bluuebunny/crossref_metadata_2025 huggingface dataset:
Prostaglandin E2 type‐1 (EP1) receptors are required for the cerebrovascular dysfunction induced by angiotensin II (AngII)
Authors: Iadecola Costantino , Capone Carmen , Anrather Josef , Wang Gang
Published: June 2021

Hypertension (HTN), a leading cause of stroke and dementia, threatens the cerebral blood supply by disrupting key cerebrovascular regulatory mechanisms (JAP 100:328, 2006). Prostaglandin E2 EP1 receptors (EP1R) contribute to AngII‐induced HTN (JCI 117:2496, 2007). Therefore, we tested whether EP1R are also involved in the cerebrovascular dysregulation induced by AngII. Cerebral blood flow (CBF) was monitored by a laser‐Doppler probe in anesthetized (urethane‐chloralose) mice with a cranial window. AngII (0.25 μg/kg/min; i.v.) elevated mean arterial pressure (MAP) from 71±5 to 93±8 mmHg (p<0.05) and attenuated (p<0.05) the CBF increase in somatosensory cortex induced by whisker stimulation (WS; from 27±6 to 16±4%), or topical application of the endothelium‐dependent vasodilator acetylcholine (ACh; from 22±6 to 13±3%), but not adenosine (from 29±4 to 26±4; p>0.05). The EP1R inhibitor SC51089 (10μg/kg; i.p.) did not alter baseline CBF responses (p>0.05) or the MAP increase induced by AngII (p>0.05), but prevented the effects of AngII on the CBF response to WS (24±3%) and ACh (23±3%; p>0.05 from vehicle; n=5/group). AngII did not attenuate the CBF response to WS or ACh in EP1R‐null mice (p>0.05; n=5/group). We conclude that EP1R are involved in the cerebrovascular dysfunction induced by AngII, and their inhibition could help counteract the cerebrovascular complications of HTN. (Supported by HL18974)
Activation of DAPK by PP2A mediates ceramide‐induced anoikis
Authors: Widau Ryan Cole , Jin Yijun , Gallagher Patricia Jane
Published: June 2021

Ceramide is a pleiotrophic sphingolipid produced by cells in response to radiation, TNF, and chemotherapeutic drugs. Ceramide is a potent activator of protein phosphatases including PP2A leading to dephosphorylation of Bcl‐2 and Bax to result in mitochondrial dysfunction and apoptosis. Previous studies have demonstrated an important role for death associated protein kinase (DAPK), a Ca2+/calmodulin (CaM)‐regulated Ser/Thr kinase, in regulating ceramide‐induced apoptosis, through an unknown mechanism. The goals of these studies are to identify the S308 phosphatase involved in activation of DAPK and the mechanism by which DAPK mediates ceramide‐induced apoptosis. Tandem affinity purification (TAP) and MS identified PP2A as a potential phosphatase. Studies using siRNA‐mediated knockdown of the regulatory B subunit of PP2A resulted in increased phosphorylation of S308 and protein stability of DAPK in response to ceramide. These data suggest that activation of DAPK by PP2A, results in an overall decrease in rLC phosphorylation which is linked to depolymerization of actin cytoskeleton and cell detachment, resulting in apoptosis (anoikis). Ongoing studies are focused at elucidating the role of rLC phosphorylation and anoikis in response to ceramide. Supported by NCI‐CA11198, HL54118, and DK062810
Exercise‐induced alterations and fibre type specific distribution of ARC protein in human skeletal muscle
Authors: Vigna Chris , Bombardier Eric , Tupling A. Russell , Quadrilatero Joe
Published: June 2021

Apoptosis repressor with caspase recruitment domain (ARC), is an anti‐apoptotic protein expressed highly in cardiac and skeletal muscle. This study characterized the fibre type specific expression pattern of ARC in human skeletal muscle for up to six days following acute isometric knee extension exercise. Eight males (age 19.5 ± 0.6 yrs; mass 81.8 ± 2.9 kg; height 182.3 ± 4.9 cm; mean ± SE) completed 30 min of intermittent (50% duty cycle, 5 sec on – 5 sec off) isometric exercise at 40% of their maximal voluntary contraction. Biopsies were taken from the vastus lateralis before and after exercise and at 1, 2, 3 and 6 days post exercise. Immunohistochemical analysis revealed fibre type specific differences in ARC expression prior to exercise with Type I fibres having ∼22% and 33% higher (p<0.05) protein content relative to Type IIA and IIAX/X fibres, respectively. Immediately post exercise, ARC protein was decreased (p<0.05) by ∼24%, 32% and 26% in Type I, IIA and IIAX/X fibres, respectively. ARC remained depressed in all fibres for up to six days following exercise. Similar results were observed with western blot analysis on whole muscle homogenates. These results suggest the anti‐apoptotic protein, ARC, is differentially expressed in skeletal muscle by fibre type, but equally responsive to exercise.

