bluuebunny/crossref_metadata_2025 huggingface dataset:
Lipopolysaccharide‐induced nitric oxide production and hypotension in Mkp‐1 deficient mice depends on L‐arginine uptake
Authors: Nelin Leif D , Calvert Thomas J , Chicoine Louis G , Liu Yusen
Published: June 2021

Mitogen activated protein kinase phosphatase‐1 (MKP‐1) limits the pro‐inflammatory response to lipopolysaccharide (LPS). We hypothesized that Mkp‐1−/− mice would respond to low‐dose LPS with a fall in blood pressure (BP) due to augmented expression of inducible nitric oxide synthase (iNOS), which would depend on L‐arginine uptake. To test this hypothesis, Mkp‐1−/− mice and their wild‐type littermates were treated with 10 μg/kg LPS iv and mean BP and exhaled NO production (exNO) were measured. Wild‐type mice had no change in BP or exNO during the experimental period, while Mkp‐1−/− mice had a fall in BP (79 ± 5% of baseline (BL)) and an increase in exNO (266 ± 50% of BL) after 150 min. Tissue levels of iNOS were greater in Mkp‐1−/− mice than in wild‐type mice. In additional experiments, 60 min after LPS Mkp‐1−/− and wild type mice were given L‐lysine (a competitive inhibitor of L‐arginine uptake) and BP and exNO monitored for 90 min. Treatment with L‐lysine prevented the LPS‐induced increase in exNO and decrease in BP in Mkp‐1−/− mice. When L‐lysine was given 150 min after LPS, L‐lysine treatment reversed both the LPS‐induced fall in BP and increase in exNO in Mkp‐1−/− mice. These results demonstrate that deficiency of MKP‐1 results in an exaggerated hypotensive response to LPS mediated by augmented iNOS expression, and the LPS‐induced increase in NO production depends on uptake of extracellular L‐arginine.
Activators of SKCa and IKCa channels enhance agonist‐evoked vasodilation and endothelial NO synthesis
Authors: Braun Andrew , Sheng JZ , Ella S , Davis MJ , Hill MA
Published: June 2021

Pharmacologic blockade of small‐ and intermediate conductance, Ca2+‐activated K+ channels (SKCa and IKCa channels, respectively) is known to interfere with agonist‐evoked vasorelaxation via nitric oxide (NO) synthesis and the pathway involving an ‘endothelium‐derived hyperpolarizing factor’ (EDHF). Given these important functional effects of vascular SKCa and IKCa channels, we examined the hypothesis that activation of these channels may augment the normal cellular pathway(s) underlying agonist‐mediated vasorelaxation. In single human umbilical vein endothelial cells (HUVECs), the SKCa/IKCa channel openers NS309 and DCEBIO in combination (1 μM each) enhanced ATP‐evoked cytosolic Ca2+ elevation and NO synthesis, as monitored by Fluo−3 and DAF‐FM, respectively. NS309 and DCEBIO also enhanced ATP‐induced membrane hyperpolarization. On their own, these channel openers produced significant membrane hyperpolarization and modest Ca2+ elevations, but did not induce NO production. In rat cremaster arterioles pressurized to ∼50 mm Hg, NS309 and DCEBIO alone evoked a significant reduction of myogenic tone and further augmented ACh‐induced vasodilation. Removing the endothelium by passage of an air bubble through the vessel lumen significantly diminished the vasodilatory responses to either ACh or NS309 and DCEBIO, indicating a predominant endothelial site of action for these agents.
Use of mono‐bromo‐bimane to derivatize sulfide in whole blood: comparison of blood sulfide levels during atmospheric hydrogen sulfide exposure and intravenous sulfide infusion
Authors: Bengtsson Asa , Johnson Jeff , Hill Paul , Mulligan Joanne , Leviten Dina , Insko Michael , Mebel Elise , Vertz Peter , VandenEkart Emily , Szabo Csaba , Deckwerth Thomas , Wintner Edward A
Published: June 2021

With the discovery of hydrogen sulfide as a signaling molecule and a potential therapeutic, measurement of free sulfide in blood – as hydrogen sulfide or hydrosulfide anion – has taken on importance. Here, we demonstrate and validate a method of free sulfide measurement whereby the free sulfide in whole blood is derivatized with excess monobromobimane. The resulting sulfide‐dibimane is subsequently extracted into ethyl acetate, followed by quantitation of sulfide‐dibimane via reverse‐phase HPLC with fluorescence detection. Reaction conditions are validated through 1) characterization of rate of conversion from sulfide to sulfide‐dibimane, 2) analysis of reaction in the presence of potential interferants, and 3) recovery of standard samples from a whole‐blood matrix. We found that reaction conditions of a mixture of acetonitrile and HEPES buffer (50 mM pH 8) gave rapid, clean conversion of sulfide to sulfide‐dibimane in the presence of excess monobromobimane. For whole blood, a 1:1:1 reaction mixture of 200 μl each acetonitrile:HEPES:blood proved optimal. Using this protocol, standard samples were consistently recovered in approximately 76% yield over the range of the assay. Baseline levels of free sulfide in rat blood were found to be about 0.3 – 0.5 μM. Subsequent work has proved the method effective in generating whole‐blood sulfide PK data in multiple species.
p38 gamma Mapk is required for exercise‐induced Pgc‐1alpha expression and mitochondrial biogenesis in skeletal muscle
Authors: Pogozelski Andrew , Li Ping , Jiang Huiping , Yan Zhen
Published: June 2021

