulation of MMP7 and FN may identify patients who have PTC with more aggressive behavior
Anti‐anxiety effects of β3‐adrenoceptor agonists may be due to selective enhancement of lateral paracapsular GABAergic interneurons in the basolateral amygdala
Authors: Silberman Yuval , Ariwodola Olesegun J , Yorgason Jordan T , Chappell Ann M , Weiner Jeff L
Published: June 2021

Extensive research suggests that stress‐related stimuli increase norepinephrine (NE) levels in brain regions that regulate anxiety, like the basolateral amygdala (BLA). NE, via α and β adrenoceptor (AR) activation, is thought to increase BLA excitability, thus contributing to increased anxiety. Surprisingly, recent studies have shown that systemic β3‐AR agonist exposure decreases anxiety measures, suggesting that β3‐ARs may actually inhibit BLA excitability. Therefore, we tested the hypothesis that β3‐AR agonist‐mediated anxiolysis may be due to increased BLA GABAergic inhibition. We first examined the effects of BRL37344 (BRL, selective β3‐AR agonist) on BLA GABAergic synapses arising from local feedback and lateral paracapsular (LPCS) (feed‐forward) interneurons in rat BLA slices using whole‐cell patch methods. BRL (0.5‐10μM) selectively increased LPCS‐evoked IPSCs (eIPSCs) with no effect on local eIPSCs. BRL potentiation of LPCS eIPSCs was blocked by SR59230A (β3‐AR antagonist) or by including Rp‐CAMPS in the patch pipette solution. BRL had no effect on spontaneous IPSCs or LPCS paired‐pulse ratios. Finally, bilateral intra‐BLA injection of BRL significantly reduced anxiety‐like behaviors on the elevated plus‐maze and open‐field assay. Collectively, these data suggest that β3‐AR activation selectively enhances LPCS‐mediated BLA inhibition and that increased LPCS inhibition is sufficient to decrease anxiety‐like behaviors.

Funded by NIH grants AA17039 and AA17056
α1‐Adrenergic Receptors Regulate Neurogenesis and Differentiation of Interneurons, Dopaminergic, and/or Noradrenergic Neurons
Authors: Perez Dianne , Gupta Manveen , Papay Robert , Jurgens Chris , Doze Van
Published: June 2021

Understanding the function of α1‐adrenergic receptors (α1‐ARs) in the brain has been limited due to a lack of specific ligands and antibodies. We approached this problem by generating transgenic mice engineered to over‐express either wild‐type receptor with enhanced green fluorescent protein tags or constitutively active mutant (CAM) α1‐ARs using isogenic promoters. We identified intriguing α1A‐AR expressing cells with a migratory morphology in the subventricular zone (SVZ) that co‐expressed markers of neural stem cells/progenitors. There was increased BrdU incorporation in vivo in the SVZ in CAM α1A mice while BrdU incorporation decreased in the migratory stream. Normal neonatal neurospheres expressed a mixture of α1A and α1B‐AR subtypes and α1‐AR stimulation resulted in increased neuronal precursors that increased the expression of tyrosine hydroxylase, dopamine‐β‐hydroxylase, and GAD 65/67. CAM‐derived neurospheres had a reduced ability to self‐renew, proliferate, and were composed of a greater number of neuronal, but reduced astrocytic precursors. CAM neurospheres also differentially increased the RNA of proneural transcription factors. Our results indicate that α1‐ARs can regulate neurogenesis and neuronal differentiation and may offer new therapies to treat neurodegenerative diseases. This work was supported by NIHRO1HL61438 (DMP) and NSF CAREER award 0347259 (VAD).
Oxidative Stress and Antiandrogen Resistance in Prostate Cancer
Authors: Schultz Michelle Anne , Abdel Mageed Asim B. , Agrawal Krishna , Mondal Debasis
Published: June 2021

