bluuebunny/crossref_metadata_2025 huggingface dataset:
The HUS‐box is required for allosteric regulation of the Sec7 Arf‐GEF
Authors: Halaby Steve , Fromme Chris
Published: June 2021

The Golgi complex is the central membrane and protein sorting station of eukaryotic cells. Activation of Arf GTPases is essential for vesicle formation via recruitment of cargo adaptors and coat proteins necessary for Golgi trafficking. Arf activation is spatially and temporally regulated by distinct guanine nucleotide exchange factors (GEFs) at different Golgi compartments. The yeast Arf‐GEF Sec7 is a conserved and essential activator of Arf1 at the trans‐Golgi network. Sec7 contains a highly conserved regulatory region, the HUS‐box, with an unknown mechanistic role. In this study we explore how the HUS‐box, which is N‐terminal to the catalytic domain, acts together with C‐terminal regulatory domains in the allosteric activation of Sec7. We show that mutation of the HUS‐box disturbs positive feedback and allosteric activation of Sec7 by the GTPase Ypt31, the yeast Rab11 homolog. Taken together, our data lead to a model in which the inter‐ and intramolecular interactions of the HUS‐box and C‐terminus are necessary for the allosteric activation of Sec7.

Support or Funding Information

This work was supported by NIH/NIGMS award R01GM09861 to J.C.F. and NIH/NIGMS training grant T32GM007273 to S.L.H.

This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Structure and function of N‐glycans in cobra venom glycoproteins: Site specific glycosylation for structural stability and terminal sialic acid for target selection
Authors: Chien Kun Yi , Wu Wen Guey , Bui Ba Han , Lin Chien Chu
Published: June 2021

Snake venoms consist of glycoproteins such as phosphodiesterase (PDE), 5′‐nucleotidase (5NT), L‐amino acid oxidase (LAAO) and snake venom metalloprotease (SVMP) with different degree of glycosylations, but their structures and functions remain largely unknown. We purify these four enzymes from Taiwan cobra (N. atra) venoms and determine their respective glycosylation patterns at peptide level using mass spectrometry. Although the detected N‐glycan structures are diverse with mainly complex and high mannose type, they are found to be site specific. For instance, cobra PDE consists of 7 N‐glycans with 4 high mannose and 3 complex types without detectable sialic acid. In contrast, K‐like SVMP consists of 2 complex type N‐glycans with different degree of sialylations. Additional 3D structural and enzymatic characterizations indicate that the high mannose N‐glycan in PDE maintain the PDE 3D structures while the terminal sialic acid of the complex type glycan in SVMP play a role in target selection of fibrinogen. We conclude that the N‐glycans of snake venom glycoproteins play both structural and functional roles and likely involve in the action mechanism of venom toxicity.

Support or Funding Information

MOST, Taiwan

This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Identification of CRISPR‐Cas9 Mutants in Arabidopsis Glutaredoxin Genes AtGRXS11, AtGRXS6, and AtGRXS3/4/5/7/8 Gene Cluster
Authors: Fernandez Francisco , Sanchez Kassandra , Escobar Matthew
Published: June 2021

Glutaredoxins are small redox enzymes that use glutathione as a substrate to reduce disulfide bonds in target proteins. Plants have a far greater number of glutaredoxins than other organisms, mostly due to a unique clade of class III glutaredoxins that is exclusively found in higher plants. Previous studies in our lab demonstrated that a small group of class III glutaredoxin genes are strongly upregulated by nitrate in Arabidopsis thaliana, and that RNA silencing of some of these glutaredoxins leads to increased primary root growth. Thus, glutaredoxins appear to link nutrient sensing with plant growth and root system architecture. To further explore this hypothesis, we are using CRISPR‐Cas9 technology to perform targeted mutagenesis of the AtGRXS11, AtGRXS6, and AtGRXS3/4/5/7/8 glutaredoxin genes in A. thaliana. CRISPR vectors have been assembled and utilized for Agrobacterium‐mediated plant transformation. Transgenic plant lines were initially screened by restriction enzyme‐mediated cleavage of glutaredoxin sequences amplified from genomic DNA, demonstrating the loss of restriction sites near presumptive Cas9 cut sites. The presence of small insertion‐deletion mutations in the AtGRXS11 and AtGRXS6 genes was then verified by Sanger sequencing. The AtGRXS3/4/5/7/8 genes are located in a ~10 kb tandem array on A. thaliana chromosome 4, and we have attempted to delete this entire region using two CRISPR guide RNAs that target the flanks of this gene cluster. PCR screening has identified transgenic lines which appear to contain the targeted large‐scale deletion. Because the nitrate‐regulated glutaredoxins appear to be important regulators of primary root growth, this work could have significant broader implications related to important agricultural traits such as root depth (drought tolerance) and nitrogen use efficiency.

