bluuebunny/crossref_metadata_2025 huggingface dataset:
μ‐opioid and NK1 receptor immunofluorescence and involvement in the neuroventilation of bullfrogs
Authors: Dykstra B L , Brundage C M , Taylor B E
Published: June 2021

The investigation of the functional homologies between anuran and mammalian respiratory rhythm generators (RRGs) has been limited by difficulty in defining the sites and neuronal composition of the RRGs. n‐opioid and neurokinin‐1 (NK1) receptor colocalization has been used as a method of identifying the pre‐Bötzinger complex RRG in mammals. We conducted immmunocytochemical staining for μ‐opioid and NK1 receptors of Rana catesebeiana brainstem slices to determine if a similar site could be identified in the bullfrog. We identified one distinct region of μ‐opioid and NK1 receptor coexpression near the facial nucleus, an area purportedly involved in anuran respiratory rhythm generation. Bath application of the μ‐opioid agonist DAMGO (100nM) to the in vitro brainstem preparation significantly depressed lung burst frequency (P<0.001). Subsequent application of the opioid antagonist naloxone significantly increased lung burst frequency (P<0.05). Bath application of substance P (5.0μM), a NK1 receptor agonist, significantly increased lung burst frequency (P<0.001) while successive application of antagonist D (750nM) significantly decreased lung burst frequency (P<0.001). These results collectively support the identification of a bullfrog RRG, one that is closely associated with lung ventilation and potentially homologous to the mammalian pre‐Bötzinger complex RRG.
Focal adhesion kinase controls load‐dependent myofibre differentiation
Authors: Flueck Martin , Desplanches Dominique , Klossner Stephan , Freyssenet Damien , D'Antona Giuseppe , Bottinelli Roberto , Durieux Anne‐Cecile
Published: June 2021

The present report tested the hypothesis that focal adhesion kinase (FAK) is a myocellular transducer of mechanical signals towards downstream control of slow, fatigue‐resistant muscle fibres. This examination was approached in an integrative setting combining somatic transgenesis for myocellular FAK overexpression with transcript profiling, morphometry and myography in two physiological rat models for load‐dependent plasticity of anti‐gravitational soleus muscle. The molecular measures identified FAK‐regulated expressional reprogramming of gene ontologies underlying the oxidative and mechanical specialisation of soleus muscle. FAK‐dependent transcript levels were suppressed with unloading but transient elevated along with muscle weight with reloading. The transcript upregulation of energy and protein metabolism with reloading corresponded to increased tyrosine 397 phosphorylation and the translocation of FAK to the sarcolemma. The load‐dependent modulation of the soleus muscle phenotype by FAK was related to fibre regeneration and became manifest as a shift towards slow contractile characteristics with functional overload. Collectively the findings expose spatially controlled FAK signalling as a key upstream element of the mechano‐dependent muscle differentiation.
Control of mitochondrial respiration in flight muscle of bar‐headed geese
Authors: Scott Graham R , Richards Jeff G , Milsom William K
Published: June 2021

We examined whether flight muscle metabolic capacity or mitochondrial regulation have evolved in the bar‐headed goose to facilitate flight at high altitude. Biopsies were collected from the pectoralis muscle of this species and several low altitude waterfowl species (greylag geese, barnacle geese, and mallard ducks), the muscle fibers were permeabilized, and their respiration was measured by high resolution respirometry. Respiratory capacities, namely maximum ADP‐stimulated respiration using single or multiple inputs to the electron transport system, as well as complex IV excess capacity, were similar between species. However, the kinetics of ADP stimulated respiration were different in bar‐headed geese than in low altitude waterfowl, due to a reduced ADP sensitivity (higher Km) under oxygen saturating conditions. The sensitivity of respiration to creatine was also enhanced in bar‐headed goose muscle. These data suggest that ATP supply and demand may be better coordinated in the flight muscle of bar‐headed geese, which should be adaptive for intense exercise in severe hypoxia. (Supported by NSERC of Canada)
Oxidant stress modulates PPARγ expression and activity in vascular endothelial cells
Authors: Blanquicett Carmelo , Ritzenthaler Jeff , Kleinhenz Dean , Hart C. Michael
Published: June 2021

The ligand‐activated nuclear transcription factor, peroxisome proliferator‐activated receptor γ (PPARγ), exerts pleiotropic effects on metabolism and inflammation and modulates vascular endothelial nitroso‐redox balance. We hypothesized that oxidant stress alters PPARγ expression and activity, an effect that could be prevented by the antioxidant, catalase. Human umbilical vein endothelial cells (HUVEC) were exposed to 100 μM H2O2 for 0.5–4 h. PPARγ activity and mRNA levels were determined ± catalase addition using a PPARγ activity assay and real‐time PCR, respectively. H2O2‐mediated alterations in PPARγ mRNA were examined using Actinomycin‐D and luciferase reporter assays. PPARγ activity in bovine aortic endothelial cells (BAEC) was also examined by altering the extracellular redox potential (Eh), using the cysteine (Cys)/cystine (CySS) couple. Thirty min. following exposure to H2O2, HUVEC PPARγ activity and mRNA levels were significantly reduced. Catalase prevented H2O2‐induced reductions in PPARγ activity and mRNA. Compared with normal (−80 mV) and reduced (−150 mV) Eh, treatment with oxidized (0 mV) Eh significantly decreased PPARγ activity. Neither H2O2 nor catalase significantly affected PPARγ mRNA half‐life. These findings demonstrate that oxidant stress modulates both PPARγ mRNA and activity in vascular endothelial cells. Supported by grants from the NIH and VA Research Service.
H2S toxicity via oxidative damage in erythrocytes of a sulfide‐tolerant marine invertebrate
Authors: Ortega Jocelynn M , Ortega Jessica A , Stein Jenny R , Julian David
Published: June 2021