We have previously shown that endurance exercise promotes peroxisome proliferator‐activated receptor γ co‐activator‐1α (Pgc‐1α) gene transcription through activation of the p38 mitogen activated protein kinase (MAPK) pathway. Here, we subjected mice with a muscle‐specific knockout (KO) of the p38α, p38β, or p38γ gene (all normally expressed in skeletal muscle) to 4 weeks of voluntary running. Exercise training resulted in increased expression of cytochrome c oxidase IV (COX‐IV) and cytochrome c (Cyt c) in plantaris muscles of wild‐type, p38α KO and p38β KO mice, but not in p38γ KO mice. In addition, we employed electric pulse‐mediated gene transfer of plasmid DNA encoding dominant negative (DN) forms of p38α, p38β, or p38γ into mouse tibialis anterior (TA) muscles followed by motor nerve‐stimulation. Neuromuscular activity resulted in a significant increase of endogenous Pgc‐1α mRNA in muscles transfected with empty control DNA, DN‐p38α and DN‐p38β, but not in muscles transfected with DN‐p38γ. Thus, these gain‐of‐function and loss‐of‐function experiments strongly support that p38γ is required for exercise‐induced Pgc‐1α gene expression and mitochondrial biogenesis in skeletal muscle.
A compartment model for optimal delivery in radioimmunotherapy (RIT) on neuroblastoma
Authors: Fu Bingmei M. , Lv Yonggang , Cheung Nai‐Kong V.
Published: June 2021

RIT can effectively treat neuroblastoma when radioactive isotope‐antibodies are administered through cerebrospinal fluid (CSF). To optimize the delivery efficacy in RIT, a compartment model was developed to quantify the time‐dependent concentrations of the bound antibodies with isotopes to the receptors on tumor cells (CIAR(t)),the free antibodies with isotopes (CIA(t)), and the unbound receptors (CR(t)), under various conditions. The criteria for the optimal delivery in RIT are to maximize the area under the curve (AUC) of CIAR(t), AUC(CIAR(t)), and minimize AUC(CIA(t)). Our results showed that: 1) The association and dissociation rate coefficients of antibodies to receptors have the same effects on optimal delivery under our clinical conditions of radioactive isotope half life ~8.5 days and CSF flow rates from 5–20 ml/hr in human; 2) The change from 10−8 M to 10−9 M in the equilibrium dissociation constant has the most improvement in optimal delivery. In contrast, further change from 10−10 M to 10−12 M has only insignificant improvement; 3) The AUC(CIAR(t)) under continuous infusion has no obvious difference from that under one dose injection although CIAR(t) profiles are different; 4) If the parameter values for the dosage, CSF volume, CSF flow rate are in proportion, the rat model can be used to predict the delivery efficacy for the human. This work was supported in part by a MSKCC research grant.
Development of a new type of biodressing for burns, human hair keratin‐collagen sponge‐poly 2‐hydroxyethyl methacrylate complex
Authors: Dong Weiren , Chen Yinghua , Chen Qinyuan , Zhao Binglei , Zou Zhongzhi , Xiao Yingqing , Le Yi
Published: June 2021

Human hair keratin (HHK) has been proved to be an ideal scaffold for tissue engineering. Guided by a hypothesis of “in vivo tissue engineering”, we developed a two‐layer scaffold complex with inner layer of HHK‐collagen sponge and outer layer of poly 2‐hydroxyethyl methacrylate (PHEMA) film containing polydatin (PD), which is also a drug carrier. We then evaluated therapeutic effects of this two‐layer complex for deep ?° burn wounds and its feasibility as a burn dressing. PD was released constantly from PHEMA with time‐lapse and fully released at 48 hours. The gross observation results demonstrated that the effusion of tissue fluid in experiment group was less than that of control groups under ideal moisture. Healing time in experimental group was shorter than that of in control group. Moreover, at 14 and 21 days, the healing rate in experiment group was higher than that in control group. These results suggest that HHK‐collagen sponge‐PHEMA/PD complex is a new scaffold candidate via in vivo construction of tissue engineered skin for burn dressing, and PHEMA could be a feasible drug‐delivery carrier.
Experimental and computational perturbation analysis of enzyme deletions in Drosophila energy metabolism
Authors: Feala Jacob Daniel , Omens Jeffrey , Paternostro Giovanni , McCulloch Andrew
Published: June 2021

The systems biology approach is complementary to the traditional reductionist approach in that it attempts to use all available data to build increasingly accurate computational models, from which specific new hypotheses can then be generated and tested classically. We use results from various experiments to refine a model of the reaction network of ATP‐generating central metabolism in Drosophila flight muscle. The network, built previously using the annotated genome and the KEGG database of genes and reactions, was made specific for flight muscle by integrating absolute gene expression data for fly thorax, which is composed mostly of flight muscle. Quantitative 1H spectroscopy and biochemical assays were used to measure accumulation of products, substrate depletion, and ATP concentration over 4 hours of hypoxia (0.5% O2) and anoxia, for wild‐type and several enzyme deletion strains, and the model was fitted to these data by constraining reaction fluxes. Based on production of ATP and protons in the steady‐state computer model, we hypothesized increased hypoxia tolerance in flies deficient in the lactate dehydrogenase (LDH) enzyme. Compared to wild‐type, heterozygous LDH deletions were found to have improved heart rate and faster recovery of mobility after 4 hours hypoxia. The refined version of the model can be used to generate other testable hypothesis regarding chronic hypoxia in Drosophila muscle tissue, or the generic model can be similarly tailored to other specific contexts of interest.
Regulation of mitochondrial free Ca2+ by metabolite and pH‐dependent Ca2+ buffering in the matrix: analysis by a computational model of mitochondrial Ca2+ handling
Authors: Dash Ranjan K , Haumann Johan , Patel Runa , Aldakkak Mohammed , Rhodes Samhita S. , Camara Amadou KS , Stowe David F , Vinnakota Kalyan C , Beard Daniel A
Published: June 2021