Support or Funding Information

This research was supported by NSF RUI‐IOS grant 1651584. We would like to thank the MARC U*STAR Program (NIH GM‐08807) for funding the student researchers Francisco Fernandez, Kassandra Sanchez

This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Reenergized Adoptive Cell Transfer Immunotherapy
Authors: Kaiser Carl , Klestinski Keith , Cui Weiguo , Chang Mika , Cowan Nelson , Smith Sean , Strom Daniel , Strom James
Published: June 2021

Immunotherapy, in treating solid tumors, has seen increased efficacy due to the development of novel strategies of implementation. One innovative strategy, in development by Dr. Weiguo Cui and his colleagues, combines adoptive cell transfer (ACT) with pathogen‐based vaccine techniques in what is called Reenergized ACT (ReACT). Tumor specific CD8 T‐Cells that already have a tumor specific T‐Cell Receptor (TCR), are genetically modified outside the body to express a second TCR that will recognize a bacterial antigen. These newly created, dual specific T cells, when introduced to mice in combination with intratumoral injection of bacterial vaccine, show a significant decrease in tumor volume than their mono‐specific T‐Cell counterparts. The Marquette High SMART (Students Modeling a Research Topic) Team used 3D printing technology, in partnership with the Milwaukee School of Engineering Center for Biomolecular Modeling, to model the TCR complex in order to study its structure and function to better understand ReACT. TCR is a heterodimeric protein consisting of highly variable alpha and beta chains. Both chains are composed of a variable region and a constant region, both of which form antiparallel beta sheets. The variable region binds to the MHC complex whereas the constant region is near the cell membrane of the T‐cell. The constant domains of the TCR consists of cysteine residues that form disulfide bonds, which form a link between the two chains. Further research into immunotherapy, TCR structure and function, and the implementation of Reenergized ACT has major promise in increasing the efficacy of treating disease in human beings now and in the future to come.

Support or Funding Information

Weiguo Cui, MD, PhD at the Blood Research Institute, BloodCenter of Wisconsin & Medical College of WisconsinDiane Muzenmaier at the Milwaukee School of Engineering Center for Biomolecular Modeling

This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
The Mechanism of RAS nucleotide exchange in the Mitogen Activating Pathway
Authors: Shannon Dan , Hall Ben , Peters Kyle , Glass Jake , Gutzwiller Leah , Buehler Sam , Shirley Connor , Lima Nick , Nassar Nicholas
Published: June 2021

RAS is a protein that is integral to the mitogen activating pathway (MAP) that controls cell growth and division. Mutations in RAS deregulate the pathway, which can result in cancer. When bound to the substrate GTP, RAS is activated; when bound to the substrate GDP, RAS is deactivated. The substrate bound to RAS catalyzes conformation changes in two switch regions, which coordinate the interaction RAS has with its substrate. To switch between GTP and GDP, RAS requires assistance from other proteins. GEFs activate RAS by exchanging GDP for GTP; GAPs deactivate RAS by assisting in GTP hydrolysis. The Moeller SMART Team in conjunction with MSOE Center for Biomolecular Modeling used 3‐D modeling and printing technology to pinpoint amino acids that play a crucial role in RAS GTP to GDP exchange. Our mentor, Dr. Nicolas Nassar, created RAS mutations and observed the effect they had on RAS. In these mutations, the two switch regions are restructured, which affects the MAP activity. This alters Mg2+ ion coordination and guanine nucleotide stabilization. These mutations also prevent the coordination of the catalytic water molecule responsible for the hydrolysis of GTP. In mutated RAS, GEFs are not able to exchange GDP for GTP. These mutants transform RAS to stay active longer compared to wt‐RAS. By learning about RAS GTP and GDP exchange, Dr. Nassar hopes to dock a small molecule into the binding site of cancerous RAS thus inhibiting the mitogen activating pathway.

This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Kolaviron, A Flavonoid From Garcinia Kola Seeds, Ameliorates Experimental Colitis In Rats Exposed To Cadmium
Authors: Adegoke Adeoti Gbemisola , Olaleye Samuel Babafemi
Published: June 2021

Cadmium has been shown to potentiate experimental ulcers in laboratory animals. Its ability to predispose humans to ulcerative colitis has also been postulated. Polyphenols and flavonoids found in plants possess antioxidant and metal binding potentials and may therefore, be beneficial in pathological conditions of oxidative stress resulting from exposure to heavy metals. In the present study, the effect of kolaviron, a biflavonoid complex from the seeds of Garcinia kola (Heckel) on colitis in cadmium‐exposed rats was investigated. Male Wistar rats (110–120 g) were randomly divided into five groups. Group 1 received distilled water, Group 2 received dimethyl sulphoxide, and served as negative and positive controls respectively. Group 3 received cadmium (10 mg/kg) while group 4 animals received Cadmium (10 mg/kg) in addition to kolaviron (200 mg/kg). The last group received kolaviron (200 mg/kg) alone. After four weeks of exposure to cadmium with or without kolaviron, colitis was induced in all animals in groups 2–5 by intrarectal administration of 2 mL of 4% Acetic acid (AA). Rats were sacrificed and colons resected at days 0, 3, 7 and 14 days of colitis induction. Assessment was based on symptoms of colitis such as weight loss and stool consistency using a diarrhoea score scale. Colon sample homogenates were analysed for concentrations of Malondialdehyde (MDA), Nitric oxide (NO), Glutathione (GSH), Sulfhydryl, activities of Superoxide dismutase (SOD), Na+‐K+ ATPase, Myeloperoxidase (MPO) by spectrophotometry and histology by hematoxylin and eosin staining. The weight loss (%) in Cadmium group by week 3 was (31.05 ± 0.04). Intrarectal administration of AA resulted in increased stool score in Cadmium group (0.80 ± 0.00) till day 14 post colitis induction. Macroscopic score was significantly (p < 0.05) increased in Cadmium group till day 14. MDA were significantly (p < 0.05) increased in Cadmium group at days 7 and 14. The notable increases in MPO and NO during AA‐induced colitis and cadmium exposure were significantly (p < 0.05) reduced by Kolaviron. Significant (p < 0.05) decreases in SOD, Na+‐K+ ATPase activities, sulfhydryl and GSH concentrations upon AA administration were attenuated in other groups except Cadmium group. Histology showed healing in all groups post colitis induction except in Cadmium group where histopathological damage was still evident at day 14. Kolaviron ameliorated the delayed healing of Acetic acid‐induced colitis during cadmium exposures in Wistar rats and may therefore, be useful as dietary intervention in cases of cadmium exposures.