Antiandrogen (AA) resistance in prostate cancer (PCa) prevents successful hormonal therapy. Aberrant activity of the androgen receptor (AR) and oxidative stress (OS) may contribute to this resistance; AR signaling induces ROS and NADPH Oxidases (NOX) correlate with PCa aggressiveness. Peroxiredoxin‐1 (Prx‐1) is an OS induced, antioxidant response element (ARE) regulated gene. Prx‐1 enhances AR transactivation, which may limit AA efficacy. AREs are regulated by two transcription factors, Nrf1 and Nrf2. ARE genes were implicated in resistance in tamoxifen resistant MCF‐7 cells. We used 4 cell lines representing 4 subsequent stages of PCa progression (RWPE1, RWPE2, LNCaP, and C42B) to establish a correlation between AA resistance and ARE gene expression. We used RT‐PCR, western, and cell viability assays to determine how levels of Prx‐1, Nrf1, Nrf2, NOX4, and NOX5 expression correlate with bicalutamide resistance. With increasing aggressiveness and resistance, enhanced expression of Prx‐1, Nrf1, NOX4, and NOX5 was observed, while decreasing expression of Nrf2 was seen. Interestingly, bicalutamide treatment also increased Nrf1 protein expression in LNCaP and C42B cells. Our results have shown a direct correlation between expression and/or activity of ARE proteins and antiandrogen resistance. Understanding these mechanisms of AA resistance may aid in developing new treatments for aggressive PCa.
Profile of prostaglandin E1 and E2 binding to four subtype prostaglandin E receptors
Authors: Chillar Annirudha Jaikaran , Mohite Anita , So Shui‐Ping , Ruan Ke‐He
Published: June 2021

Prostaglandin E1 (PGE1) and E2 (PGE2) are ligands for prostaglandin E2 receptor (EP) family, which are designated as EP1, EP2, EP3 and EP4. Interestingly, PGE2 mediates inflammation whereas PGE1 is acting as an anti‐inflammatory factor. However the molecular basis of their opposite actions on the EP receptors is poorly understood. In this study, the PGE1 and PGE2 binding affinity and signaling on the human recombinant EPs expressed in the live HEK293 stable cell lines were determined by [3H]PGE2 binding and the calcium and cyclic AMP signaling, respectively. The Kd of [3H]PGE2 binding to the EP1, EP2, EP3 & EP4 was 0.35 nM, 0.45 nM, 0.021 nM, and 0.047 nM, respectively. PGE2 showed higher affinity or preference for EP3 and EP4 as compared to that of EP1 and EP2. The IC50 thus calculated from [3H]PGE2 displacement experiments using cold PGE1 and PGE2 for the EPs is, EP1 ( PGE1 0.1mM, PGE2 5μM), EP2 ( PGE1 1μM, PGE2 1μM), EP3 ( PGE1 1μM,PGE2 100 nM) and EP4 ( PGE1 1μM, PGE2 100nM). PGE1 showed a higher calcium signal in EP1 as compared to that of PGE2. In addition, there was a one log concentration difference between PGE2 and PGE1 for generation of calcium signal in EP4. The calcium signal indicated that the EP1 is the likely dominant receptor in terms of signaling in tissues that co express the EPs. This study provides a molecular basis to understand the biological functions of PGE1 and PGE2 through their binding and signaling properties.
Engineering of a hybrid enzyme to study thromboxane A2 mediating vascular diseases
Authors: Tabatabai Mina , Ruan Ke‐He , So Shui Ping , Chillar annirudha j
Published: June 2021

Thromboxane A2 (TXA2) is synthesized by a coupling reaction between cyclooxygenase (COX) and TXA2 synthase (TXAS). However, the mechanism of this coordination is poorly understood. To uncover the details of this coupling, it is crucial to have a model showing their coordination. This study focuses on creating a hybrid enzyme to mimic the coupling between COX‐1 and TXAS in the ER membrane. The two enzymes, COX‐1 and TXAS, were joined by a 10 amino acid (aa) linker to create a hybrid enzyme, COX‐1‐10aa‐TXAS. The cDNA of the engineered COX‐1‐10aa‐TXAS was successfully cloned into the pcDNA 3.1 vector containing a cytomegalovirus early promoter and used for gene expression in HEK293 cells. The expression of the hybrid enzyme in the ER membrane, which is similar to that of the co‐expressed COX‐1 and TXAS, was demonstrated by immunostaining. The hybrid enzyme could mimic the triple catalytic functions of the wild type COX‐1 and TXAS, as confirmed by the coupling assays from HPLC and LC/MS as well as Western blot analyses. The results indicated that the biosynthesis of TXA2 is likely through the configuration of the two enzymes in which the orientation of the COX‐1 C‐terminal domain is toward the TXAS N‐terminal domain, and the distance between the two enzymes is likely very short. This study provides a model to understand the molecular mechanism of the coupling between COX‐1 and TXAS in biosynthesis of TXA2, which is involved in strokes.
Comparative preclinical assessment of angioedema risk of inhibitors of renin‐angiotensin system and neprilysin in the anesthetized rat tracheal plasma extravasation assay
Authors: Hegde Laxminarayan G , Thibodeaux Harold , Yu Cecile , Cheruvu Madhavi , Olsufka Rachael , Richardson Carrie , Villarreal Jennifer , Armstrong Scott , Hegde Sharathchandra S
Published: June 2021