Marine organisms adapted to H2S exposure utilize a variety of mechanisms, including H2S oxidation, to minimize its toxicity. However, this may lead to increased free radical production. We tested whether H2S exerts at least some of its toxicity via free radical damage in erythrocytes from the sulfide‐tolerant marine polychaete Glycera dibranchiata. We exposed erythrocytes to H2S in vitro and assessed oxidative stress, oxidative damage and cell death, and whether exogenous antioxidants reduced H2S toxicity. Exposure to 0.1–3% H2S for 0.5–2 h significantly increased oxidative stress (carboxy‐H2DCFDA labeling; p<0.0001), mitochondrial superoxide production (dihydroethidium labeling; p=0.0003), and oxidative damage to RNA and DNA (via HPLC‐EC‐UV; p<0.05), consistent with increased free radical production. Exposure to H2S caused a dose‐dependent increase in cell death (via propidium iodide and calcein green AM labeling, p<0.0001), but the antioxidants glutathione ethyl ester, dithiothreitol and N‐acetylcysteine reduced this cell death by up to 70% (p<0.0001), 78% (p=0.041), and 85% (p=0.0006), respectively. Therefore, in erythrocytes of a sulfide‐tolerant marine invertebrate, oxidative damage was increased by H2S, and H2S‐induced cell death was reduced by antioxidants, both of which are consistent with free radical damage as an important mechanism of H2S toxicity.
Effect of Sodium Nitroprusside on pHi Recovery in Porcine Non‐pigmented Ciliary Epithelium
Authors: Shahidullah Mohammad , Delamere Nicholas A.
Published: June 2021

Objective

Atriopeptin, acting through cGMP, inhibits NHE1 in the bovine ocular non‐pigmented ciliary epithelium (NPE)1. Here we examined NHE isoforms and tested whether SNP, a NO donor and a stimulator of cGMP, alters intracellular pH (pHi) responses.

Methods

NHE isoforms in native and cultured NPE were detected by immunohistochemistry and immunoblot. BCECF fluorescence imaging was used to study the effect of drugs on pHi recovery after an acid load.

Results

Native and cultured NPE expressed NHE1 and NHE4 isoforms. pHi recovery in NPE was Na‐dependent and had DIDS and amiloride sensitive components. SNP inhibited pHi recovery concentration‐dependently. L‐arginine, the NOS substrate, also slowed pHi recovery. Dimethyl amiloride (DMA), a potent inhibitor of NHE, caused substantial inhibition of pHi recovery at 100μM. In cells exposed to both SNP and DMA, the degree of inhibition of pHi recovery was similar to that elicited by SNP alone. The cyclic GMP analog 8‐pCPT‐cGMP (10μM) mimicked the SNP effect. The inhibitory effect of SNP on pHi recovery was suppressed by 1μM H‐8, an inhibitor of cGMP‐dependent protein kinase.

Conclusion

Primary cultures of porcine NPE express NHE isoforms found in native cells. NO donors slow pHi recovery through inhibition of NHE. The effect of NO donors on NHE involves a cGMP‐PKG signal transduction pathway.

Funding: NIH‐EY06915
Imaging mechanical strain in small airways before and after acid injury
Authors: Waters Christopher M. , Sinclair Scott E. , Majd Shervin , Molthen Robert C.
Published: June 2021

The purpose of this study was to measure mechanical strain in small airways in an intact animal before and after injury caused by acid instillation. Male Sprague‐Dawley rats were anesthetized and mechanically ventilated with tidal volumes of 6, 12 and 25 cc/kg with and without 10cm H2O PEEP. Real‐time tantalum bronchograms were obtained at each condition using microfocal x‐ray imaging. Images were used to calculate circumferential and axial airway strains and wall tensions. Following baseline imaging at each tidal volume, 0.2 ml of 0.1 N HCl was instilled into the airways, and the airways were imaged again within 30 min. Mechanical ventilation resulted in significant airway mechanical strain that was heterogeneously distributed in the uninjured lung and depended upon the tidal volume and the level of PEEP. In comparison, mechanical strain in injured lungs was decreased but still heterogeneous. Supported by NIH grants HL064981 and HL081297.
IL‐1 mediates persistent but not acute ozone‐induced airway hyperreactivity
Authors: Blensly Kirsten Colleen , Jacoby David B , Fryer Allison D
Published: June 2021

Ozone (O3) exposure causes airway hyperreactivity, lasting at least 3 days, and increases interleukin‐1 (IL‐1) in human and animal lungs. Eosinophil depletion prevents O3 induced airway hyperreactivity 1 day post O3 but worsens airway hyperreactivity 3 days post O3. Thus, the mechanism of hyperreactivity changes between 1 and 3 days following O3 exposure in guinea pigs. O3 increases IL‐1a in bone marrow. To test whether IL‐1β mediates airway hyperreactivity 1 and 3 days post O3 guinea pigs were treated with an IL‐1 receptor antagonist (Kineret, 30mg/kg ip) 30 minutes before exposure to filtered air or O3 (2ppm, 4 hours). One or 3 days later electrical stimulation of both vagus nerves, in anesthetized animals, caused bronchoconstriction that was significantly potentiated in O3 exposed animals compared to air exposed controls. The IL‐1 antagonist prevented airway hyperreactivity 3 days but not 1 day post O3. Acetylcholine (1‐10ug/kg, iv) induced bronchoconstriction was not changed 1 or 3 days post O3 but was increased in O3 exposed animals treated with the IL‐1 antagonist. In bronchoalveolar lavage the IL‐1 antagonist did not change inflammatory cell numbers in either O3 or air exposed animals at either time point. These data confirm the mechanism of airway hyperreactivity changes over three days following ozone to become IL‐1 dependent.