Research supported by NSERC Canada
Implication of Cyclophilin D and Permeability Transition Pore in Mitochondrial Vulnerability of Compensated Heart Hypertrophy
Authors: Matas Jimmy , Ascah Alexis , Marcil Mariannick , Deschepper Christian , Burelle Yan
Published: June 2021

The goal of the present study was to investigate the mechanisms involved in the development of vulnerability to permeability transition pore (PTP) opening in the hypertrophied but compensated heart. Hypertrophy was induced by an aorto‐caval fistula (ACF) in WKHA rats. Mitochondria were isolated 12 weeks post‐surgery for measurement of respiratory function, PTP sensitivity to Ca2+, ROS production, endogenous matrix Ca2+ and expression/localization of the PTP regulating protein Cyp‐D. Sensitivity to Ca2+‐induced PTP opening was increased in ACF vs sham in absence of basal respiratory dysfunctions. Total expression of Cyp‐D and the membrane/soluble ratio was superior in the ACF group. Increased membrane binding of Cyp‐D, which is known to facilitate PTP opening, was not reversed by in vitro pre‐incubation with high levels of the PTP inhibitor and Cyp‐D ligand CsA. However, ACF mitochondria also produced more ROS in vitro and displayed increased levels of matrix Ca2+, both of which being factors that favor the recruitment of Cyp‐D from the soluble to the membrane fraction. These results suggest that early modifications of key PTP regulators, including an increase in Cyp‐D expression and membrane recruitment, contribute to the development of mitochondrial vulnerability in the hypertrophied heart. Those changes may predispose compensated hypertrophied hearts to further cellular dysfunction, ultimately leading to the activation of cell death typically observed in the transition to heart failure. Funded by CIHR and FRSQ.
Enhanced apoptotic propensity in diabetic cardiac interfibrillar mitochondria
Authors: Williamson Courtney L , Dabkowski Erinne R , Hollander John M
Published: June 2021

Cardiovascular complications, including diabetic cardiomyopathy, are the leading cause of mortality among diabetic patients. Enhanced mitochondrion‐dependent apoptosis is associated with diabetic cardiomyopathy pathogenesis. Two spatially distinct mitochondria populations exist in the cardiac myocyte, interfibrillar mitochondria (IFM), which situate between the contractile apparatus, and subsarcolemmal mitochondria (SSM), which exist beneath the plasma membrane. The goal of this study was to determine the impact of diabetic insult on the apoptotic propensity of spatially distinct mitochondrial populations. Swiss Webster mice were made diabetic by streptozotocin injection and sacrificed following diabetes onset. Our findings indicate that diabetic IFM display a greater propensity for undergoing apoptosis as compared to SSM. Specifically, mitochondrial permeability transition pore time to Vmax was decreased in diabetic IFM as compared to control (P<0.05), with no difference in SSM. Mitochondrial membrane potential was decreased in diabetic IFM as compared to control (P<0.05) with no difference in SSM. Mitochondrial cytochrome C content in IFM was decreased as a result of diabetic insult. These results indicate that cardiac IFM are more susceptible to diabetes‐associated apoptosis, and may be more contributory to the pathogenesis of diabetic cardiomyopathy.

(Supported by AHA 0665237B)
Sphingosine 1‐phosphate (S1P) induces autophagy of PC‐3 human prostate cancer cell‐line
Authors: Chang Chi‐Lun , Hsu Chi‐Yei , Ho Ming‐Chih , Lee Po‐Huang , Lee Hsinyu , Huang Wei‐Pang
Published: June 2021