Angiotensin converting enzyme inhibitors (ACEi) and dual acting ACEi‐neprilysin (NEP) inhibitors are associated with risk of angioedema in the clinic. In the present study, we used a rodent model to assess the angioedema risk of administration of angiotensin receptor blocker (ARB) and NEP inhibitor, dosed alone or in combination, at antihypertensive doses. Intravenous effects (mg/kg) of ACEi (lisinopril, LIS 0.03‐10), ACEi‐NEPi (omapatrilat, OMPT‐0.03‐10; ilepatril, ILPT‐0.3‐30), AT1 antagonist (valsartan VAL‐0.3‐10), neprilysin inhibitor (CGS‐24592, CGS‐0.3‐30) or a combination (AT1‐NEPi) and vasodilator‐ sodium nitroprusside (SNP) on tracheal plasma extravasation (TPE) was tested in anesthetized rat (Sulpizio et al., JPET 309:1411,2004). Treatment with LIS, OMPT, ILPT or ACEi‐NEPi (LIS + CGS) resulted in a significant, dose related increases in TPE (peak values 48, 91, 89 and 57 ng/mg respectively vs. 1‐10 ng/mg, vehicle). Pretreatment with HOE 140 (bradykinin B2 antagonist, 30 ìg/kg, iv) completely antagonized TPE observed with LIS, OMPT and ILPT. In contrast, VAL, CGS, VAL+CGS combination or SNP treatment produced no increase in TPE. These results indicate that unlike ACEi or ACEi‐NEPi, ARB or ARB‐NEPi did not result in increased TPE (hence carry minimal or no risk of angioedema). Additionally, bradykinin mediated angioedema risk can be reliably detected pre‐clinically using this simple assay.
Cangrelor (ARC69931MX) produces global inhibition of human platelet function by increasing cAMP levels through a Gi‐independent mechanism.
Authors: Srinivasan Subhashini , Mir Fozia , Huang Jin‐Sheng , Khasawneh Fadi , Lam Stephen C‐T , Le Breton Guy C
Published: June 2021

The recent use of antagonists against the P2Y1 and P2Y12 ADP receptors has significantly contributed to the characterization of ADP signaling in human platelets. Specifically, the results have suggested that while P2Y1 activation can initiate platelet aggregation, P2Y12 signaling accounts for the bulk of the ADP‐mediated effects. On this basis, efforts have focused on the development of a new class of P2Y12 antagonists (separate from clopidogrel and ticlopidine) as potential antithrombotic agents. The present work examined the molecular mechanisms by which one such adenosine‐based P2Y12 antagonist, i.e., Cangrelor (ARC69931MX), inhibits human platelet activation. It was found that the ability of ARC69931MX to block aggregation was associated with significant increases in platelet cAMP levels. It was also found that this elevation of cAMP did not require Gi signaling or even P2Y12 receptors, but was mediated through a separate G protein‐coupled pathway, presumably involving Gs. However, additional experiments revealed that ARC69931MX does not increase cAMP through activation of known platelet Gs‐coupled receptors, i.e., A2a, IP, DP or EP2. Collectively, these findings provide evidence that ARC69931MX interacts with an unidentified platelet G protein‐coupled receptor that stimulates substantial cAMP elevation and significant inhibition of platelet function through a P2Y12‐independent mechanism.
Metabolism of the analgesic flupirtin in man
Authors: Scheuch Eberhard , Methling Karen , Ullrich Anett , Runge Dieter , Terhaag Bernd , Siegmund Werner
Published: June 2021