This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Berberine hydrochloride induces brown adipogenesis
Authors: Li Lixin , Poudel Anil , Zhou Joseph
Published: June 2021

Brown adipose tissue (BAT) dissipates energy in the form of heat through uncoupled respiration. Brown adipocytes originate from different precursor cells and can be induced by various stimuli. Pharmacologically induction of BAT thermogenesis has been shown to be a promising treatment for obesity‐related metabolic disease. In this study, we investigated the potential role of berberine hydrochloride (BBR), a naturally occurring plant alkaloid, in activating brown fat adipogenesis.

3T3L1 preadipocyte cells, C2C12 myoblasts, and stromal vascular cells isolated from perigonadal fat of C57BL/6 mice were cultured in cell differentiation induction media for three days followed by differentiation maintenance media for another three days in the presence or absence of 10 μM of BBR. C2C12 differentiation media was also supplemented with PPARγ agonist rosiglitazone (0.5μM). Adipocyte differentiation was confirmed by oil red staining. Western blot analysis was performed on the differentiated cells to measure brown fat specific protein markers including UCP1, PCG‐1α, PPARγ, PRDM16 and CIDE‐A.

BBR treatment significantly induced all of the above brown‐fat specific markers (p<0.005; n=4) in 3T3L1 cells, C2C12 myoblasts and stroma vascular blast cells. Specifically, UCP‐1 expression is increased 30.3 fold in 3T3L‐1, and 2.00 fold in primary adipocytes extracted from WAT respectively.

Our results thus indicate that BBR induces brown adipogenesis, and is a promising treatment for obesity and obesity related metabolic diseases.

Support or Funding Information

Start Up fund (Central Michigan University )

This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Sox9‐independent Control of Intestinal Paneth Cell Differentiation and Function
Authors: Bankaitis Eric , Dajee Devesh , Magness Scott
Published: June 2021

The genetic and signaling mechanisms controlling the generation and function of differentiated cell types are of continued interest. The intestinal Paneth cell (PC) is a post‐mitotic and dedicated secretory cell type with reported functions in supporting intestinal stem cell (ISC) maintenance in the crypt stem cell niche, and in secreting antimicrobial peptides to protect the crypt from potentially pathogenic luminal microbiota. Previous studies have reported a near‐complete loss of PCs (as defined by morphological and marker‐based criteria) in epithelium where the transcription factor Sox9 is genetically ablated, leading to the proposition that Sox9 is required for PC differentiation. Currently, however, the mechanisms through which Sox9 controls PC differentiation are not known. We have found evidence in Sox9 knockout (KO) small intestine (SI) that cells at the crypt base frequently express PC‐markers such as Lysozyme (Lyz) and Cryptdin, exhibit apical granules, and are post‐mitotic, all of which are features indicative of PC differentiation. The numbers of Lyz+ cells are highest in the distal Sox9 KO SI but are significantly reduced relative to control, and are essentially absent in the proximal SI, suggesting that regionally patterned signals may drive PC differentiation independent of Sox9. Apical granularity and expression levels of Lyz/Cryptdin are also altered, suggesting that while Sox9 KO ISCs can initiate PC differentiation, they are unable to reach full maturity. Antibody and qPCR based detection of PC gene products (including secreted antimicrobials, growth factors, and transcriptional regulators proposed to drive PC differentiation downstream of Sox9) are maintained in Sox9 KO cultured intestinal organoids and are elevated in organoids treated with pharmacological agents that drive the PC differentiation program. These results indicate that Sox9‐deficient ‘Paneth‐like’ cells are continually generated in culture and can be pushed toward PC differentiation with factors known to stimulate the PC differentiation program. We conclude that Sox9 is not required for the activation of at least sub‐sets of gene products associated with PC differentiation and function. Ongoing experiments are designed to investigate Sox9‐ dependent and independent pathways toward PC fate, and to characterize the functionality of Sox9−/− ‘Paneth‐like’ cells compared to controls.