Sphingosine 1‐phosphate (S1P) is a platelet‐enriched lysophospholipid (LPL) which regulates various cellular functions through binding and activating a specific family of G protein‐coupled receptors. During early cancer development, cells experience serum starvation, which is a stress known to induce autophagy. Autophagy is a sub‐cellular membranes trafficking process which leads to the degradation of cellular proteins and cytoplasmic organelles. In our previous study, we found that S1P inhibited cell proliferation in PC‐3 cells independent of apoptosis. However, the detailed mechanism is currently unclear. Through monitoring the modification patterns of LC‐3 by Western blots, we demonstrated that S1P treatments induced autophagy of PC‐3 cells. In addition, S1P enhanced the EGFP labeled autophagosome formation in EGFP‐LC3‐expressed stable cell line. Our results suggested that in serum‐starved condition, S1P might inhibit proliferation of PC‐3 cells through activation of autophagy.
Protective effect of curcumin against high glucose‐induced endoplasmic reticulum stress by downregulation of COX‐2 expression in human umbilical vein endothelial cells
Authors: Fan Ying , Meng Xiang‐hong , Chen Wen‐liang , Zhu Li , Shen Yue‐liang , Chen Ying‐ying
Published: June 2021

OBJECTIVE: The aim of this study is to investigate whether curcumin could protect against high glucose‐induced apoptosis by suppressing endoplasmic reticulum (ER) stress pathway in human umbilical vein endothelial cells (HUVECs). METHODS: HUVECs were exposed to low glucose (5.5mmol/L) or high glucose (30mmol/L) for 12~48 h. The expression of ER stress‐related markers (glucose‐regulated protein 78 (GRP78/Bip) and CAAT/enhancer binding protein homologous protein (CHOP)) and cyclooxygenase‐2 (COX‐2) were also evaluated by western blotting analysis. RESULTS: (1) Annexin V‐FITC/PI flow cytometry analysis revealed a time‐dependent increase of apoptotic cells in HUVECs treated with high glucose. Curcumin (10~100 μ mol/L) inhibited the high glucose‐induced apoptosis in a dose‐denpendent manner. (2) High glucose induced a time‐dependent increase in the levels of COX‐2, GRP78 and CHOP proteins expression in HUVECs. (3) Curcumin and NS‐398 (a COX‐2 inhibitor) downregulated high glucose‐induced overexpression of COX‐2 and CHOP expression in HUVECs. CONCLUSION: These data suggest that curcumin can protect the HUVECs against high glucose‐induced apoptosis by downregulation of COX‐2 and inhibition of endoplasmic reticulum stress.

(This work was supported by the National Natural Science Foundation of China (No. 30400094))
Effects of BITC on TRAIL Sensitization in Human Pancreatic Adenocarcinomas
Authors: Wicker Christina Ann , Kulkarni‐Datar Kashmira , Srivastava Sanjay K. , Brown Thomas L.
Published: June 2021

Pancreatic adenocarcinoma is an aggressive cancer with a greater than 95 % mortality rate and an average survival period of 4–6 months after diagnosis. Treatment options for pancreatic cancer are limited. Chemotherapeutic resistance has been associated with mutations within codon 12 of the K‐Ras gene (K‐Ras 12). These mutations are present in over 90 % of all pancreatic adenocarcinomas and leave Ras in a constitutively‐active state leading to continual cellular proliferation.

Benzyl isothiocyanate (BITC) is a component of cruciferous vegetable extract, and a reported cell cycle inhibitor. TNF‐related apoptosis inducing ligand (TRAIL) is a novel cytokine, whose receptor is highly expressed on tumor cells but generally absent on normal cells. Thus, TRAIL can preferentially target tumor cells to induce apoptosis while leaving non‐tumor cells relatively unharmed.

Our study determined if TRAIL resistance in pancreatic adenocarcinoma with codon 12 K‐Ras mutations can be overcome by first sensitizing the cells using BITC. Western blot analysis revealed activation of early apoptotic pathways but failure to activate later apoptotic pathways resulting in little increase in apoptosis between TRAIL alone and BITC with TRAIL. These results reveal the need to further study alternative survival mechanisms that contribute to chemotherapeutic resistance.
TNF signaling induces the tumor necrosis factor receptor‐1 (TNFR1) complex with the members of death‐inducing signaling complex (DISC) to localize the nucleus in hepatocytes
Authors: Eum Hyun‐Ae , Vallabhaneni Raghuveer , Loughran Patricia A. , Billiar Timothy R.
Published: June 2021