Mitochondrial free Ca2+ (mfCa2+) is regulated by ion fluxes through Ca2+ uniporter (CU), Na+/Ca2+ exchanger (NCE), Na+/H+ exchanger (NHE), and Ca2+/H+ exchanger (CHE) as well as by mitochondrial protein buffering. Regulation of mfCa2+ by metabolite and pH‐dependent dynamic Ca2+ buffering in the matrix during transient respiration, however, is not well known. To gain a quantitative understanding of these phenomena, we developed a biophysical model of mitochondrial Ca2+ handling and bioenergetics by developing and integrating kinetic models of the CU, NCE, NHE, and CHE into our well‐validated model of mitochondrial bioenergetics. Experiments were performed to spectrofluorometrically measure mfCa2+, pHm, membrane potential, and NADH redox state in guinea pig heart mitochondria suspended in Na+ and Ca2+ free buffer with 2.5 mM EGTA and 0.5 mM pyruvic acid (PA). Dynamics were inferred with addition of CaCl2, ADP, and CCCP with and without ruthenium red (CU blocker). Model analysis of the data on a) decreased mfCa2+ with addition of Na+‐independent substrate PA, and b) transient increases of mfCa2+ with added ADP and CCCP, implicates metabolite (ATP, ADP, Pi and TCA cycle intermediates) and pH‐dependent dynamic Ca2+ buffering in the cardiac mitochondrial matrix. The model will be helpful to understand mechanisms by which mfCa2+ both regulates, and is modulated by, mitochondrial energy metabolism.
Evolutionary and functional variation of PGC‐1α in vertebrates
Authors: Le Moine Christophe M.R. , Genge Christine E. , Lougheed Stephen E. , Moyes Christopher D.
Published: June 2021

In mammals, PGC‐1α is a central regulator of oxidative metabolism through its interactions with NRF‐1 and the PPARs. We examined the role of PGC‐1α in the evolution and development of physiological variations in vertebrates’ oxidative capacity. First, we sought to determine the regulators of the temperature and dietary‐induced metabolic remodeling in goldfish. NRF‐1 and PPARα, respectively, had typical roles in orchestrating mitochondrial proliferation and fatty acid oxidation. In contrast, while goldfish PGC‐1α seemed to be involved in the regulation of the PPAR axis, it did not appear to play a role in NRF‐1 dependent mitochondrial proliferation. To assess the structural basis of these divergent roles in mammals and fish, we investigated the evolutionary trajectory of PGC‐1α in representative vertebrate lineages. The analysis revealed a good conservation of the activation/PPAR interaction domain across vertebrates, whereas the NRF‐1 interaction domain experienced accelerated rates of evolution in Actinopterygians (fish lineages) compared to Sarcopterygians (tetrapod lineages). Furthermore, protein sequence analysis of this variable domain in lineages giving rise to modern teleosts revealed successive serine and glutamine residues insertions with important functional repercussions on PGC‐1α control of mitochondrial proliferation in teleosts. Funded by NSERC (Canada).
Migrating blackcaps huddle at night at stopover sites
Authors: Pinshow Berry , Wojciechowski Michal S , Jefimow Malgorzata
Published: June 2021

Small migratory birds employ a wide range of behavioral and physiological strategies to accelerate their fuel accumulation rate at migratory stopovers. Although they could potentially reduce the cost of night‐time rest by huddling with conspecifics, to date only anecdotal information exists about this behavior. We found that resting blackcaps (Sylvia atricapilla) regularly huddled in groups of two to six individuals in aviaries during migratory stopover. This occurred in spring migration in the Negev Desert and in autumn migration in central Poland. On some nights, huddling blackcaps used rest‐phase hypothermia, but on other nights, huddling birds maintained higher body temperatures at night than solitarily roosting individuals. Since migrating birds congregate at migratory stopovers, we anticipate huddling to be a common energy saving phenomenon among small migrating passerines.
IRES mediated initiation of translation during mammalian hibernation
Authors: Pan Peipei , van Breukelen Frank
Published: June 2021

Concordant with energetic constraints, protein synthesis is depressed during hibernation in golden‐mantled ground squirrels, Spermophilus lateralis, to values of 0.13 to 0.5% of active rates. Roles for both passive and active mechanisms in the regulation of translational initiation have been demonstrated. Phosphorylation events appear to regulate cap‐dependent translation during torpor. However, a subset of mRNAs contain internal ribosome entry sites (IRESs) which allow initiation to occur independent of the 5? mRNA cap. IRES regulated translation may be important in stress responses. We asked if there was a preference for IRES mediated initiation in one particular state of the torpor cycle e.g. as animals begin to arouse from torpor. Initiated mRNAs associate with polysomes. We used sucrose gradient fractionation to isolate polysomes from the animals in various stages of a torpor bout, including summer active (SA), entrance to torpor (ENT, at 10°C, 20°C, and 30°C), early torpor (ET), late torpor (LT), arousal from torpor (ARO, natural and alarm arousals at 10°C, 20°C, and 30°C), and interbout arousal (IBA). We performed quantitative real time PCR to investigate whether known IRES transcripts preferentially associate with ribosomes at the various states as compared to known cap‐dependent transcripts. Current efforts are underway to analyze the data.
Testing The Hypothesis That “Methionine Residues In Proteins Are Antioxidants”
Authors: luo shen , Levine Rodney L
Published: June 2021