Support or Funding Information

RO1 DK091427‐05

F32 DK115016‐01

This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Relationship Between Sympathetic Baroreflex Sensitivity And Vascular Transduction
Authors: Taylor Chloe E , Hissen Sarah L , Brown Rachael , Macefield Vaughan G
Published: June 2021

Sympathetic baroreflex sensitivity (BRS) is a tool used to quantify how effectively the baroreflex buffers beat‐to‐beat changes in blood pressure. However, current methods of assessment do not take into account vascular transduction, i.e. the response of the peripheral vasculature to vasoconstrictor drive. The aim of the study was to investigate the relationship between sympathetic BRS and vascular transduction. Muscle sympathetic nerve activity (MSNA, microneurography), blood pressure, heart rate and superficial femoral artery blood flow (Doppler ultrasound) were recorded at rest for 5 min in 28 healthy adults (18–31yrs, 13 males, 15 females). Sympathetic BRS was quantified by plotting MSNA burst incidence against mean diastolic pressure using 3 mmHg bins. For vascular transduction, the % change in leg vascular conductance was determined for 15 cardiac cycles following each MSNA burst and the nadir of the response was calculated. Linear regression analysis was used to examine the relationship between sympathetic BRS and vascular transduction in males and females separately. Sympathetic BRS was not significantly different between males (−2.9± 1.8) and females (−3.2 ± 1.6 %bursts/mmHg, p=0.68). However, vascular transduction was significantly lower in males (−5.5 ± 4.1) compared with females (9.1 ± 4.7 %change in ml/min/mmHg, p=0.04). In males there was a significant inverse relationship between sympathetic BRS and vascular transduction (r=0.78, p=0.002), but in females there was no significant relationship (r=0.34, p=0.21). The results suggest that in males those with high sympathetic BRS have low vascular transduction, and vice versa. This may represent a compensatory effect to ensure that blood pressure is regulated effectively. However, this relationship between sympathetic BRS and vascular transduction was not apparent in young females.

This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Evidence Supporting a Role for Autonomic Modulation of C‐Low Threshold Mechanoreceptors
Authors: Provost Makalele , Goolsby Bill , Hochman Shawn
Published: June 2021

C‐low threshold mechanoreceptors (LTMRs) are a functionally distinct class of cutaneous sensory fibers believed to facilitate the sensing and coding of pleasurable touch. These afferents have also been implicated in neuropathic pain states including allodynia, in which normally innocuous tactile stimuli elicit painful sensations. The mechanisms that mediate changes in C‐LTMR function are incompletely understood. Autonomic activity has been implicated in the maintenance of various pain syndromes, yet increases in autonomic neural activity do not seem to influence the firing properties of cutaneous nociceptors. Autonomic modulation of cutaneous C‐LTMRs may contribute to allodynia and neuropathic pain, but this has not been studied. We developed an in vitro isolated truncal skin‐nerve preparation to study the recruitment properties of C‐LTMRs following optical stimulation of skin (TH::CHR2 mice) and the actions of modulatory transmitters when bath applied to the subcutaneous interior side of the skin. Extracellular spike recordings were obtained from dorsal cutaneous nerves. Optogenetic recruitment of C‐LTMRs was temperature sensitive with lower temperatures recruiting more units. Recruited C‐LTMRs had typical conduction velocities (ranging from 0.35 to 0.5 m/s) and an average spike half‐width of 0.53 ms (+/− 0.2ms). We assessed fatigability in response to repetitive optical stimuli at 10 Hz. Consistent with previous reports C‐LTMRs were highly fatigable, especially at higher temperatures. Application of acetylcholine resulted in increased spontaneous afferent activity, reduced C‐LTMR recruitment, and increased fatigability. Application of the cholinesterase inhibitor neostigmine also depressed C‐LTMR recruitment. Norepinephrine also decreased recruitment and increased fatigability but reduced the incidence of spontaneous activity. The norepinephrine transport inhibitor clomipramine also reduced C‐LTMR recruitment. Serotonin did not result in changes to recruitment or fatigability of C‐LTMRs, but increased spontaneous activity. However, the serotonin transport inhibitor citalopram did reduce C‐LTMR recruitment. Similarly, dopamine administration had no effect, but application of the dopamine transport inhibitor buproprion depressed C‐LTMR recruitment. We conclude that C‐LTMR activity can be modified by neuromodulatory transmission and studies are underway to further characterize this effect.

This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Ion Channel Activity in a 3‐hydroxyacyl‐coA dehydratase 1 (HACD1) Deficient Muscle Cell Line
Authors: Morgan Rhiannon Sarah , Walmsley Gemma , Dyer Steven , O'Brien Fiona , Staunton Caroline , Blondelle Jordan , Tiret Laurent , Piercy Richard , Barrett‐Jolley Richard
Published: June 2021

Centronuclear myopathy in Labrador retrievers is the most common inherited neuromuscular disorder in dogs: this is caused by a mutation in a fatty acid processing gene, HACD1. The mutation is carried by 15–20% of the breed worldwide (Pele et al., 2005). These dogs present with generalised progressive weakness. A congenital myopathy due to a HACD1 mutation has also been documented in a human family, where infants have hypotonia and delayed motor milestones (Muhammed et al., 2013). The cellular mechanisms by which HACD1 mutation lead to muscle weakness are unknown, but since fatty acids interact with membrane ion channels we hypothesised that dysregulation of membrane function could be involved. To investigate this we have used a muscle cell line (C2C12) with shRNA knock down (KD) of HACD1 in combination with ion channel gene qPCR and patch‐clamp electrophysiology.

To quantify changes in C2C12 myogenic differentiation following HACD1 KD, we used a fusion index calculation on day 8 myotubes stained for the differentiation marker MF20 and counted nuclei that were present in MF20 positive myotubes against those that were in undifferentiated, MF20 negative cells. No significant difference in myotube fusion was observed in HACD1‐KD cells compared to control (n=2).