Activation of TNFR1 triggers a cascade of signaling events, including assembly of the DISC, that culminate in cellular apoptosis. Although key elements of DISC are well defined, the molecular mechanisms DISC formation and its subcelluar localization in TNFR1 signaling are poorly defined. Therefore, the aim of this study is to investigate the subcellular localization of TNFR1 and components of the DISC on TNF‐induced apoptosis. We hypothesized that the TNFR1 and the DISC components would be located within the nucleus and the mitochondria after initiation of TNF induced signaling. Sprague‐Dawley rat hepatocytes were used for all experiments. TNF (2000 units/ml) and actinomycin D (Act D; 200 ng/ml) were utilized to induce apoptosis. We demonstrated that TNFR1 recruits TRADD, FADD, and caspase‐8 to establish the DISC in the cytoplasm by both TNF and TNF/Act D stimulation. We also evaluated whether TNFR1 complex with DISC components are localize in the nucleus. The stimulation of TNF by itself resulted in caspase‐8 immunoprecipitation (IP) with TNFR1 into the nucleus within 30 min. However, the TNF/Act D treatment caused the caspase‐8 to Co‐IP with TNFR1 to even more quickly localize into the nucleus. Although TRADD is presented in the nucleus, it did not Co‐IP with TNFR1 in the nucleus with either TNF or TNF/Act D stimulation. These novel findings point to the importance of nucleus in the early TNF cell signaling cascade.
Identification of intestinal bicarbonate transporters involved in formation of carbonate precipitates to stimulate water absorption in marine teleost fish
Authors: Kurita Yukihiro , Nakada Tsutomu , Kato Akira , Doi Hiroyuki , Mistry Abinash C , Chang Min‐Hwang , Romero Michael F , Hirose Shigehisa
Published: June 2021

Marine teleost fish precipitate divalent cations as carbonate deposits in their intestine to minimize the potential for excessive calcium entry and to stimulate water absorption by removing osmolyte ions. This carbonate deposit formation therefore plays an important role in osmoregulation in the seawater environment and also requires controlled secretion of HCO3− to match the amount of calcium entering the intestinal lumen. Despite this physiological importance, the process of carbonate precipitate formation has not been characterized at the molecular level. We first isolated 24 candidate cDNA clones representing all possible membrane proteins capable of transporting HCO3− from the euryhaline pufferfish, mefugu Takifugu obscurus based on the DNA sequence information on the T. rubripes genome, then performed Northern blot analyses to select clones highly expressed in the intestine of seawater mefugu, and finally determined the cellular locations by immunohistochemistry. Based on these observations, we proposed a model for HCO3− excretion across the intestinal epithelium of marine teleost fish that includes a basolateral NBCe1 (Na+/bicarbonate cotransporter) and apical Slc26a6 (Cl−/HCO3− exchangers).
Relative expression of sarco‐endoplasmic reticulum calcium ATPase (SERCA) protein in crayfish, Procambarus clarkii during molting and in response to cold temperature
Authors: Jamal Zeenat , Gao Yongping , Wheatly Michele G.
Published: June 2021

Molting is a prerequisite for development and growth in crustaceans and other arthropods. In the past we have used the natural molting cycle as a non‐mammalian model system to study calcium (Ca2+) homeostasis and the genes encoding for the Ca2+ handling proteins. Ca2+ pumps are integral membrane proteins having 1000 amino acid residues and three cytoplasmic domains (ATP‐binding site, phosphorylation site and a transduction domain). In both epithelial and non‐epithelial tissues of Procambarus clarkii, the Ca2+ pumping ATPase of sarcoplasmic reticulum (SERCA) performs the critical function of sequestering Ca2+ from the cytoplasm at the expense of ATP hydrolysis. In this study, we have specifically looked at the expression of mRNA and protein of SERCA in cardiac muscle during the molting cycle and cold acclimation at 4ºC for 3 weeks by using in‐situ hybridization and immunofluorescence. During intermolt and postmolt stages, a dynamic upregulation of SERCA expression was revealed in cardiac tissues as compared with premolt stage. Tissues of animals cold acclimated at 4ºC for 3 weeks also exhibited a significant increase in SERCA expression. These two experimental conditions will be further employed to better examine the mechanisms of regulation of expression of this important Ca2+ sequestration mechanism (Supported by Grant IBN 0445202 to MGW, YG and CMG).
Expression of Cl/HCO3 Exchanger AE2 and Na‐K‐2Cl Cotransporter NKCC1 in Amphibian Stomach
Authors: Talbot Colleen , Ballesteros Jessica , Kindervater Ryan , Lytle Christian
Published: June 2021