Flupirtin (FLU) is a non‐opiotic analgetic drug used to relieve acute and chronic pain. Recently, FLU was suspected to cause liver damage as detected by an elevation of hepatic transaminases. However, the biochemical mechanism behind this is unknown; furthermore, the metabolism of FLU is known only in part. Therefore, we evaluated drug metabolism in vitro and metabolic disposition of the drug in man using a metabolomics approach. Healthy volunteers received an oral administration of FLU (100 mg/day) over a period of 5 days. The metabolic pattern in urine collected within this interval was compared with a blank urine sample before treatment. Mass spectrometric techniques were performed using urine fractions at pH = 6 and pH = 10. For estimating the influence of glucuronidation of FLU and its main metabolite D‐13223 in detail, recombinant expressed human UGT isoenzymes were used for competition assays between FLU and their specific substrates. In urine, FLU, D‐13223, the N oxidized metabolites of FLU and D‐13223 as well as two, so far not described, N‐glucuronides and mercapturic acid derivatives were detected. Furthermore, FLU did not substantially inhibit the enzymatic activity of UGT1A isoenzymes, however, 300 µM FLU lead to a small but significant reduction of UGT2B7 and 2B15 activities. In addition to known metabolites Flupirtin is metabolized to glucuronides and glutathione adducts in man.
Qualitative Analysis of the Role of Metabolites in Inhibitory Drug‐Drug Interactions: literature evaluation based on the Metabolism and Transport Drug Interaction Database
Authors: Yeung Catherine K. , Isoherranen Nina , Hachad Houda , Levy Rene H.
Published: June 2021

FDA guidance is specific in the characterization of metabolites of new drugs that may be involved in toxicities, but does not address the potential for metabolites to cause inhibitory drug‐drug interactions. This work was conducted to evaluate what fraction of inhibitors of common drug metabolizing enzymes (CYPs 1A2, 2E1, 2D6, 2C9, 2C19, 2C8, 2B6 and 3A4) have circulating metabolites that may contribute to in vivo interactions. A literature analysis was conducted using the Metabolism and Transport Drug Interaction Database to identify all precipitants (i.e. inhibitors) that cause >20% increase in the AUC of marker substrates. Of 130 inhibitors identified, 106 were confirmed to have metabolites that circulate in plasma. Only 7% of the inhibitors did not have circulating metabolites. Of the 21 potent inhibitors, 17 had circulating metabolites; the remaining 4 were all extensively metabolized. Based on available in vitro data, 21 of the inhibitors were mechanism‐based inactivators of P450 enzymes while 111 were reversible inhibitors. In vitro evaluation of inhibition potential was conducted for only 32% of the circulating metabolites of the inhibitors. We conclude that circulating metabolites are often present with inhibitors of P450 enzymes suggesting a need for increased efforts to characterize the inhibitory potency of metabolites of candidate drugs, and for newer models for in vitro‐in vivo extrapolations.
Bioavailability of oversulfated chondroitin sulfate in rats. Pharmacological implications in contaminated heparins
Authors: Losada Angel , Hoppensteadt Debra , Litinas Evangelos , Emanuele Joe , Mostert Michael , Linhardt Robert , Fareed Jawed
Published: June 2021

The main contaminant in recalled heparins were characterized to be oversulfated chondroitin sulfate (OSCS). The molecular weight profile of the contaminants was similar to the unfractionated heparin (14,000‐16,000 daltons). However, OSCS exhibited distinct biochemical and pharmacologic profiles. The purpose of this study is to determine the pharmacokinetic profile of OSCS in rats after intravenous (IV) and subcutaneous (SC) administration. Individual groups of rats were administered with a 2 mg/kg dosage of OSCS (15.8) via IV or SC route. Blood samples were drawn within 15‐30 minutes after the IV administration and within 2‐3 hours after the SC administration. The absolute concentration of OSCS was measured using a modified version of a highly specific GAG binding assay (Lifespan, Salt Lake City, UT). PT, aPTT, Heptest and thrombin time were measured. The OSCS was also measured in the urine. The circulating level of OSCS after IV ranged from 20‐30 ug/ml (22+4 ug/ml). The urinary levels were higher after IV administration ranging from 25‐40 ug/ml (31+7 ug/ml). In the SC studies, the circulating levels of OSCS exhibited wide variations ranging from 0‐7 ug/ml (4+3 ug/ml). In the urine from SC administered rats, 7/10 rats did not have any OSCS, whereas the other 3 rats exhibited detectable amounts ranging from 4‐12 ug/ml. These results demonstrate that OSCS is capable of being absorbed after SC administration with an apparent bioavailability of 0.15. The presence of OSCS in urine indicates that some of its components are cleared through the kidney.
2‐Phenyl‐naphtho[2,3‐d]oxazole‐4,9‐dione is Cytotoxic against Androgen‐dependent and ‐independent Prostate Cancer Cell Lines.
Authors: Ononiwu Innocent Maduabughichukwu , Bakare Oladapo , Adedeji Dolapo , Copeland Robert , Kanaan Yasmine , Warrick Cynthia , Gwebu Ephraim
Published: June 2021