Before beginning patch‐clamp studies on C2C12 cells we used qPCR to identify potential ion channel targets in control C2C12 myoblasts. We found expression of (<30ct) of 21 ion channels, including those of particular interest to calcium homeostasis such as the voltage‐gated Ca2+ channels; Cacna1c, cacna1g, the non‐selective cation (NSC) channels; TRPV4, TRPV2, TRPC1 and a Ca2+‐activated K+ channel; Kcnn3.

To functionally identify channel gating we used cell‐attached patch‐clamp electrophysiology of control and HACD1‐KD C2C12 myoblasts. K‐means clustering revealed five statistically significant clusters of ion channel activity in these cells (p<0.05). Assuming an RMP of −15mV (Tanaka et al., 2017) clusters had centeroids of;(i) conductance 21 ± 0.4pS, Vrev 30 ± 6mV (ii) 28 ± 6pS, −38 ± 4mV (iii) 29 ± 0.7pS, −78 ± 4mV (iv) 40 ± 6pS, 5 ± 3mV (v) 94 ± 13, 16 ± 44mV.

In conclusion, the phenotype of the smallest ion channel conductance (28pS) would be consistent with the Ca2+‐activated K+ channel KCNN3, and the 94pS consistent with any of the three TRP channels identified by qPCR. These will be further characterised with pharmacological inhibitors and in the future we will examine if the populations change as the control and HACD1‐KD myoblasts differentiate into myotubes.

Support or Funding Information

Authors would like to thank J Blondelle & L Tiret from the Alfort Veterinary School for the cells used in these experiments.

This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
The Effects of Leucine Supplementation on Skeletal Muscle Mitochondrial Content and Protein Turnover in Tumor Bearing Mice
Authors: Lee Harold , Persinger Aaron , Lee Kevin , Jones Michelle , Puppa Melissa
Published: June 2021

Introduction

Many forms of cancer are associated with loss of lean body mass, a significant indicator of increased mortality. This effect is commonly attributed to decreased protein synthesis and stimulation of proteolytic pathways within the skeletal muscle due to metabolic disturbances from the tumor burden. The branched chain amino acid (BCAA) leucine has been shown to improve protein synthesis, insulin signaling, and mitochondrial biogenesis, key signaling pathways influenced by tumor signaling.

Purpose

Our study aimed to elucidate the effects of leucine supplementation on mitochondrial biogenesis, within the Lewis Lung Carcinoma (LLC) implantation model. We hypothesized that LLC implantation will impair mitochondrial biogenesis and protein synthesis leading to a loss of muscle mass, with leucine attenuating these effects.

Methods

Twenty male C57BL/6 mice were divided into four equal groups (n=5) of equivalent body weight: Chow, leucine (Leu), LLC, LLC+Leu. At the age of 9–10 weeks, mice received a subcutaneous injection of 1×106 LLC cells or phosphate buffered saline (PBS). Leu groups were then switched to diet supplemented with 5% leucine (w/w). Upon euthanasia, muscle and tumors were weighed and collected. Measures of protein synthesis, mitochondrial biogenesis, and inflammation in the gastrocnemius muscle were assessed via western blot analysis.

Results

While body mass was not different between groups, gastrocnemius mass was decreased in the LLC+Leu group relative to the LLC group (p=0.040). Protein synthesis, quantified through the SUnSET technique, was decreased in LLC mice (p=0.001). Phosphorylation of STAT3, an indicator of inflammation was decreased in the Leu group relative to the control (p=0.019), but did not significantly attenuate the inflammatory effect of LLC implantation (p=0.619). Peroxisome proliferator‐activated receptor Gamma Co‐activator 1‐α (PGC‐1α), a marker of mitochondrial biogenesis, was increased in LLC+Leu relative to LLC (p=0.001). Finally, LLC implantation decreased mitochondrial content as measured by Cytochrome C (p=0.015), mitochondrial complexes III (p=0.006) and V (p=0.041).

Conclusion

Within our study, leucine supplementation was unable to preserve protein synthesis or mitochondrial content associated with LLC implantation. However, Leucine supplementation was able to increase mitochondrial biogenesis signaling.

Support or Funding Information

University of Memphis School of Health Studies Faculty Grant

This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Comparative Pharmacokinetic Profile of Three Batches of Ovine Enoxaparin and One Batch of Branded Enoxaparin
Authors: Rangnekar Varun , Jeske Walter , Yao Yiming , Kouta Ahmed , Hoppensteadt Debra , Duff Rick , Fareed Jawed
Published: June 2021

Background

Heparins can be derived from tissues of a variety of different species. Although current pharmaceutical grade heparin is derived almost exclusively from porcine intestinal mucosa, there is interest within the FDA for diversifying the sourcing of heparin in the US to protect against supply shortages and economically motivated adulteration as was seen in 2008. Because sheep‐derived (ovine) heparin is structurally similar to porcine heparin, it is expected that low molecular weight heparin (LMWH) derived from ovine‐heparin will be comparable to porcine‐derived LMWH. This study will compared the pharmacokinetic behavior of ovine LMWH with that of enoxaparin in non‐human primates.