The gastric mucosa secretes Cl− with or without H+ (acidic and nonacidic Cl− secretion) by macroscopically electroneutral and electrogenic processes, respectively, that utilize 2 mechanisms for basolateral Cl− uptake: Cl/HCO3 exchange via AE2 and Na‐K‐2Cl cotransport via NKCC1. The relative contribution these transporters to acidic and nonacidic Cl− secretion and the anatomical origin of these secretions in different animal models remains unclear. We localized AE2 and NKCC1 proteins in the corpus and antrum of the anuran frog Rana catesbeiana and urodele salamanders Necturus maculosus and Ambystoma spp by quantitative confocal immunofluorescence microscopy. In the salamanders, basolateral labeling of AE2 and NKCC1 was exceptionally prominent in mucus cells at the base of antral glands and in oxyntopeptic cells in gastric glands throughout the corpus. In the frog, mucus cells in the antral gland base likewise contained abundant AE2 and NKCC1, whereas oxyntopeptic cells contained high levels of only AE2. Our results support the concept that antral gland base cells contribute importantly to nonacidic Cl− secretion and suggest that this process in amphibians, but not in rodents, involves AE2 in addition to NKCC1. Our data reconcile previous functional evidence that in the frog, in contrast to urodele amphibians and rodents, NKCC1 plays a minor role in gastric Cl− secretion. Supported by NIH GM076981.
Functional analysis of phospholipid hydroperoxide glutathione peroxidase by targeting its expression
Authors: Yoo Min‐Hyuk , Patterson Andrew D , Xu Xue‐Ming , Carlson Bradley A , Gladyshev Vadim N , Hatfield Dolph L
Published: June 2021

Phospholipid hydroperoxide glutathione peroxidase (also known as PHGPX or GPx4) is an essential mammalian protein that functions as both an oxidoreductase enzyme and a structural protein. It acts on a range of substrates from hydrogen peroxide to lipid hydroperoxides and has increased affinity for phospholipid hydroperoxides. To better elucidate the function of GPx4, we used RNAi technology to knockdown its expression in NIH3T3 cells. GPx4 knockdown cells grew more than 70% slower than control cells, developed a dendrite‐like structure and died abruptly after several passages providing evidence that GPx4 is essential for cell survival. The dendrite‐like phenotype suggests that the knockdown cells underwent significant changes in their plasma membrane structure. Since lipid hydroperoxides are known to be potent cytotoxins, we examined the cytoprotective mechanism of GPx4 against these compounds by investigating membrane lipid composition of GPx4 knockdown and control cells. The data provide important insights into GPx4 function beyond its role as a detoxification enzyme.
Role of Se‐glutathione peroxidase‐1(GPX1) in catalyzing nitrotyrosine formation in murine hepatic proteins
Authors: Yang Fang , Lei Xin Gen
Published: June 2021

We reported that primary hepatocytes of GPX1 null mice (GPX1−/−) were more resistant to peroxynitrate (PN)‐induced cell death than those of the wild‐type mice (WT). The present study was conducted to determine if and how GPX1 catalyzed nitrotyrosine formation in murine hepatic proteins in vitro. In Exp. 1, liver samples of GPX1−/− and WT (9 to12 wk old) were homogenized in 100 mM Tris HCl, 0.25M sucrose buffer, pH 7.2, and centrifuged (105,000 g) for 1 h at 4°C. The supernatant (500 μg protein) was treated with 0.18 μmol peroxynitrite (Calbiochem, San Diego, CA) at 37°C for 5 min. Samples from the WT, but not the GPX1−/−, showed strong bands of nitrotyrosine by Western analysis using mouse anti‐nitrotyrosine antibody (Upstate Biotechnology, Lake Placid, NY). In Exp. 2, bovine GPX1 enzyme (Sigma‐Aldrich, St. Louis, MO) was added to liver homogenates of both genotypes before the PN treatment. There was a GPX1 activity‐dependent increase in nitrotyrosine formation in hepatic proteins of both genotypes. In Exp. 3, nitrated proteins of liver homogenates were immuno‐precipitated by anti‐nitrotyrosine agarose beads and subject to nanoLC‐MS/MS after the PN treatment. Presence of intrinsic or extrinsic GPX1 activity in the reaction mixture resulted in five unique nitrated proteins. In conclusion, GPX1 enzyme was able to catalyze or promote the PN‐mediated mouse hepatic protein nitration in vitro [NIH DK 53018 to XGL].
A key molecular switch specifies species barriers in prion disease
Authors: Sigurdson Christina J. , Nilsson Peter , Hornemann Simone , Manco Giuseppe , Draganova Kalina , Castilla Joaquin , Schwarz Petra , Goebel Hans H. , Wüthrich Kurt , Aguzzi Adriano
Published: June 2021