Funding: Steinberg Fellowship; NIH: HL‐55543, HL54659, HL071795, ES‐014601
Ca2+/Calmodulin‐dependent protein kinase regulation of sarcoplasmic reticulum Ca2+ uptake in airway smooth muscle
Authors: Sathish Venkatachalem , Leblebici Figen , Kip Sertac N , Pabelick Christina M , Prakash Y S , Sieck Gary C
Published: June 2021

In airway smooth muscle (ASM), the state of sarcoplasmic reticulum (SR) Ca2+ stores is an important determinant of [Ca2+]i response to agonist. We have previously shown that acetylcholine (ACh) induces sustained [Ca2+]i oscillations representing repetitive SR Ca2+ release and reuptake. Thus, SR refilling is a key component in refilling of SR stores. We hypothesized that, like cardiac and vascular smooth muscle, ASM SR refilling is regulated by calmodulin (CaM)‐CaM kinase (CaMKII) dependent phosphorylation of phospholamban (PLB). In ASM cells from fresh porcine trachea, effects of W7 (CaM antagonist) and KN‐93/KN‐62 (CaMKII antagonists) on [Ca2+]i responses to ACh were measured using fluorescence microscopy. Separately, SR‐enriched membranes were isolated to monitor ATP‐energized Ca2+ uptake and in vitro phosphorylation of PLB in the presence of CaMKII inhibitors. KN‐93 significantly slowed decay of [Ca2+]i responses, and slowed or inhibited ongoing [Ca2+]i oscillations, with visible slowing of fall‐times of individual oscillations. Pre‐exposure to W7 or KN‐62 did not prevent initiation of oscillations. In SR‐enriched membranes, addition of KN‐93/KN‐62 resulted in significantly slower Ca2+ uptake, accompanied by a matching decrement in the in vitro phosphorylation of Thr 17‐PLB, indicating the influence of CaM‐CaMKII on this protein. These data suggest that agonist‐induced [Ca2+]i regulation in ASM involves the CaM‐CaMKII pathway which indirectly influences Ca2+ release and reuptake, likely through PLB.

Supported by NIH grant HL74309 (GCS)
Dexamethasone‐stimulation of Na‐transport differs across lung epithelia
Authors: Mairbaurl Heimo , Schuler Akelei , Güney Sevin , Zuegel Stefani , Bartsch Peter
Published: June 2021

Dexamethasone (DEX) stimulates alveolar reabsorption by stimulating the expression of ENaC and Na/K‐ATPase. Nasal potentials (NP) have been used repeatedly to estimate changes in alveolar reabsorption. However, in a clinical trial on DEX‐treatment (Maggiorini AIM 2006) we found no effect of DEX on NP. Therefore we tested whether a similar DEX‐treatment of rats affects both NP and the expression of Na‐transporters in nasal epithelium, trachea, primary alveolar epithelial cells (AII), and in leukocytes. Rats were treated with DEX (2mg/kg/day; i.p.) for up to three days. NP was measured after anesthesia, and tissues were removed for mRNA preparation. In a separate series of experiments lung reabsorption was measured after fluid instillation. Total NP was increased (+2mV) after 48hDEX but the amiloride‐sensitive component was not affected. DEX did not affect. α,β,γ‐ENaC in nasal epithelium and leukocytes but increased it in trachea and AII cells (50–100%). DEX did not affect α1,β1‐Na/K‐ATPase mRNA in nasal epithelium but increased α1 mRNA in trachea (100%). Leukocyte α1 mRNA was decreased by 50%. CFTR, nedd‐4 and sgk‐1 mRNAs were not affected. DEX stimulated alveolar fluid reabsorption by increasing its amiloride‐sensitive component. These results indicate that a) NP do not reflect alveolar ion transport activity upon treatment with DEX, and that b) DEX stimulation of Na‐transport varies across lung epithelia.
CFTR function of dendritic cells reveals a novel mechanism of inflammation in the cystic fibrosis lung
Authors: Di Anke , Gao Xiappei , Xue Jiaping , Liu Guilan , Patel Kevin , Bachmaier Kurt , Naren Anjaparavanda P. , Tiruppathi Chinnaswamy , Malik Asrar B.
Published: June 2021

Airway inflammation is a common feature of cystic fibrosis (CF) lung disease. Major histocompatibility complex class II (MHCII) molecules expressed on dendritic cells (DCs) play a pivotal role in presenting bacterial antigens to T lymphocytes to trigger the immune response. We show here that the cystic fibrosis transmembrane regulator (CFTR) is expressed in DCs where it participates in regulating plasma membrane MHCII expression. DCs from CFTR−/− mice showed enhanced plasma membrane expression of MHCII as well as stimulatory effect on CD4+ T cell proliferation. Metabolic labeling studies showed that degradation of invariant chain (Ii) in MHCII‐Ii complex was significantly greater in DCs from CFTR−/− mice than CFTR+/+ mice. Lysosomal acidification in DCs from CFTR−/− mice was also greater and acidification was prevented by amiloride, an epithelial sodium channel (ENaC) inhibitor. Thus, CFTR may regulate MHCII plasma membrane expression and antigen presentation by inhibiting lysosomal ENaC, and thus modulate lysosomal acidification. These results suggest a novel pathogenic mechanism of inflammation in cystic fibrosis patients based on the hyper‐activation of the immune response. This work was supported by Parker B. Francis Fellowships in Pulmonary Research and US National Institutes of Health grants P01 HL77806.
Interactive clicker technology in teaching nutrition and metabolism in the medical curriculum
Authors: Feinman Richard D. , Accurso Anthony , Makowske Mary
Published: June 2021

Audience response technology via wireless handheld devices (clickers) allows polling of students and provides immediate feedback. We have used this methodology in a first year medical school course to assess students’ preconceptions about nutrition and to address a unique problem: the inconsistency between biochemical facts and commonly accepted recommendations of health agencies. Representative questions from the first class meeting that provided information on student background, led to critical discussion and were used for focus of the course:

During the epidemic of obesity and diabetes, the macronutrient that increased most: (CHO, fat, protein):

In general, the effect of a low‐fat diet on plasma triglycerides? (increase, decrease, no effect)

The dietary component most likely to raise plasma triglycerides: (CHO, fat, protein).