A variety of reactive oxygen species (ROS) react readily with methionine (Met) residues to form methionine sulfoxide (MetO) and most cells contain methionine sulfoxide reductases that can reduce MetO back to Met. Thus, Met residues may serve as antioxidants and scavenge ROS. To investigate this hypothesis directly, we used an E. coli Met auxotroph to substitute Met with its analog norleucine (Nle), in which ‐CH2‐replaces the ‐S‐ of Met. Amino acid analysis of cellular proteins demonstrated an ~40% reduction of Met residues and equivalent increase in Nle. The intracellular free Met and S‐adenosylmethionine pools were not reduced by Nle substitution. The activity of glutamine synthetase was also unchanged in the cells grown on Nle, consistent with the prevalent view in the literature that protein function is little changed by substitution of Nle for Met. When challenging both cells with hypochlorite, hydrogen peroxide, or ionizing radiation, Nle‐containing cells died more rapidly than the control cells. Protein carbonyl levels were not significantly affected by these oxidative stresses, possibly because carbonyl levels are already elevated as a consequence of carbon starvation. MetO contents were also higher in Nle‐containing cells than in the control cells after hypochlorite and hydrogen peroxide challenges. Our results support the hypothesis that Met residues act as endogenous antioxidants in cells.
Oxidative stress induced mitochondrial dysfunction leads to age related loss of muscle mass via myonuclear apoptosis and proteolysis
Authors: Jang Young Charles , Muller Florian L. , Hayworth Chris , Liu Yuhong , Richardson Arlan , Van Remmen Holly
Published: June 2021

Aging is accompanied by the loss of skeletal muscle mass and function (sarcopenia). Mice lacking the antioxidant enzyme CuZnSOD (Sod1−/−) exhibit high oxidative stress in skeletal muscle and an accelerated sarcopenia. Mitochondria isolated from Sod1−/− muscle generates significantly elevated levels of H2O2 and show a decline in mitochondrial function. We hypothesized that the accelerated sarcopenia in Sod1−/−mice, is due to increased mitochondrial oxidative damage which results in apoptotic deletion of myonuclei and proteolysis. Sod1−/− muscle mitochondria show increased levels of cytochrome c and apoptosis inducing factor (AIF) release. These data are supported by an increase in downstream events; caspase‐9, caspase‐3 activity, DNA fragmentation, and cell‐free apoptosis. However, the myonuclear domain (myofiber volume/myonuclei) is not affected by myonuclear apoptosis. Therefore, it is likely that muscle cells respond to apoptosis by reducing its size via proteolysis. Indeed, cysteine protease, calpain and the 26S proteasome activity is increased by approximately 15~20% along with increased overall ubiquitinylation in Sod1−/− skeletal muscle. Overall, our data support the hypothesis that oxidative stress induced mitochondrial dysfunction leads to myonuclear apoptosis and proteolysis. This could be a possible underlying mechanism of age related muscle loss and function.
Protective role of calreticulin in HFE hemochromatosis
Authors: Pinto Jorge P , Ramos Pedro , de Almeida Sérgio , Oliveira Susana , Breda Laura , Michalak Marek , Porto Graça , Rivella Stefano , de Sousa Maria
Published: June 2021

Background/Aims: HFE mutations are associated with over 80% of cases of Hereditary Hemochromatosis (HH), an iron‐overload disease in which the liver is the most frequently affected organ. Research on HFE has traditionally focused on its interaction with the transferrin receptor. Recent studies suggested a more complex function for this non‐classical MHC‐I protein. The aim of this study was to examine how HFE and its two most common mutations affect the expression of selected genes in a hepatocyte‐like cell line.

Methods: Gene expression was analyzed in HepG2 cells over‐expressing wild‐type and mutant HFE. The effect of HFE in iron import and oxidative stress levels was assessed. Unfolded protein response (UPR)‐activated gene expression was analyzed in peripheral blood mononuclear cells from HH patients.

Results: C282Y‐HFE down‐regulated hepcidin and enhanced calreticulin mRNA expression. Calreticulin levels correlated with intracellular iron increase and were associated with protection from oxidative stress. In C282Y+/+ patients calreticulin mRNA levels correlated with the expression of the UPR marker BiP and showed a negative association with the number of HH clinical manifestations.

Conclusions: The data show that expression of C282Y‐HFE triggers a stress‐protective response and suggest a role for calreticulin as a modifier of the clinical expression of HH.
Splice Cassette II Within The N Terminus Of The Electroneutral Na+‐Coupled Bicarbonate Transporter NBCn1 Includes A Functional Calcineurin Aβ Binding Site
Authors: Parker Mark Daniel , Boron Walter F
Published: June 2021

In human heart and skeletal muscle cells, alternative splicing of SLC4A7 transcripts produces a subpopulation of NBCn1 protein that includes an additional and unique 124‐amino acid ‘cassette II’ within the transporter's cytosolic N terminus. Others report that the presence of cassette II does not alter the activity of NBCn1 expressed in Xenopus oocytes (Cooper et al., JBC, 2005) and suggest instead that the cassette may function as a binding domain for protein partners. In a yeast two‐hybrid screen of human skeletal muscle cDNA—using isolated cassette II as bait—we identified calcineurin Aβ (CnAβ) as a candidate binding partner of cassette II. Calcineurins—Ca++‐calmodulin activated Ser/Thr phosphatases—bind and modulate the activities of many ion transporters and channels. Using a quantitative X‐α‐Gal assay, we confirmed the specificity of the CnAβ‐cassette II interaction. In 1‐on‐1 interaction screens with truncated baits, we identified residues 68–124 of cassette II as being necessary for the interaction with CnAβ. This region includes three sequences that are similar to a Cn‐binding consensus site ‘PxIxIT’. Mutation of the most carboxy‐terminal of these motifs ‘PTVVIH’ is sufficient to eliminate CnAβ binding. Our data indicate that cassette II forms a binding domain with the potential to place subpopulations of the transporter under the control of Cn signaling pathways.
Extremely alkaline intracellular pH in the anterior stomach of larval yellow fever mosquitoes (Aedes aegypti)
Authors: Onken Horst , Parks Scott K , Goss Greg G , Moffett David F
Published: June 2021