Prion diseases are caused by the conformational conversion and aggregation of the cellular prion protein, PrPC. The disease‐associated isoform, PrPSc, can exist in a multitude of structurally distinct variants which are believed to be directed by the incoming infectious prion conformation. Here we use “rigid loop” (RL) transgenic mice that express a mouse prion protein variant with two amino acid substitutions (S170N, N174T), similar to the deer and elk prion protein, to study the effect on species barriers. These residue replacements result in a profound effect on species barriers and prion conversion depending on whether the incoming prion strain primary sequence shared an asparagine at position 170. Although wild type mice do not develop a clinical infection from cervid chronic wasting disease (CWD) or hamster scrapie, the RL mice overcome these species barriers and convert both CWD and hamster scrapie. Moreover, wild type mice are highly susceptible to BSE and sheep scrapie, however the RL mice resist both BSE and sheep scrapie infection. The prion strain and primary PrP sequence influenced the myotropism of the pathogenic prion. Thus the amino acid sequence at the prion loop region (aa165–175) appears to be critical for templating and propagating incoming strains, and may be relevant for predicting species barriers in CWD.
CD98hc is required for the formation of intimal hyperplasia following arterial injury
Authors: Fogelstrand Per , Feral Chloe , Ginsberg Mark H
Published: June 2021

CD98 heavy chain (CD98hc) is a transmembrane protein that mediates integrin signaling and functions as a chaperone for amino acid transporters (CD98 light chains). It is highly expressed in proliferating tissues; hence we hypothesized that CD98hc has a role in intimal hyperplasia that causes restenosis after vascular injury. Using flow cytometry, we found that smooth muscle cells (SMCs) in culture express CD98hc at high levels and that CD98hc is upregulated in the vessel wall after carotid injury in mice. To specifically delete CD98hc in SMCs, CD98hc conditional knock out mice (floxed CD98hc) were crossed with SM22alpha‐Cre mice. CD98hc was deleted in >97% of vascular SMCs. The mice were healthy and the morphology of the vasculature was the same as in littermate controls. Following carotid injury there was a dramatic decrease in neointimal formation in the CD98hc deleted mice (74% reduction). Ex vivo, the number of SMCs growing out from CD98hc deleted aortic explants was reduced with 73%. BrdU and TUNEL analyses showed reduced proliferation and increased apoptosis in the CD98hc deleted explants. In conclusion, deletion of CD98hc in SMCs leads to a dramatic reduction of neointimal formation following vascular injury, but does not affect normal vascular morphology.
Osteoprotegerin modulation of immunefunction during of the atherosclerotic lesion progression
Authors: Scatena Marta , Rosenfeld Michael
Published: June 2021

Osteoprotegerin (OPG) is a secreted TNF‐like decoy receptor shown to regulate bone homeostasis. Evidence indicates that serum levels of OPG correlate with the severity of cardiovascular disease. We have shown that in ApoE−/− mice, OPG deficiency worsen the lesion burden, suggesting that OPG is an athero‐protective factor. Recent studies indicate that OPG also plays a role in adaptive immunity. B cells subsets are expanded in OPG−/− mice, and in vivo OPG induces increase of IgM and IgG antibodies titers. Furthermore, DCs isolated from OPG−/− mice were more effective in stimulating T‐cells than WT. Thus, we hypothesize that in ApoE−/− mice OPG stimulates the production of athero‐protective antibodies and dampens the production pro‐atherogenic Th1 cytokines, thus explaining the increased lesion size observed in ApoE−/− mice also lacking OPG. To test this hypothesis we have measured serum levels of total IgG and IgM, and of anti‐ox‐LDL antibodies. We have also measured serum levels for the pro‐atherogeneic IL12 cytokine. We have found that ApoE−/− OPG−/− mice have lower serum levels of IgM and IgG and lower serum levels of the atheroprotective anti‐oxLDL antibodies compared to ApoE−/− OPG+/+. ApoE−/− OPG−/− mice have higher serum levels of IL12 than ApoE−/− OPG+/+. In vitro we found that ApoE−/− OPG−/− DCs and macrophages express more IL12 than ApoE−/− OPG+/+. These results indicate that OPG may modulate key immune factors and cells during the atherosclerosis process. Future studies will be aimed to determine whether OPG in immune cells subsets causatively modulate the atherosclerotic lesion progression.