The major stimulus for insulin secretion: (glucose, fatty acids, amino acids)

The dietary requirement for carbohydrate: ( ~130 g/d, ~ 50 % of calories, none)

CHO intake recommended by USDA, American Diabetes Association, others: ( ~130 g/d, ~ 50 % of calories, none).

A major effect of insulin: (glucose uptake, inhibition of lipolysis, fatty acid uptake, all)
Case‐based learning for understanding cardiovascular development
Authors: Severson Arlen R , Overgaard Joshua , Maust Thomas , Sauter Jennifer , Lofgren Jonathan , Alder Jason , Glawe Brett
Published: June 2021

Many medical school curricula have moved from discipline‐based courses to integrated system‐based courses, and from passive lecture‐based learning to active student‐centered learning. The student‐centered formats utilize peer learning and clinical examples to emphasize clinical application of basic science material. Problem‐based learning (PBL) and team‐based learning (TBL) are the most widely used formats for active student‐centered learning. In learning about cardiovascular development, students were given a reading assignment and provided with a handout, learning objectives, and an audio‐overlaid PowerPoint lecture on normal and abnormal development. A brief discussion of cardiovascular development during the scheduled class period was followed by the students taking an Individual Readiness Assessment Test (IRAT) and a Group Readiness Assessment Test (GRAT). Prior to the final class session on the topic, students were given clinical cases with multiple choice questions to review, and these cases with their questions were discussed during a scheduled class session. Questions related to the cases were included on the final examination. Students completed a survey regarding the effectiveness of the active learning format in understanding cardiovascular development. The survey indicated that students prefer the active, student‐centered learning format to the traditional lecture format.
XBP‐1, a novel HTLV‐1 Tax binding protein, activates HTLV‐1 basal and Tax‐activated transcription
Authors: Chao Sheng‐Hao , Ku Sebastian , Lee Jialing , Lau Joanne , Gurumurthy Meera , Ng Raymond , Lwa Siew Hui , Lee Joseph , Klase Zachary , Kashanchi Fatah
Published: June 2021

X‐box binding protein 1 (XBP‐1) is a CREB/ATF (cyclic AMP response element binding protein/activating transcription factor) family transcription factor. XBP‐1 plays a major role in the cellular unfolded protein response (UPR), which is triggered by accumulation of unfolded or malfolded proteins in the endoplasmic reticulum. There are two XBP‐1 isoforms in cells, the spliced XBP‐1S and the unspliced XBP‐1U. In 1990, XBP‐1U (also known as TREB5) was identified as one of the cellular proteins binding to the Tax‐responsive element of human T‐lymphotropic virus type 1 (HTLV‐1). However, this study was carried out before the discovery of XBP‐1S. Here we identify XBP‐1S as a transcription activator of HTLV‐1. Compared to XBP‐1U, XBP‐1S demonstrates stronger activating effects on both basal and Tax‐activated HTLV‐1 transcription. Our results show that both XBP‐1S and XBP‐1U interact with Tax and bind to HTLV‐1 LTR in vivo. In addition, elevated mRNA levels of XBP‐1 and several UPR genes are detected in the HTLV‐1‐infected T cell lines, suggesting that HTLV‐1 infection may trigger UPR in the host cells. We also identify Tax as a positive regulator of XBP‐1 gene expression. Collectively, our study demonstrates a novel host‐viral interaction between a cellular factor XBP‐1 and HTLV‐1 transcription.
Repression of EGR target genes by NAB2 and the NuRD complex
Authors: Srinivasan Rajini , Ward Rebecca M. , Svaren John
Published: June 2021

The Early Growth Response (EGR) family of transactivators are critical regulators of immune system regulation, peripheral nerve myelination and prostate carcinogenesis. The NAB1 and NAB2 (NGFI‐A/EGR1‐binding) transcriptional corepressors directly interact with three EGR family members (Egr1, Egr2/Krox20, and Egr3) and repress activation of their target promoters. In particular, NAB proteins appear to play an important role in regulating growth factor and cell cycle control genes, although the underlying molecular mechanisms have been elusive. We previously uncovered two independent repression domains in NAB2, one of which uniquely associates with the CHD4 (Chromodomain Helicase DNA‐binding protein 4) subunit of the repressive NuRD (Nucleosome Remodeling and Deacetylase) chromatin remodeling complex. To probe the role of the NuRD complex in physiological regulation of endogenous EGR target genes, we identified several genes that are regulated by NAB2 and the NuRD complex using a combination of gene expression and chromatin immunoprecipitation assays. Moreover, repression by NAB2 of at least some target genes requires the histone deacetylase subunits of the NuRD complex. Overall, the results indicate that NAB‐mediated recruitment of the NuRD complex is required for repression of EGR target genes that promote proliferation.