The anterior stomach of larval Aedes aegypti is an alkalinizing organ where basolateral V‐ATPase drives luminal alkalinization to a pH of up to 12. However, the apical mechanisms involved are unknown. We hypothesized that luminal alkalinization involves absorption of H+ via apical H+ channels. Such a system would require increased intracellular pH (pHi) to function effectively. pHi was monitored using live cell imaging and BCECF as a pH‐dependent fluorophore. Serotonin (0.2 ìM), which stimulates luminal alkalinization, increased pHi in the anterior stomach from 7.0 ± 0.1 to 7.7 ± 0.1 (N = 64), whereas pHi was not affected in the posterior stomach (6.7 ± 0.1 before and 6.8 ± 0.1 after serotonin; N = 19). Perfusing the lumen with pH 10 saline increased pHi to 8.6 ± 0.1 (N = 41), suggesting a marked apical H+ permeability. However, in presence of luminal Zn2+ (10 ìM) similarly high pHi values were recorded (8.2 ± 0.1; N = 15), indicating that an apical proton channel, if present at all, cannot be blocked with Zn2+. The rate of pH change after switching to pH 10 in the lumen in the presence or absence of luminal Zn2+ was also not different. Together these results show that remarkably alkaline pHi values can be observed in the anterior stomach of larval Aedes aegypti. However, further investigation is required to determine the transporters involved in this unique physiological system. This work was supported by NIH (1R01AI063463‐01A2).
Phenotypic Characterization of Acute Systemic and Renal Vascular Responses to Angiotensin II, Norepinephrine, and Endothelin‐1 in Mice Lacking the ADP Ribosyl Cyclase CD38
Authors: Thai Tiffany L , Arendshorst William J
Published: June 2021

Vasoconstriction is mediated by increases in cytosolic calcium concentration ([Ca2+]i). ADP ribosyl cyclases regulate [Ca2+]i via metabolite activation of Ca2+‐induced Ca2+ release. We tested the hypothesis that mice lacking CD38, the predominant mammalian ADP ribosyl cyclase, have attenuated acute hemodynamic responses to angiotensin II (Ang II), norepinephrine (NE), and endothelin‐1 (ET‐1). Despite similar body wt, kidney wt, heart wt, anesthetized mean arterial pressure (MAP), and hematocrit (P>0.1, n≥8), CD38−/− mice (from Dr. Frances Lund) had decreased heart rate and urinary excretion (P<0.01) compared to wild type controls. Using an ultrasonic flow transducer and pressure transducer, we measured renal blood flow (RBF) and MAP in anesthetized mice during iv bolus injections of vasoconstrictor agents. Maximum reductions in RBF produced by Ang II (4–40 ng), NE (2–20 ng), or ET‐1 (20 ng) were attenuated 30–70% in CD38−/− mice (P<0.05) vs. control mice. A trend was seen toward a weaker peak MAP response to Ang II (0.05<P<0.1), but not NE or ET‐1 (P>0.2). We conclude that CD38 contributes to roughly 50% of acute renal vasoconstriction elicited by Ang II, NE, and ET‐1. Our studies indicate participation of CD38 in RBF responses to all agonists tested, while an impaired systemic pressor response was selective to Ang II. CD38 appears to play a more important role in the renal vasculature than other vascular beds.
Carbon dioxide movement from residual volume (RV) toward expiratory reserve volume (ERV)
Authors: Rueda Luis Antonio , Cruz Julio Cesar , Caucha Luis Jhony
Published: June 2021

The expired CO2 reflects the dilution of alveolar CO2 present in the ERV as shown by Cruz (FASEB J. 21:609.4, 2007). Three young male subjects consented to these experiments. The breath was held at the end of a normal expiration and exhaled to RV at the end of apnea. The mass of CO2 in the ERV was computed and plotted as a function of time of apnea (time of expiration plus time of apnea). The figure belongs to one subject and shows that CO2 movement from RV to ERV increases during the first 25s and then plateaus. The ERV=2.2L decreases with time of apnea 13mL per second (F<0.05). It is concluded that 30mL of CO2 moves from RV to ERV in 25s and stops thereafter.

Supported in part by ONG‐Centro de Enseñanza, Investigación y Servicios.
Ion transport properties of small airways
Authors: Shamsuddin A K M , Quinton Paul M
Published: June 2021

The ion transport properties of small airways have been neglected because intact bronchiolar epithelia are small and extremely difficult to obtain without trauma. To accommodate the small size of the tissue, we designed a special micro‐Ussing chamber (Area=1mm2). Constant current pulses (0.5–1.0 μA) were passed across pig airway tissue and basic electrophysiolgical properties were determined. In bilateral 150 mM NaCl Ringers Vt was −1.1±0.2 mV and Rt was 89.4±9.7 Ω‐cm2. When gluconate replaced luminal chloride, Vt (−33.6±2.7 mV) and Rt (239.0±37.8 Ω‐cm2) increased significantly. Adding Fsk plus IBMX further hyperpolarized Vt (−43.0±2.1 mV) and decreased Rt (229.1±33.7 Ω‐cm2). Anion selectivity sequences are grossly the same as for CFTR. The 2:1 NaCl dilution potentials indicate that Cl− is significantly more permeable than Na+. Cl− conductances were inhibited by CFTR inhibitors. Luminal amiloride depolarized Vt and significantly increased Rt. GNa was not affected by Fsk. Bi‐ionic diffusion potentials for NMDG vs. Na+ indicate a slightly Na+ selective paracellular shunt. Small airways are highly conductive due to a major constitutively active Cl− conductance, probably CFTR. These results give the first measurements of transepithelial properties of isolated native small airways <1.5 mm (i.d.).