Supported by AHA Grant in Aid #0655677Z
Retinoic acid gives limb development a hand
Authors: Duester Gregg , Zhao Xianling
Published: June 2021

Retinoic acid (RA) signaling has been suggested to play an essential role during induction and patterning of both forelimb and hindlimb buds. Here, we utilize Raldh2−/− and Raldh3−/− mouse embryos lacking RA synthesis to demonstrate that RA is not required for hindlimb induction or patterning, but that RA is required for forelimb induction. RA generated by Raldh2 in somitic mesoderm acts as a proximal source of RA required during forelimb induction, but RA signaling in limb mesoderm itself is not required. Under conditions in which RA activity is absent in mesoderm during limb induction, genes required for limb induction and patterning are induced in both forelimbs and hindlimbs. RA action outside the limb field is sufficient for mesodermal induction of Tbx5 in the forelimb field, suggesting that RA reduces expression of a diffusible forelimb inhibitor produced in nearby tissues during the stage when forelimb induction occurs. As one example of this mechanism we demonstrate that RA signaling functions to repress Fgf8 expression in the intermediate mesoderm adjacent to the forelimb field. Thus, rather than playing an instructive role in forelimb and hindlimb mesoderm, our findings indicate that endogenous RA plays a forelimb‐specific permissive role via its action on tissues situated outside the forelimb field that influence limb induction but not patterning. Supported by NIH grant GM62848.
BMP signaling participates in the regulation of Shh's domain of expression during limb development
Authors: Ros Marian , Bastida María Félix , Sheth Rushikesh
Published: June 2021

The secreted factor Sonic hedgehog (Shh) plays a critical role during limb development controlling patterning in the anterior‐posterior axis. It is expressed by a group of cells at the posterior border of the bud that constitute the Zone of polarizing activity (ZPA). Interestingly, the domain of Shh expression in the posterior border of the bud, once activated, remains of a similar size during further development despite the fact that cell lineage analysis has demonstrated that Shh descendants extend beyond the expression domain of Shh expression. This indicates the existence of mechanisms controlling the number and location of Shh–expressing cells. Here we show that BMP signaling participates in the control of Shh domain of expression by negatively regulating Shh expression and that this function is not mediated by interfering with the known positive regulation exerted by FGFs from the Apical ectodermal ridge or by WNT7A signaling from the dorsal ectoderm. We also show that this BMP function is achieved indirectly requiring protein synthesis and that the proposed mechanism based on Shh induction of cell‐death is mediated by BMP signaling. Supported by grant BFU2005–09309–CO2–01 from Spanish Ministry of Education and Science.
Hormone receptors in the brain and relevance to reproductive aging
Authors: Gore Andrea C , Wu Di , Yin Weiling , Chakraborty Tandra R
Published: June 2021

The control of reproductive physiology and behavior involves a complex hypothalamic circuitry that both drives, and responds to, gonadal steroid hormones. To determine effects of aging on expression of steroid hormone receptors, we quantified estrogen receptor alpha (ER) and androgen receptor (AR) immunoreactive cells in two key neuroendocrine brain regions: anteroventral periventricular nucleus (AVPV) and medial preoptic nucleus (MPN). Female and male Sprague‐Dawley rats were perfused at young (4 mo), middle‐aged (12 mo) and old (20–24 mo) ages. Females were ovariectomized and treated with estradiol (E2) or vehicle. Male rats were intact. In female rats, numbers of ER cells increased modestly with age in AVPV and MPN, with few effects of hormone treatment. In male rats, AR cell numbers and density increased robustly (3–6x), and ER increased slightly, in both regions during aging. These results are surprising because in females, E2 treatment down‐regulates ER mRNA levels, but we observe little effect of E2 on ER cell numbers, suggesting a disconnect between transcriptional and post‐transcriptional processes. In males, androgen levels decrease with age, but AR immunoreactivity increases dramatically, despite evidence that testosterone up‐regulates its receptors. These results suggest a dysregulation of nuclear receptors in the aging hypothalamus. Funding: AG16765 and AG028051.
Protein biomaterial systems for tissue engineering strategies
Authors: Kaplan David
Published: June 2021