This work is supported by a grant (HD41590) from NIH.
Overexpression of FAT10 upregulates p53 transcriptional activity
Authors: Li Tianwei , Santockyte Rasa , Yu Shiqin , Yang David C. H. , Chock P. Boon
Published: June 2021

FAT10, also called diubiqutin, has been implicated in the regulation of diverse cellular processes, including mitosis, chromosome stability, immune response, and apoptosis. To date, no target protein has been identified for this ubiquitin‐like modifer. We investigated the FAT10 target proteins by over‐expressing human FAT10 or its non‐conjugatable derivative, FAT10[Delta]GG, in HEK293 cells. Compared to cells expressing FAT10[Delta]GG, we observed a number of high molecular weight FAT10 conjugates in cells expressing wild‐type FAT10. The FAT10 conjugates are inducible by TNF‐? and significantly accumulated when cells were treated with proteasome inhibitor, MG132. We identified p53 as the first FAT10 target protein. Over‐expression of FAT10 in HEK293 cells reduced the mutant conformation of p53 and greatly enhanced p53 transcriptional activity. FAT10 in the nucleus was found co‐localized with p53 and altered its subcellular compartmentalization. Furthermore, over‐expressing FAT10 also led to a reduction in the size of Promyelocytic Leukemia nuclear bodies (PML‐NBs) and altered their distribution in the nucleus. Based on these observations, a potential mechanism which correlates FATylation of p53 to its translocation and transcriptional activation is discussed.
Assay for monitoring in real time RNA reverse transcription and measurement enzyme inhibition.
Authors: Sun Ye , Golovlev Val
Published: June 2021

The objective of this study is to demonstrate a novel approach to the detection of RNA transcription in real time. This new technique offers alternative to real time PCR for the quantitative measurement of RNA without transcription to cDNA. The elimination of the transcription to cDNA step became possible by using the Reverse Transcription Quantitative Bioluminescent Assay (RT‐qBLA). We previously reported RT‐qBLA as a new, sensitive method of detection and quantification of RNA in biological samples (1,2). In current research, the use of RT‐qBLA was expanded to the monitoring of reverse transcription reaction (RT) kinetics and the measurement of RT inhibition and reaction constants. The use of RT‐qBLA for measurement of the efficiency of the inhibition of HIV‐1 RT by nucleoside and non‐nucleoside inhibitors (NRTI and NNRTI) was demonstrated for the first time. RT‐qBLA provides a fast, accurate, and cost‐efficient alternative to the established methods in various applications in research and antiviral drug discovery.

This project is supported by NIH grant R43 CA126647.
Antrodia camphorate extract preferentially degrades eukaryotic translation initiation factor 5A
Authors: Huang Jenq‐Kuen , Wang Yinglu , Murtaugh Megan L , Soustek Meghan S , Lin Chi‐Tsai , Wen Lisa
Published: June 2021

Antrodia camphorate (A. camphorate) is a medicinal mushroom found only in Taiwan. The fruiting bodies of A. camphorate have been used for a long time as a remedy for various diseases in folk medicine, including cancer treatment. The objective of this research was to search for inhibitors in A. camphorate extract that can reduce the activity of eukaryotic initiation factor 5A (eIF5A) modification which is essential for its function in cell proliferation and survival.

The maturation/hypusination of eIF5A is a two‐step posttranslational modification catalyzed by deoxyhypusine synthase (DHS) and deoxyhypusine hydroxylase (DOHH). The eIF5A modification has been implicated to be a suitable target for anticancer therapy. We wish to find ingredient(s) in A. camphorate extract that can inhibit eIF5A modification. We report that the presence of aqueous extract of A. camphorate capsules reduced the bovine DHS and DOHH activities. The inhibition toward DOHH was in a dose‐dependent manner. Further analysis suggested that the reduced activities were due to preferential degradation of eIF5A protein over seven other proteins tested, presumable by a novel protease present in the extract.

Supported by University Research Council, Western Illinois University.
Identification of novel β‐secretase inhibitors through the inclusion of protein flexibility in virtual screening calculations
Authors: Cosconati Sandro , Huey Ruth , Marinelli Luciana , Novellino Ettore , Goodsell David S. , Olson Arthur J.
Published: June 2021

Alzheimer's disease is a neurodegenerative disorder featuring both the aggregation of extraneuronal amyloid plaques and intraneuronal neurofibrillary tangles. The deposition of Aβ peptides in the brain plays a key role in the onset and progression of this disease, and β‐secretase (BACE‐1) is critical in this process. Thus, the inhibition of this enzyme is an attractive approach to anti‐Alzheimer therapy. So far, the rational design of new BACE‐1 inhibitors has been hampered by the intrinsic flexibility displayed by the enzyme which is not included in standard molecular modeling methods.

Here, we incorporated protein flexibility into Virtual Screening (VS) in order to discover new BACE‐1 inhibitors in two ways. First, we used an ensemble of static Xray BACE‐1 structures representing the receptor flexibility in a VS of the NCI database. Also, the different conformations of BACE‐1 were combined to generate a unified description of the protein motion. To do this, a set of maps was computed for each protein conformation, combined in a single set using an energy weighting scheme and finally used in a second VS of the NCI database.

The enrichment factors from these two VS approaches were used to assess their respective predictive power. The in vitro evaluation of the best ranking ligands finally led to the discovery of new leads which will be subjected to further structural optimization.
Kinetic and Structural Analysis of Tight Complexes of TEM‐1 Beta‐Lactamase and BLIP mutants
Authors: Mai Tu Thanh , Nguyen Toan Duc , Wang Jihong , Pham Ngoc , Vo De , Zhang Zhen , Palzkill Timothy , Chow Dar‐Chone
Published: June 2021

The S. clavuligerus ß‐Lactamase Inhibitor Protein (BLIP) is an effective inhibitor of class A ß‐lactamases, including the TEM‐1 β‐Lactamase (TEM1). A previous report from our groups shows that Y143A mutant of BLIP binds to TEM1 with similar affinity as the wild type, but with a larger entropy contribution to binding. The Y50A mutant binds to TEM1 50 fold stronger than wild type, which is the result of increased enthalpy driving forces (Wang J., et al. JBC. 2007; 282:17676–84). In addition, binding of BLIP Y50A with TEM1 exhibits a less negative ΔCp than that of the wild type complex. These findings suggest that binding between BLIP Y50A and TEM1 is enhanced by hydrophilic interactions. Interestingly, our stopped‐flow kinetic measurements show that the association rate of BLIP Y50A with TEM1 is similar to that of the wild type, demonstrating the increased affinity is mainly due to the slower dissociation. To understand the changes in binding thermodynamics and kinetics, we initiated structural studies of these complexes. After extensive crystallization screens, some high quality crystals were obtained for the BLIP Y50A/TEM1 complex and the BLIP Y143A/TEM1 complex crystallized in micro‐needle form. We are currently optimizing conditions to obtain high‐quality crystals. Future X‐ray diffraction analysis will determine if the crystals are suitable for structure determination.