Abstract:

In the United States of America, prostate cancer is the second most common cancer which affects thousands of men each year. Treatment options for prostate cancer are limited and the search for compounds with significant efficacy against prostate cancer is on‐going. 1‐4 naphthoquinones have been reported to possess significant effects against cancer cell lines and solid tumors. In the present study, we evaluated the efficacy of 2‐Phenyl‐naphtho[2,3‐d]oxazole‐4,9‐dione against two androgen‐dependent (LNCaP, CW‐22R) and two androgen‐independent (PC‐3, DU‐145) prostate cancer cell lines using the MTT‐antiproliferation assay.

Our data show that 2‐Phenyl‐naphtho[2,3‐d]oxazole‐4,9‐dione possesses potent antiproliferative activity against the prostate cancer cell lines with IC50 values of 0.19 μM, 0.37µM, 0.89µM and 1.78µM, for LNCaP, PC‐3, CWR‐22R and DU‐145 respectively.

2‐Phenyl‐naphtho[2,3‐d]oxazole‐4,9‐dione is a potent antiproliferative compound against prostate cancer cell lines and could serve as a template for synthesizing a library of compounds with significant efficacy against prostate cancer.
Ischemic injury, hemorrhagic transformation and plasma MMP‐9 profile in experimental diabetes vs. hyperglycemia
Authors: Elgebaly Mostafa , Mezzetti Erin M , Fagan Susan C , Ergul Adviye
Published: June 2021

Diabetes increases stroke risk. We recently showed cerebrovascular remodeling associated with higher matrix metalloproteinase (MMP) activity and increased hemorrhagic transformation (HT) following 3 h middle cerebral artery occlusion /21 h reperfusion in Type 2 diabetes. Plasma MMP‐9 may be predictive of HT following tPA treatment in stroke. Thus, the goals of this study were to measure plasma MMP‐9 before and after stroke in control and Type 2 diabetic Goto‐Kakizaki (GK) rats and in acutely hyperglycemic rats during stroke. The hypothesis was that baseline plasma MMP‐9 activity is higher in GKs and is further elevated after ischemia. Plasma MMP‐9 activity was determined at baseline and at sacrifice after 3/21 MCAO/reperfusion by zymography. Basal MMP‐9 activity was higher in GKs (11.2 ± 4.1 vs. 0.7 ± 0.3, n=6, p<0.001). MCAO/reperfusion significantly increased plasma MMP‐9 in control, diabetic and acute hyperglycemia rats (103 ± 23.2, 113 ± 15, 124 ± 9, respectively, n=3‐6, p<0.0001 vs. baseline) but there was no difference between groups. Infarct size was smaller in GK (11 ± 4%, n=3, p<0.01) vs. control (42 ± 6%, n=4) and hyperglycemic (47 ± 6%, n=4) rats. Control animals had no HT yet there was macroscopic bleeding in GK and hyperglycemic rats. These results suggest that 24 h plasma MMP‐9 levels are not associated with higher bleeding in diabetic and hyperglycemic rats and earlier time points merit evaluation.
Selenium Imaging in Mouse Tissues
Authors: Malinouski Mikalai , Kehr Sebastian , Finney Lydia , Vogt Stefan , Gladyshev Vadim
Published: June 2021

Imaging of trace elements in cells and tissues provides an opportunity to understand their functions. Distribution of selenium under either physiological or pathophysiological conditions remains unknown due to the lack of sensitive analytical techniques. We applied X‐ray fluorescence microscopy (XFM) to map selenium distribution in various tissues of mice. High spatial resolution of XFM revealed cellular topography of selenium as well as other trace elements. Selenium distribution and content were analyzed in mouse brain, thyroid, liver, kidney, prostate, and testes. In mouse testes, selenium was primarily localized in elongating spermatids. Testes samples from mitochondrial glutathione peroxidase 4 knockout mice reduced selenium content. Removal of Selenoprotein P (SelP) also led to a significant decrease in this element in mouse testes. The highest concentration of selenium was observed in the glomerulus layer of olfactory bulb. This layer was associated with high expression of SelP. In SelP knockout olfactory bulb, selenium was decreased almost 10 fold. In kidney, selenium localized to basal membrane of proximal tubules and was not affected by SelP knockout. In conclusion, the use of XFM allowed visualization and analysis of specific cellular distribution of selenium and other trace elements in mouse tissues.
Dietary n‐6 PUFA deprivation in adult rats increases docosahexaenoic acid metabolism and consumption by brain
Authors: Igarashi Miki , Kim Hyung‐Wook , Chang Lisa , Ma Kaizong , Bell Jane M , Rapoport Stanley I
Published: June 2021