Support: The Nancy Olmsted Trust and NIH (RO1‐HL084042).
An efficient approach to teaching the anatomy of the middle ear cavity to first year medical students using digital images of prosected cadavers
Authors: Snow Mikel Henry , Albrecht Gene H.
Published: June 2021

Dissection of the middle ear cavity is challenging and time consuming, often resulting in a frustrating learning experience for students. Consequently, courses in gross anatomy may omit dissection of the middle ear, or revert to dissections on decalcified temporal bones, or rely on time consuming, faculty‐prepared prosections. At our school, we have developed an efficient laboratory experience to teach middle ear anatomy using faculty‐prepared prosections and digital imaging. Prosections are done in 15–20 minutes using a quick dissection technique (Anat. Rec. 181:685, 1975) that involves fracturing through the petrous part of the temporal bone. Thus, multiple specimens can be prepared in a short time. This exposure reveals the malleus and incus, the geniculate ganglion and course of the facial nerve, the chorda tympani and greater petrosal nerves, the tensor tympani, the tympanic membrane, the opening of the auditory tube, and the mastoid area. High resolution photos, taken using a macro lens on a digital camera, are enhanced, colorized, and labeled using Photoshop. Color prints (8” × 11”) are placed adjacent to the prosections so students can study the specimens without faculty assistance. This approach is curriculum friendly since it does not require additional lab time as students can exam the middle ear prosections during other scheduled labs. Our approach has proved successful for teaching clinically relevant anatomy of the middle ear in an effective, efficiently manner with neither students nor faculty spending large amounts of time in the laboratory setting.
Sharing Anatomy With Elementary Students Via Two‐way Videoconferencing
Authors: Reeves Rustin E. , Sheedlo Harold J.
Published: June 2021

Sharing human anatomy with elementary age children is often a difficult task to undertake. Logistics of bringing students to your campus, and then the ethics of bringing young students into human anatomy facilities often prohibit this age group from inclusion in outreach programs. This study shows how using live, two‐way videoconferencing technology is an effective solution to this problem, and it can reach much larger audiences of children than traditional anatomy outreach programs. A Tandberg II Intern module was used to transmit video images from the human gross anatomy laboratory at the University of North Texas Health Science Center, Department of Cell Biology and Genetics, to 54 elementary schools across the state of Texas. Approximately 3000 students watched the program on the cardiopulmonary system, and saw demonstrations on healthy and diseased human heart and lung tissue. Four schools were interactive and students could ask questions directly to the moderator. Models and skeletons were used to show relationships of the heart and lung in the human body, and these were related to state of Texas’ science standards on how systems work together. Teacher feedback was very positive and plans are already being made for future presentations. In conclusion, use of live, two‐way videoconferencing is a very efficient method of sharing human anatomy to elementary students for science outreach programs.
Axillary artery and branch variations in an 83 year‐old male Caucasian
Authors: Goldman Evan Michael
Published: June 2021

Results: A male cadaver was found to have a left subscapular artery that branched early off of the axillary artery (at the inferior border of the second rib). The cadaveric subscapular artery was 30% larger in diameter than the continuation of the axillary artery. Additional variants appearing on the left side included (1) the origin of the thoracoacromial artery from the proximal subscapular artery, (2) the origin of the posterior circumflex humeral artery from the subscapular artery and (3) the origin of the lateral thoracic artery from the subscapular artery immediately proximal to the bifurcation into the circumflex scapular and thoracodorsal arteries. Additional variations were seen on the left arm of the cadaver including a high origin of the radial artery in which the radial artery arose from a superficial branch of the brachial artery at mid‐humerus. The superficial branch of the brachial artery was noteworthy by its tortuous route. In comparison, in the right arm, no variants appeared in the axillary, subscapular, or brachial arteries.
A new approach to identification of nuclear proteins associated with chromosomal location of interest
Authors: Zhang Huiping , Wang Yuexi , Quinones Gerri , Ligon Lee A , McGown Linda B
Published: June 2021

Chromatin immunoprecipitation (ChIP) is an important tool for testing whether proteins bind to a particular region on chromatin. We have developed a new method to determine proteins associated with a specific DNA sequence in living cells. Rather than targeting a known protein to determine its chromosomal location using ChIP, a particular chromosomal location of interest is targeted with oligonucleotide probes in order to recover the entire complement of proteins associated with that location. Either chromatin antisense rehybridization (CAR) or chromatin sense rehybridization (CSR) can be used to discover new protein interactions on chromatin.

To demonstrate feasibility of this new approach to analysis of proteins in chromatin, the insulin‐linked polymorphic region (ILPR) located upstream of the human insulin gene was probed using both CAR and CSR techniques. Enzyme‐linked immunosorbent assays or mass spectrometry were then performed to determine what proteins were associated with the ILPR. Insulin and insulin‐like growth factor II, which have been shown to bind to G‐quadruplex structures formed in ILPR in vitro, were detected using CAR and CSR in the ILPR region in live cells. These newly developed CAR and CSR assays are important additions to the chromatin analysis toolbox for identification of DNA‐binding proteins without the need for prior selection of protein targets.
A protein‐directed riboswitch modulates translational repression of VEGF in response to hypoxia
Authors: Fox Paul L , Ray Partho S
Published: June 2021