Support from AHA & UH to D.C. and NIH to T.P.
Role of stearoyl‐CoA desaturase‐1 expression in cancer proliferation
Authors: Paton Chad M , Ntambi James M
Published: June 2021

Tumors are dependent on endogenous saturated fatty acids (SFA) which can be lipotoxic unless desaturated via stearoyl‐CoAdesaturase‐1 (SCD‐1). While inhibiting SCD‐1 activity induces apoptosis in cancer cells, the link between SFA and proliferation via SCD‐1 has not been shown. We hypothesize that desaturation of SFA through SCD‐1 is necessary for proliferation of cancer cells. Cancer cell lines were assessed for viability in the presence of SFA or oleate +/− Cerulenin. The variance in cell death following SFA +/− Cerulenin treatment was correlated with SCD‐1 expression. Next, HeLa cells overexpressing SCD‐1 were assessed for SFA and MUFA sensitivity +/− Cerulenin: SCD‐1 overexpression increased viability in the presence of SFA and Cerulenin. To determine the downstream effects of SCD‐1 expression variance, classification of lipogenic pathway members as well as lipid raft expression of Cav‐1, Akt, and cSrc was determined. Increased SCD‐1 was associated with higher SREPB‐1c and lower Cav‐1. Furthermore, SCD‐1 overexpression in HeLa cells resulted in a loss of Cav‐1 in lipid rafts. Oleate treatment increased Akt and p53 signaling and SCD‐1 expression. We can conclude that cancer cells rely on SCD‐1 for SFA mediated proliferation. Furthermore, SCD‐1 expression is associated with a loss of Cav‐1 expression that may augment membrane composition and lipid raft regulation of signaling. NIH RO1 DK62388 JM Ntambi (PI)
THE ASSOCIATION OF XENOBIOTIC METABOLISM GENES POLYMORPHISM IN BLADDER CANCER
Authors: Altayli Ertan , Gunes Sezgin , Yilmaz Ali Faik , Irkilata Hasan Cem
Published: June 2021

The aim of this study was to investigate the relationship between bladder tumor and variants of cytocrom p450 1A2 (CYP1A2) C734A, CYP2D6 G1934A, glutathione s‐transferase P1 (GSTP1) I105V, GSTM1 null, and GSTT1 null which play an important role in xenobiotic metabolism. We investigated the distribution of these polymorphisms in 135 bladder cancer patients and 128 age‐matched healthy individuals as controls. The polymorphisms were analyzed using polymerase chain reaction‐restriction fragment length polymorphism assay and multiplex PCR method. Genotype and allele frequencies were calculated, and their associations with bladder cancer risk are assayed, and association with bladder cancer risk or demographic factors, smoking status, and tumor stage was investigated. The prevalence of GSTT1 null genotype in cases were 23%, compared to 7% in the control group (OR, 0.254, %95 CI, 0.115–0558, p = 0.001). No association was observed between CYP1A2, CYP2D6, GSTM1, and GSTP1 genes polymorphisms and bladder cancer. GSTT1 gene polymorphism may be associated with bladder cancer in a Turkish population studied. This research was supported by the Ondokuz Mayis University.
Relationship of the intravascular and pericellular (interstitial) oxygen pressures in normal and tumor tissue
Authors: Wilson David F , Vinogradov Sergei A , Apreleva Sofia , Lee William M F
Published: June 2021

Objective: To determine the influence of vessel walls and diffusion distance in delivery of oxygen to cells in normal and K1735 murine melanoma tissue of mice.

Methods: Oxygen pressures were measured by oxygen dependent quenching of phosphorescence, using oxygen probes contained in each compartment. Oxyphor G2 was added to the blood plasma and Oxyphor G3, Pd‐tetrabenzoporphyrin encapsulated with a gen. 2 poly‐arylglycine dendrimer and then coated with oligoethylene glycol residues (Av. MW 350), was microinjected into the interstitial (pericellular) space. The oxygen distributions in the two spaces were compared resting skeletal muscle and K1735 tumors in both awake and anesthetized mice.

Results and conclusions: For skeletal muscle of awake mice, the lowest 10% of oxygen pressures for the interstitial and intravascular spaces (believed to represent capillary bed) were not different (23.8 ± 4.5 and 25 ± 4.3 mm Hg, respectively) whereas in isoflurane anesthetized mice there was a small but significant (p =0.01) difference (20.4 ± 6.3 and 27.9 ± 3.5 mm Hg, respectively). In K1735 tumors, compared to skeletal muscle, the oxygen pressures are more heterogeneous and averages lower. In addition there are larger differences between the two compartments, consistent with lower vascular density in the tumors. Supported by NS‐31465, HL081273, and U54 CA105008
Proteomic analysis of serum from visceral leishmaniasis (kala azar) patients
Authors: Rukmangadachar Lokesh Ankenahally , Kataria Jitender , Manral Pallavi , Das Utpal , Samantaray Jyotish C , Kaur Punit , Singh Tej Pal , Srinivasan Alagiri
Published: June 2021

Background: Visceral leishmaniasis is vector borne disease caused by the protozoa, Leishmania donovani. Approximately 500,000 cases are reported annually. The diagnosis is conventionally made by the demonstration of the parasite in the aspirated fluid from the bone marrow or the spleen. We used proteomics technologies to discover protein biomarkers for the disease.