We reported that dietary n‐6 PUFA deprivation in post‐weaning rats increases DHA (22:6n‐3) concentration in the brain, whereas decreasing the concentration of AA (20:4n‐6). We hypothesized that the DHA increase would be accompanied by altered kinetics of brain DHA. In this study, we quantified incorporation and turnover rates of DHA into brain phospholipids of unanesthetized male rats fed an n‐6 PUFA adequate diet (27.6% LA, 4.5% ALA) or deficient diet (2.3% LA, 4.8% ANA) for 15 weeks. Rats were infused intravenously with [1‐14C]DHA, arterial plasma was collected, and the brain was microwaved at 5 min and analyzed. n‐6 PUFA deprivation did not change the concentration of any stable brain lipid. Rats fed the deficient compared with adequate diet had less brain AA in brain phospholipids (‐28%~‐11%) and increased DHA in only EtnGpl. DHA incorporation rates from plasma were significantly increased 1.4‐1.8 fold in brain phospholipids, and DHA turnover rates were increased in ChoGpl, PtdIns, and PtdSer. Thus, n‐6 PUFA deprivation increased DHA incorporation and turnover in rat brain. The higher incorporation rates indicate that brain DHA was being lost by metabolism at an accelerated rate, since the incorporation rate of DHA is stochiometrically equal to its rate of metabolic loss from brain. This may indirectly increase dietary demand for DHA.
Protein turnover during pregnancy
Authors: Levesque Crystal L , Moehn Soenke , Pencharz Paul B , Ball Ron O
Published: June 2021

The response of whole body protein turnover in sows to increasing dietary threonine (THR) during pregnancy was determined using continuous oral delivery of L[1‐13C]phenylalanine (PHE) in six multiparous sows. Pregnancy (115 d) was divided into early (35 to 53 d) and late (93 to 110 d) gestation. Sows received diets with 4.9 ‐ 16.1 gd−1 THR. Blood was sampled every 30 min for 4 h. Data was analyzed with a nonlinear Mixed model.

In early pregnancy, THR intake did not affect protein turnover. However, when expressed as a percent of flux, protein synthesis increased (R2=0.84) and PHE oxidation decreased (R2=0.79) at THR intakes below the dietary requirement. In late pregnancy, PHE oxidation decreased (R2=0.40) and protein synthesis increased (R2=0.40) linearly. The THR requirement based on protein synthesis in early pregnancy was 2‐fold lower (6.0 gd−1) than in late pregnancy (>14.0 gd−1). There was no difference between early and late pregnancy in total protein breakdown (p=0.27, 44.1 ± 2.2 vs 46.2 ± 2.3 gd−1, respectively) and PHE flux (p=0.12, 57.0 ± 2.5 vs 60.0 ± 2.6 gd−1, respectively). However, PHE flux/kg BW was higher in early pregnancy compared to late pregnancy (82.2 ± 2.1 vs 71.2 ± 2.2 gd−1, respectively, p=0.0001).

Funding: AB Pork, ON Pork, AB Livestock Industry Development Fund, ACAAF, Ajinomoto.
Effects of Cheonggukjang Added with Job's Tears Diet on Anticoagulation Activity and Serum Lipid Profile in Hyperlipidemic Rats
Authors: Han Chan‐Kyu , Kim Sung‐Soo , Lee Hye‐Jeong , Lee Bog‐Hieu
Published: June 2021