Materials and Methods

Enoxaparin (Lovenox, Sanofi‐Aventis, Bridgewater, NJ) and three batches of ovine LMWH produced by benzylaztion/alkaline hydrolysis (Suzhou Ronnsi Pharma, Suzhou, China) were used in this study. Following anesthesia, a baseline blood sample was collected from non‐human primates (Macaca mulatta; n=4/LMWH). LMWH was administered subcutaneously (SC) at a dose of 1 mg/kg. Additional blood samples were collected at 2, 4 and 6 hours post‐administration. Circulating drug concentrations determined using anti‐Xa and anti‐thrombin assays were used to calculate pharmacokinetic parameters such as half‐life, area under the curve, systemic clearance and volume of distribution using the PKSolver add‐in for Microsoft Excel. Tissue factor pathway inhibitor (TFPI) levels were measured by ELISA.

Results

In vitro studies demonstrated that the ovine LMWHs met pharmacopoeial requirements for enoxaparin in terms of potency and molecular weight distribution. In the samples collected at 2, 4, and 6 hours, comparable levels of branded enoxaparin and the three batches of ovine enoxaparin were observed using both anti‐Xa and anti‐thrombin assays, with the concentration vs. time curves being nearly superimposable. Consistent with the similarity observed with the plasma drug concentrations, no significant differences were observed between pharmacokinetic parameters calculated for branded enoxaparin and ovine LMWH. TFPI levels returned to baseline levels by 6 hours in ovine LMWH‐treated animals, but remained slightly elevated in animals treated with branded enoxaparin.

Discussion

At a 1 mg/kg SC dosage, three ovine enoxaparin preparations produced comparable anti‐Xa and anti‐IIa effects which were similar to the ones observed with branded enoxaparin. The PK parameters calculated based on this data were also comparable for both groups. It is concluded that the SC pharmacokinetics of the three batches of ovine enoxaparin were not only comparable between batched, but were also similar to the branded product.

This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Molecular Insights into Tachyphylaxis Phenomenon in the Angiotensin II Type 1 Receptor
Authors: Duarte Diego Angelo , Parreiras‐e‐Silva Lucas Tabajara , de Oliveira Eduardo Brandt , Bouvier Michel , da Costa‐Neto Claudio Miguel
Published: June 2021

Tachyphylaxis is a phenomenon characterized as a progressive decrease in drug response after prolonged or repetitive administration; however, the precise mechanism behind such phenomenon remains unclear. Here, we assessed the molecular bases of tachyphylaxis using the Angiotensin II (AngII) type 1 receptor (AT1R) and two AngII analogs, named #001 and #002, which function as non‐tachyphylactic agonists. The affinity of both analogs for the AT1R was obtained by radioligand competition binding assays, and they were pharmacologically characterized by prolonged and short stimulation using bioluminescence resonance energy transfer (BRET) assays in Human Embryonic Kidney (HEK) 293T cells expressing the AT1R. Analogs #001 and #002 presented low affinity for the AT1R as compared with AngII, but behaved as full agonists for Gq protein activation, intracellular calcium mobilization, as well as concerning to β‐arrestin recruitment, with potencies similar to those of AngII. We also showed that the activation of Gq protein and the translocation of β‐arrestins to the plasma membrane triggered by the analogs were rapidly reversed either by washout or addition of the antagonist Losartan, whereas AngII induced a sustained response even after washout or addition of Losartan. Furthermore, the internalization of the AT1R induced by analogs #001 or #002 showed a faster recycling and to be more sensitive to washout or addition of Losartan than when induced by AngII. Our results suggest that the molecular bases of AngII tachyphylaxis are linked to distinct kinetics of sustained signaling, which we believe could be due to a higher dissociation rate of the analogs. According to our hypothesis, the non‐tachyphylactic AngII analogs activate the AT1R in a fast but transient manner, consequently leading the AT1R to be quickly recycled to the plasma membrane, becoming available to a new cycle of activation. On the other hand, the sustained activation of AT1R by AngII and the low dissociation rate of AngII seem to lead to a sustained internalization of the AT1R in endosomes, retarding their recycling to the plasma membrane. We believe that understanding the molecular mechanisms involved in tachyphylaxis may ultimately contribute to improve the efficiency of drugs and therapies.

Support or Funding Information

FAPESP (process numbers 2014/09893‐0 and 2012/20148‐0)

This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Saturated fat‐enriched diet decreases SIRT1 expression in the mouse hippocampus ‐ The SIRTain effects of saturated fat in the brain
Authors: Marwarha Gurdeep , Ghribi Othman
Published: June 2021