Methods: We compared the serum proteome of visceral leishmaniasis patients with normal healthy controls using proteomics techniques. Immunodepleted serum samples were separated using 2D DIGE. Biological variations of the protein expression level were analyzed with DeCyder software and evaluated for reproducibility and statistical significance. Differentially expressed proteins were identified by tandem mass spectrometry.

Results: The profile of differentially expressed protein spots between patients and controls revealed α1 anti‐chymotrypsin, Leucine rich glycoprotein, Complement C3, Complement C3α as up regulated and Haptoglobin and Transthyretin as down regulated in the disease.

Conclusions: Identification of differentially expressed proteins throws light on the host parasite interaction and these proteins can be potential biomarkers for noninvasive diagnosis and monitoring response to therapy.

The work has been supported from a research grant from Department of Biotechnology, Govt. of India to AS
Observing biophysical properties of swine myocardium and mitochondria with tapping mode scanning probe microscope
Authors: Zhu Jie , Guo Lian Hong , Lan Li , Wang Guodong , Guo Liang
Published: June 2021

Single ventricular myocytes and mitochondria were isolated from swine hearts follow enzymatic dissociation procedures. Experiments were carried under tapping mode AFM with 125&55¦ÍÌm scanner and Si3N4 probes at 340–430 kHz. High resolution AFM data of single myocytes and mitochondria were collected simultaneously which show ultrastructure features including striated pattern of contractile proteins, sarcoplasmic reticulum and mitochondria. From the images, we reached longitudinal columns of contractile proteins which separated by valleys, and surface invaginations separated by transverse tubules which dip into the fiber interior and mitochondria were compressed between the contractile machinery which abundant in myocardium to provide energy. These images show that there is a consistent relationship between grooves and Z lines which clue to Z line must be attached firmly to plasmolemma. At last, we have tried to do some research by putting the samples into different physiological buffer and the similar results can be collected but lacking of enough equilibrium. And the CM‐AFM experiments intended to appear the damage of cantilever and the biological sample, so we selected the TM‐AFM rather than the CM‐AFM.
Purification of a Precursor to a Potential Phospholipid Analog of Phosphatidylserine
Authors: Amato Nicholas Jay
Published: June 2021

The purpose of this project is to develop a method to purify the protected cyclic phosphatidylserine (PcycPS), an intermediate in the multi‐step synthesis of a proposed phospholipid analog (PLA). The PLA is important because it will allow us to study the structure and function of the naturally occurring phospholipid, phosphatidylserine (PS). The PLA will also allow us to study the structural interactions characteristic of the lipid bilayer and proteins in the presence of calcium by mimicking the structure and function of PS.

Chromatographic purification methods investigated have used silica gel, reverse‐phase, and cation exchange solid phases. Following the purification, the product was characterized by using 1H and 31P nuclear magnetic resonance (NMR) and thin layer chromatography (TLC) utilizing molybdenum and ninhydrin reagents for visualization. PcycPS was purified to greater than 85% purity. The final step of the synthesis uses hydrogenolysis to yield the final deprotected compound. This research is supported by The College of Wooster.
Overexpression of Desnutrin in Adipose Tissue Attenuates Diet Induced Obesity
Authors: Ahmadian Maryam , Duncan Robin Elaine , Sul Hei Sook
Published: June 2021

During energy deprivation, triacylglycerol (TAG) stored in adipose tissue is hydrolyzed by the action of lipases. We have identified a novel TAG lipase in adipocytes, induced upon fasting, and designated it desnutrin. Hormone sensitive lipase is now considered to be a diacylglycerol lipase, while desnutrin functions as TAG lipase in adipose tissue. Desnutrin is expressed most highly in adipose tissue, but is also found at lower levels in other tissues. Desnutrin null mice die early due to defects in the heart. To examine the adipose specific effect of desnutrin, we have generated transgenic mice overexpressing desnutrin in adipose tissue. Desnutrin transgenic mice have lower body weights, decreased adipose tissue mass and increased lipolysis in adipose tissue. Desnutrin transgenic mice also exhibit a more favorable metabolic profile, with improved serum lipid parameters and glucose tolerance. Despite the increased lipolysis in adipose tissue, these mice show an absence of ectopic TAG storage and do not have increased fatty acid levels in the blood. These mice exhibit increased thermogenesis associated with higher energy expenditure and genes involved in oxidative metabolism are upregulated in white adipose tissue. These results suggest that increased lipolysis may promote fatty acid oxidation in adipose tissue, contributing to higher energy expenditure and a leaner phenotype in desnutrin transgenic mice.
Lipid and lipoprotein metabolism in primary human hepatocytes
Authors: Ling Nancy , Kneteman Norman M. , Vance Dennis E.
Published: June 2021

Dysfunctions in liver lipid metabolism have been implicated in prevalent diseases such as atherosclerosis, diabetes, obesity, and hepatic steatosis. Most studies were done using human hepatoma cell lines or rodent model systems, whereas there were minimal studies on human hepatocytes in these diseases. We have shown that isolation of primary human hepatocytes is a valid model for studies on human liver. After 3 days of culturing of human hepatocytes in RPMI + 20% FBS, the cells are 70% viable. In addition, cells still maintain the ability to secrete albumin, apoB, apoA1, and apoE. Notably, the activity of phosphatidylethanolamine N‐methyltransferase (PEMT), a liver specific phosphatidylcholine biosynthetic enzyme, is found to decrease only 40% after 3 days; whereas in rat hepatocytes, PEMT activity decreases by 50% during the initial 24 hours of culture. Moreover, total phospholipid level and CTP:phosphocholine cytidylyltransferase activity are found to be constant throughout the initial 3 days of culture. With our work in the characterization of lipid/lipoprotein metabolism of primary human hepatocytes, we hope to provide new insight into lipid metabolism in humans as well as to relate knowledge gained from mouse metabolism to the situation in humans. This research is supported by Canadian Institutes of Health Research.
Structural Features of Galectin‐9 and Galectin‐1 that Determine Distinct T cell Death Pathways
Authors: Bi Shuguang , Earl Lesley A. , Jacobs Linsey , Baum Linda G.
Published: June 2021