The effects of Cheonggukjang (Korean traditional fermented soybean soup) and Cheonggukjang added with Job's tears were investigated on anticoagulation activities and lipid metabolism in adult male rats fed on high fat diet for 4 weeks. Five treatment groups(n=12 each) were the high fat control group(A) fed on AIN‐93G added with lard(5%) and cholesterol (0.5%), high fat control diet added with 9%(B), 18%(C), 27%(D) and 36%(E) of Job's tears, respectively. There were no significant changes in body weight gain and body fat amount, but food intake was significantly lower in Job's tears added groups than that of the control(p<0.05). In terms of anticoagulation activities, bleeding time was not different, but activated partial thromboplastin time and prothrombin time were siginificantly different(p<0.05). Serum total‐cholesterol and free cholesterol, and phospholipid levels in Job's tears added diet groups were lower(p<0.05). HDL‐cholesterol level was the lowest in group E(p<0.05), but triglyceride and LDL‐ cholesterol levels did not differ among the groups. Atherogenic index tended to be lower in Job's tears added groups except for group E. From the findings, cheonggukjang diet added with Job's tears seemed to be effective in improving blood lipid profile and anticoagulant activity.
Can coca leaves contribute to improving the nutritional status of the Andean population?
Authors: Penny Mary Edith , Zavaleta Alfonso , Lemay Melanie , Liria Maria Reyna , Huaylinas Maria Luisa , Alminger Marie , McChesney James , Alcaraz Franklin , Reddy Manju
Published: June 2021

Coca leaves (Erythroxylum coca) have been promoted as a food that could address the dietary deficiencies of the Andean population but this is based on analyses of a small sample of leaves. We assessed the nutritional potential of 8 samples of coca leaves from different regions of Peru. We used AOAC to measure nutrients, nutrient inhibitors (phytate, polyphenols, oxalic acid and fiber) and alkaloid concentrations per 100gm dry weight of the ground leaves. Minerals were measured by inductively coupled plasma‐mass spectrometry in two independent laboratories. Leaves contained protein 20.28g/100g, lysine was the limiting amino acid; beta‐carotene 3509 mg/100g; vitamin E 16.72/100g; traces of Vitamin D; calcium 990.18 and1033.17 mg/100g, iron 29.16 and 29.16 mg/100g, zinc 2.71and 2.63 mg/100g and magnesium 225.19 and 196.69mg/100g, labs 1 and 2 respectively. Cocaine was the principal alkaloid, 0.56mg/100g, other alkaloids were also identified. Results were compared with other edible leaves and the nutrients in coca powder (5g) and bread made with coca were compared with normal portions of alternative foods. Two spoonfuls of coca leaf flour would satisfy less than 10% of dietary intakes for schoolchildren and adults for critical commonly deficient nutrients in the diet. Coca leaves do not provide significant nutritional benefits and the presence of absorbable cocaine and other alkaloids maybe potentially harmful.
Efficacy of Medifast's 5&1 meal replacement program compared to a food‐based diet for weight loss and weight maintenance: 4‐month weight loss results
Authors: Coleman Christopher , Davis Lisa M. , Rampolla Joni , Kiel Jessica , Hutchisen Tammy , Ford Laura , Andersen Wayne S.
Published: June 2021

Objective

A RCT was conducted to determine the effectiveness of Medifast's 5 & 1 meal replacement program on body weight and body composition compared to an isocaloric food‐based diet plan for a 4‐month period of weight loss.

Methods

Subjects 90 obese adults randomly assigned to 2 groups: Medifast (MD) (n=45; 30 women, 15 men; BMI 38.5 ± 6.8) and Food‐based (FB) (n=45; 34 women, 11 men; BMI 37.8 ± 4.5). Subjects met biweekly with RDs to have anthropometrics measured and for dietary and behavioral counseling. Weight and bp were measured biweekly. Waist circumference (WC), % body fat, lean muscle mass (LMM), visceral fat, and pulse were measured every 4 weeks.

Results

Subjects lost 2x more body weight on Medifast [MD ‐29.8lbs, FB ‐14.4lbs (p=0.000); % wt loss: MD ‐7.0, FB ‐1.2 (p=0.002)]; 5x more body fat [% Δ in body fat: MD ‐13.6, FB ‐2.7 (p=0.001)]; improved WC [WC Δ: MD ‐13cm, FB ‐8.2cm (p=0.047); % Δ WC: MD ‐11.2, FB ‐7.2 (p=0.069)]; and had 7x greater % reduction in visceral fat: MD ‐25.4, FB ‐3.7 (p=0.001), while maintaining LMM [% wt loss as LMM: MD 16.6, FB 47.3 (p=0.002)]. No significant between groups differences were found for bp or pulse. Retention rates (64% vs. 44%) and program adherence was higher in the MD group.

Conclusion

At 4 months, Medifast's 5&1 meal replacement program out‐performed an isocaloric food‐based diet in weight loss and body composition. This research was funded by Medifast, Inc., Owings Mills, MD.