A multitude of prospective cohort epidemiological studies have posited a direct positive correlation between saturated fat‐enriched diets and an increased risk for cognitive impairment, deficits in learning and memory, leading to enhanced susceptibility for the development and progression of neurodegenerative disorders such as Alzheimer's disease (AD). Our previous studies have shown that saturated‐fat enriched diet cause an increase in BACE1 expression and activity leading to an increase in Amyloid‐beta burden in the brains of the C57BL/6J wild‐type mice (1–3). The histone deacetylase and the sirtuin SIRT1, is a master regulator of molecular processes that regulate lifespan and aging, the primary risk factor for AD. A plethora of laboratory studies have demonstrated the indispensable role of SIRT1 in regulating and maintaining synaptic integrity as well as synaptic plasticity at the molecular level and cognitive processes that underlie learning and memory. Herein, we show that feeding nine‐month old C57BL/6J wild‐type mice, a palmitate‐enriched diet for three months, elicits a profound attenuation of SIRT1 expression in the hippocampal region of the brain. Exogenous palmitate treatment of human SH‐SY5Y neuroblastoma cells also results in an attenuation of SIRT1 expression. We further elucidated and delineated the underlying signaling and molecular mechanisms involved and discovered a deleterious role of the palmitate‐evoked Endoplasmic Reticulum (ER) stress‐induced transcription factor C/EBP Homologous Protein (CHOP) in mediating the palmitate effects on SIRT1 expression. Homozygous Chop−/− knock‐out mice fed a palmitate‐enriched diet did not exhibit the same degree of loss of basal SIRT1 expression in the brain compared to their C57BL/6J wild‐type littermates. Also, RNA‐interference mediated knock‐down of CHOP expression in SH‐SY5Y neuroblastoma cells rescued the palmitate‐induced deficits in SIRT1 expression. Further insight into the mechanisms unveiled the role of CHOP in transcriptional repression of SIRT1 expression executed through the negative regulation of the transcription factor C/EBPα. Our study unveils SIRT1 as a unique molecular target that is negatively regulated by palmitate‐enriched diets. Further studies are warranted to determine the potential biochemical and physiological impact of this palmitate‐induced attenuation of SIRT1 expression, a histone deacetylase that plays an indispensable role in the regulation of cognitive processes and aging at the molecular level.

Support or Funding Information

This work was supported by a Grant from the NIH (R01AG045264) to Dr. Othman Ghribi. The authors declare that they have no competing interests.

This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Calcium‐induced apoptosis in cultured, primary corneal endothelial cells from monkey
Authors: Nakajima Emi , Gleason Ellen K. , Walkup Ryan D. , Shearer Thomas R. , Azuma Mitsuyoshi
Published: June 2021

Apoptosis plays a key role in the loss of corneal endothelial cells (CEC). Losses can occur during corneal storage and after transplantation. Unlike the epithelium, CEC do not regenerate. CEC stretch to compensate for dead cells. This reduces cell density, allows influx of water, and promotes formation of opacities. Several studies confirm that staurosporine and mitomycin cause apoptosis in CEC. However, molecular mechanisms responsible for apoptosis in CEC have not been well studied. The purpose of the present study was to determine if CEC apoptosis is initiated by calcium uptake.

To increase calcium levels, cultured primary CEC from monkey were treated with either A23187 (A23) or thapsigargin (TG). The ionophore A23 allows influx of Ca2+ across cell membranes, while TG inhibits the outward directed Ca2+‐ATPase pump in endoplasmic reticulum (ER).

We found that both A23 and TG caused: increased cytosolic calcium levels, up‐regulation of ER stress markers BiP and CHOP, and positive staining for TUNEL. Apoptosis was further confirmed by positive immunostaining for activated caspase‐3. The known CEC maker/calcium binding protein S100 was decreased after A23 treatment but not after TG. (Further testing is needed as to why only A23 down‐regulates S100). We suggest that apoptosis in CEC occurs when elevated cytosolic calcium stresses the ER, the stressed ER release calcium, and the calcium flows into the mitochondrial matrix to trigger the apoptotic response. The unfolded protein response (UPR) may also contribute since Ca‐mediated ER stress is known to cause CEC degeneration.

Support or Funding Information

Dr. Shearer receives consulting fees from, and Drs. Azuma and Nakajima are employees of, Senju Pharmaceutical Co., Ltd.

This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
In‐Silico Comparative Structural Analysis of Human and Murine Cyclooxygenase‐2 (Cox‐2)
Authors: Hari Saran Krishnan , Boluwaduro Adeyemi , Gautham Sakthi , Villanueva James Amador
Published: June 2021

Cyclooxygenase 2 (COX2) is a major enzymatic mediator of inflammation that catalyzes the formation of a host of prostaglandins, leukotrienes and thromboxanes. Inflammation is a principal component of a vast range of pathologic processes, ranging from simple infections to malignant neoplasms. Since this physiologic response is sometimes more damaging than beneficial to the human body, curbing its excesses by controlling the activity of COX2 is of great clinical importance. In‐silico studies have utilized murine, bovine, or other forms of COX2, as the human COX‐2 crystal structure has only been very recently elucidated. Using molecular docking and alignment studies, we found differences in the bonding interactions of murine COX2, and a recently resolved human COX2 crystal structure with their substrate, arachidonic acid, as well as piceatannol. Piceatannol is a metabolite of resveratrol, which is found in red wine. Our experiments show that although the sequence and structures of human and murine COX2 are largely similar with variation of 12%, there are important differences in the way their binding sites interact with ligands, with respect to binding energies and amino acid involvement. These differences may become significant in the search and design of small molecules that can function to inhibit the activity of COX‐2. Clinically, this would translate to better control over the deleterious effects of inflammation.