Galectin‐9 and galectin‐1 both kill thymocytes, peripheral T cells and T cell lines. However, differences in target cell specificity, glycoprotein receptor requirements, and intracellular death pathways for these two galectins are not well understood. We found that galectin‐9 and galectin‐1 required different glycan ligands and glycoprotein receptors to trigger T cell death. They also triggered different intracellular death pathways, as only galectin‐9 T cell death was blocked by intracellular bcl‐2, while only galectin‐1 T cell death was blocked by intracellular galectin‐3. Galectin‐9 and galectin‐1 were both expressed in murine thymus, but killed different thymocyte subsets, with mature thymocytes more susceptible to galectin‐9. To define the structural features responsible for distinct activities of galectin‐1 and galectin‐9, we created a series of bivalent neo‐galectins with galectin‐1 and galectin‐9 carbohydrate recognition domains on different peptide linkers. The N‐terminal carbohydrate recognition domain and linker peptide contributed to potency of the galectins. Strikingly, the C‐terminal carbohydrate recognition domain determined receptor recognition, death pathway signaling and target cell susceptibility. Thus, carbohydrate recognition domain specificity, presentation, and valency all contribute to specific effects of different galectins in regulating T cell death.
Recombinant expression, purification, and construction of fluorescently labeled alpha‐hemolysin for single‐molecule optical studies
Authors: Paulson Ashley R. , Jones Brynna H. , Bailey Derek J. , Hubbell Kathleen E. , Burden Daniel L. , Keranen‐Burden Lisa M.
Published: June 2021

Single‐molecule optical and electrical measurements of transmembrane ion channels can reveal disparate behavioral subpopulations that are not detectable via conventional approaches. However, such measurements require fluorescently labeled materials of high purity and functional integrity. We produced eight mutants of Staphylococcus aureus α‐hemolysin (αHL), introducing single cysteine residues at strategic locations on the extracellular cap of the protein. Expression in E. coli and purification by affinity chromatography were optimized to produce protein with greater than 95% purity at concentrations of more than 1 mg/ml. Mutant proteins were derivatized with one of two fluorescent labels (BODIPY‐TMR or TMRIA) followed by proteolytic removal of the affinity tag and further purification. The resulting fluorescently labeled αHL proteins are devoid of contaminating free dye and demonstrate pore‐forming capacity in planar lipid membranes.
Transcriptomics and proteomics of drought tolerance in peanuts
Authors: Kottapalli Kameswara Rao , Burrow Mark D , Puppala Naveen , Payton Paxton
Published: June 2021

A peanut mini‐core accession relatively tolerant to drought was previously identified from our physiological screening. In the present study we employed a combined transcriptomics and proteomics analysis to study both the primary transcriptional networks and functional proteins involved in the drought tolerance mechanism. Peanut microarrays developed with 14352 genes in a high density (8 x 15k) format were used for transcript profiling. One‐ and two‐dimensional gel electrophoresis (1‐ and 2‐DGE) was performed on leaf soluble protein extracts of selected tolerant and susceptible accessions. Combined transcriptomics and proteomics analysis reveals that lipoxygenase, an enzyme of jasmonic acid biosynthesis induced under drought may be involved in drought signaling. Similarly, induction of the fatty acid biosynthesis enzyme, acetyl‐CoA carboxylase carboxyl transferase, in the tolerant accession suggests a possible role of lipid biosynthesis in the drought tolerance phenotype. Suppression of lectin proteins and induction of aldolases were also noticed and probably are consequences of general drought response in the tolerant peanut accession. Some of these identified proteins and their corresponding genes could contribute a physiological advantage under drought, making them potential candidates for marker‐assisted breeding.
Long‐term ethanol uptake in mice results in loss of peripheral B cells without affecting B cell production in the bone marrow
Authors: Tygrett Lorraine Tunink , Coleman Ruth A. , Cook Robert T. , Waldschmidt Thomas J.
Published: June 2021

Chronic alcoholics suffer increased susceptibility to infection pointing to defects in immunity. Alcoholics exhibit loss of circulating B cells and diminished Ab responses after vaccine challenge. To better understand the effects of ethanol on B cell survival, a murine model of long‐term alcohol intake was established. Adult mice are administered 20% w/v ethanol in drinking water while given full access to rodent chow. Using this model, mice do not lose weight nor do they show evidence of systemic stress, even after months on alcohol. C57BL/6 mice treated for 32 wks exhibit a progressive loss of peripheral B cells, including the transitional, marginal zone and follicular subsets when compared with water drinking controls. Of interest, none of the developing B cell subsets in the BM (Hardy fractions A‐F) were found to be reduced (total cells recovered) suggesting B cell development to be unaffected by long‐term alcohol exposure. To substantiate this finding, 32 wk mice were provided BrdU for an additional 2 wks during which time the percent of BrdU incorporation was determined in both BM and splenic B cell subsets. There was no difference in BrdU incorporation at any stage of maturation confirming BM B cell development and export to be normal in ethanol consuming mice. Taken together, the findings strongly suggest B cell loss in the periphery results from alcohol‐induced lesions in the secondary lymphoid organs.