Riboswitches are found in bacteria, but similar switching mechanisms are rare in eukaryotic cells. We have shown vascular endothelial growth factor‐A (VEGF), a potent angiogenic factor produced by macrophages, is translationally silenced by the IFN‐gamma‐activated inhibitor of translation (GAIT) pathway. Translation is repressed by binding of the GAIT complex to a structural RNA element (GAIT element) in the 3′UTR. Remarkably, cell exposure to hypoxia specifically abrogates GAIT‐mediated translational repression of VEGF mRNA. The GAIT element is within the previously identified hypoxia stability region (HSR) in the VEGF 3′UTR which binds heterogenous nuclear ribonucleoprotein L (hnRNP L) during hypoxia and stabilizes the mRNA. RNA secondary structure prediction indicates the HSR might exist as two alternative conformers, only one of which permits formation of the GAIT structural element. We show binding of hnRNP L switches the HSR to the translation‐permissive conformer that does not bind the GAIT complex, and permits translation of VEGF mRNA even in the presence of IFN‐gamma. During normoxia, IFN reduces the level of hnRNP L, permitting the HSR to switch to the translation‐repressive conformer that allows GAIT complex binding. Our results describe a unique hypoxia‐responsive, protein‐directed riboswitch that regulates macrophage VEGF expression. Supported by NIH grants P01 HL29582 and P01 HL76491.
Unique High Molecular Weight Proteins expressed in Cytoplasmic Male Sterile Wheat (Triticum aestivum) as determined by Two‐Dimensional Electrophoresis (2DE)
Authors: Wang BaiChen , Brush James , Wang TongChang
Published: June 2021

When careful computer analysis of the patterns produced during 3 separate growth years by 2DE was performed the separation of expressed proteins at the young spike stage of growth of male sterile wheat plants indicated there were 18 proteins missing when compared to male fertile plants at the same growth stage. Surprisingly, there were found 12 high molecular weight (over 90 kDa) proteins present in the male sterile plant patterns that were not present in the male fertile plant patterns. Molecular weight and isoelectric points of the affected proteins will be presented. Leaf 2DE patterns at the corresponding developmental growth stages indicated there were no differences to be found. This suggested that the protein pattern differences seen are specifically expressed at the spike stage of growth. Searching of the literature suggests this is the first study of expressed proteins by 2DE of the rather common cytoplasmic male sterility phenomenon in nature. Because of this it is tentatively proposed that male cytoplasmic sterility in wheat at least may have a quite unique genetic program controlling expressed proteins in specific structures of wheat in at least part of its life cycle. Supported in part by institutional research grants from Harbin Normal University.
Identification of new boronic acids as inhibitors against AmpC Beta‐lactamase
Authors: Kubiak Rachel L , Tomlinson Jenna M , Powers Rachel A
Published: June 2021

Antibiotic resistance has emerged as the leading public health crisis of the 21st century. The most prevalent resistance mechanism to commonly prescribed β‐lactam antibiotics like penicillin is the β–lactamase. These enzymes break apart the lactam ring that is crucial for the antibiotic to be effective. One way to overcome this resistance is to block the activity of this enzyme. Boronic acids are a class of molecules known to inhibit the activity of the class C β–lactamase AmpC. Inhibition is achieved through specific interactions between the boronic acid and amino acids in the active site of AmpC, thus preventing the destruction of the β‐lactam. In this study, we chose several boronic acids that differed in size and chemical functionality to investigate specific molecular interactions in the active site of AmpC. Each boronic acid was tested experimentally for inhibition of AmpC in kinetic assays to determine IC50 and KI values, which indicate how effective a molecule is at blocking AmpC activity. All inhibitors were co‐crystallized with AmpC and the structures of these complexes were visualized using x‐ray crystallography. Data obtained from these complexes provides information on binding site interactions in the active site and may be useful for future drug discovery against AmpC.
Directly Cardioprotective Effects of Angiotensin Converting Enzyme (ACE) Inhibitor
Authors: Aizaki Yoshimi , Yoshikawa Ayumu , Maruyama Kei , Muramatus Toshihiro , Nishimura Shigeyumi , Senbonmatsu Takaaki
Published: June 2021

ACE or chymase plays a pivotal role in the conversion from Ang I to Ang II in renin‐angiotensin system. ACE inhibitor (ACEI) prevents Ang II through ACE not chymase, but has pleiotropic effects for cardiovascular protection resulting in great benefits for cardiovascular patients. However, how much participation of pleiotropic effects of ACEI to engage with the cardioprotective effects is controversial.

Method and results: SD rats treated with Ang II (200ng/kg/min, 40days) by the osmotic pump plus 1%NaCl in drinking water, or Ang II and imidapril (40mg/kg/day in 1%NaCl drinking water, Mitsubishi Tanabe Pharma CO., LTD) showed significantly higher blood pressure and cardiac hypertrophy than those of sham operated rats. There were no significant differences between both groups. By contrast, SD rats treated with Ang II and imidapril showed normal cardiac function despite cardiac hypertrophy, and these cardiac function was significantly higher than those of SD‐rats treated with Ang II alone.

Conclusion: Imidapril may prevent development of pathologically cardiac hypertrophy without reduction of blood pressure. This effect may be independent of inhibition of Ang II.
Design Novel Selective Inhibitors of Class II Fructose‐1,6‐bisphosphate Aldolase
Authors: Wang Liangchen , Labbé Geneviève , Krismanich Anthony , Dmitrienko Gary I. , Guillemette J. Guy
Published: June 2021

Fructose‐1,6‐bisphosphate (FBP) aldolase (E.C. ) catalyzes the reversible aldol condensation of dihydroxyacetonephosphate (DHAP) and glyceraldehyde‐3‐phosphate in glycolysis, gluconeogenesis and Calvin cycle. FBP aldolases are categorized into two groups based on different catalytic mechanisms: Class I aldolases form a Schiff‐base intermediate with the substrate through an active site lysine residue, whereas Class II aldolases contain a divalent metal which coordinates and stabilizes the carbanion intermediate. Noticeably, Class II aldolase does not exist in animals or plants, making this enzyme a potential drug target for pathogenic microbial organisms such as Mycobacterium tuberculosis, Magnaporthe grisea, Pseudomonas aeruginosa and Bacillus anthracis. Our research group has found that a commercially available antidote for heavy metal poisoning is a good competitive inhibitor of Class II FBP aldolase. In order to improve the binding affinity of this inhibitor for the enzyme activate site, new molecules based upon this compound were designed, synthesized and tested against recombinant Class II FBP aldolases from aforementioned microorganisms using kinetic assays developed by our group.