This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Utilizing Structure‐Activity Relationships to Design Non ‐ Oxime Reactivators
Authors: Cadieux C. Linn , Canter Zachary , Martin Kevin , Hepperle Michael , Cerasoli Douglas M.
Published: June 2021

Organophosphorus nerve agents are highly toxiccompounds that pose a threat worldwide. These compounds induce toxicity bycovalently binding to the active site serine of acetylcholinesterase, whichresults in inhibition of the native enzyme function. Without functionalacetylcholinesterase, levels of the neurotransmitter acetylcholine inneuromuscular junctions rise quickly, causing overstimulation of the nervoussystem, which can quickly culminate in death if not treated. Current treatmentsrely on small molecules that interact with inhibited enzyme to disrupt thecovalently bound phosphorus moiety at the active site. The most effectivemolecules incorporate a pyridinium oxime which acts via direct nucleophilicattack on the phosphorus to achieve reactivation of the enzyme. These compoundshave limited effectiveness because the charged portion of the molecule does notallow them to cross into the central nervous system where acetylcholinesteraseinhibition is most harmful. We recently characterized a small moleculereactivator (4‐amino‐2‐((diethylamino)methyl)phenol [ADOC]) that does not utilizean oxime but is capable of reactivating nerve agent‐inhibited enzyme as well asor better than current treatments; these results have been used to inform thedesign of additional novel compounds. This study describes the in vitro characterization of these novelcompounds as reactivators of phosphonylated human acetylcholinesterase.

Support or Funding Information

*Thisresearch was supported in part by an appointment to the Postgraduate ResearchParticipation Program at the U.S. Army Medical Research Institute of ChemicalDefense administered by the Oak Ridge Institute for Science and Educationthrough an interagency agreement between the U.S. Department of Energy and theU. S. Army Medical Research and Materiel Command.

Thisresearch was supported by the Defense Threat Reduction Agency – Joint Scienceand Technology Office, Medical S&T Division.

The views expressedin this abstract are those of the author(s) and do not reflect the officialpolicy of the Department of Army, Department of Defense, or the U.S.Government.

This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
The structural diversity and the biological meaning of antibody
Authors: HIFUMI EMI , AKIYOSHI YUKO , UDA TAIZO
Published: June 2021

Along with the development of antibody drugs and catalytic antibodies, the structural diversity issue has attracted much attention. Structural diversity(or structural heterogeneity) is generated caused by the modification of different charges, different molecular sizes, and/or modified amino acid residues. Namely, an antibody takes multi‐molecular forms in the solution. It is desirable that the antibody and/or the subunits must have a defined structure for practical use. Regarding with the difficult issue on the structural diversity of antibody, we found the interesting phenomena in which copper ion can convert the multi‐molecular forms of antibodies to mono‐molecular form (1). The ion hugely contributed to the enrichment of the dimer‐form and the homogenation of the differently charged full‐length of the light chain. The role of copper ion must be significant for preparing a single, defined, not multiple, isoform structure.

In addition, we found that the constant region domain (CL) of the antibody light chain also plays an important role in the structural heterogeneity of the light chain (2), which was clarified by expressing the constant domain. Considering the chemical features of the full length light chain (VL+CL) and CL, note that the CL domain can influence to generation of the structural diversity of the VL domain. This finding is important, because, even at the present, the function of CL is still unknown. It is considered that the generation of the structural diversity of a protein may influence on the binding ability as well as the specificity.

In the presentation, the authors will discuss not only the reason why antibodies take such structural diversity but also their biological meanings.

This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Effect of CNTM on high‐fat diet‐induced obesity in C57BL/6J mice
Authors: Cho Bong‐Yeon , Choi Sun‐Il , Jung Tae‐Dong , Choi Seung‐Hyun , Sim Wan‐Sup , Han Xionggao , Ra Moon‐Jin , Kim Sun‐Young , Kang Il‐Jun , Han Kyoung Chan , Kim Young‐Cheul , Lee Ok‐Hwan
Published: June 2021

Cirsium setidens Nakai, a wild perennial herb, grows mainly in Gangwon province, Korea, and it has been reported to contain bioactive ingredients with various medicinal activities including treat edema, bleeding and hemoptysis. In our previous studies have demonstrated that C. setidens nakai ethanolic extract had an anti‐adipogenic effect during the adipogenesis of 3T3‐L1 cells and beneficial effect on high‐fat diet (HFD)‐induced obesity in C57BL/6J mice. The purpose of this study describes a preliminary evaluation of the anti‐obesity activity on Cirsium setidens Nakai ethanolic extract containing test material (CNTM). Also, this study was to evaluate the effects of CNTM high‐fat diet‐induced obesity in C57BL/6J mice. To study for anti‐obesity agents effectively, we first examined the effect of CNTM on body weight gain and relative weight of the adipose tissue. In addition, we investigated the beneficial effect of CNTM on high‐fat diet (HFD)‐induced obesity in C57BL/6J mice. C57BL/6J mice were fed a high‐fat diet to induce obesity and were orally administered CNTM (160, 320 mg/kg) for 8 weeks. CNTM‐treated groups were reduced body weight, adipose tissue weight and improved serum lipid profiles and adiponectin protein expression in obese C57BL/6J mice fed a high‐fat diet. These results suggested that CNTM may have an anti‐obesity effect through the regulation of transcription factors related to adipogenesis, lipogenesis, and lipolysis. metabolism., hypertension, and diabetes. This study suggests that CNTM are potential candidates as anti‐obesity agents for treatment of obesity as non‐toxic natural resources.

This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.