This research is supported by NSERC (J.G.G. and G.I.D.)
Phosphorylation of the calpains
Authors: Vazquez Randy , Wendt Amanda S , Thompson Valery F , Novak Stefanie M , Ruse Cristian , Yates John R , Goll Darrel E
Published: June 2021

The calpains are phosphorylated and phosphorylation affects their activities, but no attempts have been made to determine which calpain resides are phosphorylated. Ca2+ requirements of the calpains are much higher in in vitro assays than would ever be encountered in living cells, and cells must contain a mechanism to reduce this requirement; the crystallographic structure of m‐calpain suggests that phosphorylation at selected residues in either domain IIa or IIb of the calpains may lower their Ca2+ requirement. We have used anti‐phospho‐Tyr, anti‐phospho‐Ser, and antiphospho‐Thr antibodies and mass spectrometry to characterize phosphorylation of calpains purified from bovine skeletal muscle or human placenta. Anti‐phospho antibodies show that both μ‐ and m‐calpain have at least 6 phosphorylated sites; 2 each phospho‐Tyr, phospho‐Ser, and phospho‐Thr. The N‐terminal part of the 80‐kDa subunit from residues 6‐266 (μ‐calpain) or residues 9‐265 (m‐calpain) have at least 1 each phospho‐Tyr, phospho‐Ser, and phospho‐Thr. The C‐terminal part of the 80‐kDa subunit from residues 245 to 714 (μ‐calpain) or from residues 265‐700 (m‐calpain) also have at least 1 phospho‐Tyr, 1 phospho‐Ser, and 1 phospho‐Thr. Mass spectrometry has thus far identified 4 phosphorylated sites in the 80‐kDa subunit of μ‐calpain (only 1 in the N‐terminal 266 residues), 8 phosphorylated sites in the 80kDa subunit of m‐calpain (5 in the N‐terminal 265 residues), 1 phosphorylated site in the small subunit of μ‐calpain, and 3 phosphorylated sites in the small subunit of m‐calpain. Four of the sites identified are in domains IIa or IIb.

Supported by NIH and MDA
Proline ‐ A Critical “Stress Substrate” for Bioenergetics and Programmed Cell Death
Authors: Phang James Ming , Pandhare Jui , Borchert Greg , Donald Steven P. , Liu Yongmin
Published: June 2021

Cancer can use “stress substrates” for survival or apoptosis. A “stress substrate” must be readily available and its metabolism responsive to stress challenges. Proline is abundant in collagen, and collagen makes up 25% of total body protein. Importantly, matrix metalloproteinases (MMP) degrading ECM collagen are upregulated during tumor invasion, chronic inflammation and starvation. Thus, proline satisfies the first criterion for a stress substrate. Secondly, we showed that proline oxidase (POX) a.k.a. proline dehydrogenase, the first enzyme in proline degradation, is upregulated by genotoxic stress (p53), inflammatory stress (PPARγ and its ligands), and nutrient stress (AICAR and Rapamycin). POX is an FAD‐dependent enzyme tightly bound to mitochondrial inner membranes. Electrons from proline can be used to generate ATP or they can reduce oxygen to superoxide. The former is used for bioenergetic maintenance under nutrient stress, and the latter can be a mechanism inducing both intrinsic and extrinsic mechanisms for apoptosis. Relevant to cancer, the apoptotic effects of PPARγ ligands are markedly inhibited by knockdown of POX by its siRNA, strongly suggesting that the upregulation of POX may play a critical role in PPARγ‐dependent anticancer effects. Thus, the metabolism of proline as a critical “stress substrate” is a potential target for the prevention and treatment of cancer.
The metabolism of the R and S epimers of sulindac and their ability to enhance the selective killing of cancer cells by oxidative stress
Authors: Brunell David , Marchetti Maria , Resnick Lionel , Gamliel Edna , Kantorow Marc , Binninger David , Weissbach Herbert
Published: June 2021

Sulindac is a non‐steroidal anti‐inflammatory (NSAID) prodrug which requires reduction to sulindac sulfide in order to become an active cyclooxygenase (COX) inhibitor. Recent studies have demonstrated that sulindac and its derivatives have anti‐cancer activity, although the mechanism remains unknown. Sulindac has a chiral sulfur center and exists as R and S epimers. We have isolated the individual epimers and found that both have similar activity against human colon, lung and skin cancer cell lines, providing enhanced killing in the presence of the oxidizing agent tert‐butylhydroperoxide (TBHP), while not adversely affecting normal cells. The death of the cells is accompanied by loss of mitochondrial membrane potential, indicating an apoptotic pathway. Previous in vitro enzymatic studies have indicated that methionine sulfoxide reductase A (MsrA) can reduce the S epimer. In the current studies with whole cells, we have observed that the S epimer was much more rapidly reduced than the R epimer in normal cells, while in cancer cell lines the reduction of both epimers occurred at similar rates. These results suggest that the R epimer may have a higher safety profile when used in the treatment of human cancers, as it is not as rapidly converted to a COX inhibitor in normal cells. Studies are ongoing to elucidate the enzymes and tissue‐specific conditions responsible for the differential metabolism of the R and S epimers.