Improved understanding of matrix‐cell interactions has provided new direction to the design and study of biomaterial scaffolds to direct cell and tissue outcomes in vitro and in vivo. The ability to deliver appropriate topography, structure and chemistry to cells to direct their fate is emerging as a useful strategy relevant to functional tissue formation. Toward this goal, we utilize fibrous protein scaffold systems (e.g., collagens, silks) and control material features during biosynthesis, assembly, processing and functionalization. Specific examples of cell‐material interactions and outcomes include rates and modes of protein scaffold remolding related to protein structure, protein matrix structure related to control of stem cell fate, protein matrix designs for the delivery of cell signaling factors, and genetically tailored protein matrix designs to add functional features to regulate cell function via scaffold signaling. In these cases, the ability to tailor the protein matrix structure, morphology or chemistry leads to new insight into modes to control cell fate, and thus tissue outcomes. The control of these features also mimics, to some extent, the information context found in cell‐matrix communication in native tissues, in abnormal tissues or in tissue development. Thus, highly controlled matrix designs offer new options with which to gain insight into tissue regeneration, disease states and many related areas of biomedical need such as drug discovery. Our specific areas of interest include hard and soft tissue regeneration. In most cases, different human stem cell sources are utilized in defined bioreactor environments to engage with the matrix‐cell interface toward desired functional tissue outcomes. Specific examples of tissue systems will be reviewed, along with current strategies aimed at understanding and utilizing vascularization in these systems to improve functional outcome. Disease models will also be discussed in the context of matrix design and cell signaling requirements.
Coupling between osteogenesis and angiogenesis
Authors: Feng Jian , Ye Ling
Published: June 2021

Recent progresses clear show that osteogenesis, which requires a coupling between angiogenesis and osteogenesis, is not a cell autonomous process. The new findings suggest that pericytes, arising from a vessel‐associated stem cell progenitor, represent an osteoprogenitor capable of bone formation in a “right” microenvironment. Second, hypoxia environment where bone cells are located greatly affect osteogenesis. Recent studies showed that hypoxia‐inducible factor 1α (HIF‐1α), a transcriptional regulator of vascular endothelial growth factor (VEGF) expression, represents rate‐limiting components of osteogenic‐angiogenic coupling and trabecular bone formation. Last but not the least, the bone does not have a lymphatic system to transport the excessive fluid back to the circulation system. Our studies using a scanning electronic microscope combining with a resin‐casted‐acid etching method reveal that the entire blood vessel system in bone, including arterioles, venules and capillaries are connected with dendritic osteocytes. We propose that osteo‐canalicular system, in addition to its mechanical function, may play a role in facilitating fluid flow back to circulation system. (This work is partially supported by NIH grants: AR51587 and DE 016977).
Effects of Oat Beta‐Glucan and Exercise Training on Tumor Burden in APC‐Min Mice
Authors: Zielinski Mark R. , Gambone Julia E. , Davis J. Mark
Published: June 2021

Both oat beta‐glucan (OBG) and exercise training (EXT) can enhance macrophage function. We hypothesized that EXT and OBG would reduce intestinal polyp burden in APC‐Min mice by reducing macrophage‐mediated inflammation and that the combination of the two treatments would have an added beneficial effect. Eighty male APC‐Min mice were randomly assigned to the following treatment groups at 4 wks of age: EXT, OBG, EXT and OBG, and control (CON). EXT mice ran on a treadmill (18–22 m/min, 60 min/d, 6 d/wk, 11 wks). OBG mice received 6 mg/ml of the soluble fiber in their drinking water daily. At 15 wks of age, mice were sacrificed and tissues collected. Polyps were counted based on size, crypt‐to‐villi (CV) ratios were measured, and macrophages in polyps and lamina propria were determined immunohistochemically. Statistical analysis was done using ANOVA and Tukey post‐hoc analysis. Compared with CON, there was a significant reduction in polyp size, polyp number, CV ratios, and macrophage numbers in the EXT and OBG groups. However, the combination of EXT and OBG resulted in a significant increase in deaths, polyp size, polyp number, and CV ratios compared with CON. Therefore, both OBG and EXT were beneficial in this mouse model, in part, through reduced macrophage tumor infiltration and inflammation. However, their combination was detrimental through an as‐yet‐undetermined mechanism.
Untwisting and untangling DNA: Symmetry breaking by topoisomerases
Authors: Neuman Keir Cajal
Published: June 2021

Replication wreaks topological havoc on the genome. Ahead of replication forks DNA is over‐wound, while nascent DNA molecules are intertwined behind them, leading to linked chromosomes. Replication and chromosome segregation depend on the resolution of these topological tangles. Topoisomerases solve these problems by relaxing positive supercoils ahead of replication forks and decatenating replicated chromosomes. Consistent with these roles, members of the type II family of topoisomerases asymmetrically relax positive and negative supercoils. Topoisomerase IV (Topo IV), a bacterial type II topoisomerase, removes positive supercoils ~20 fold faster than negative supercoils. Current models explaining this asymmetry suggest that Topo IV binds DNA segments juxtaposed at an acute crossing angle. Single‐molecule experiments permitted us to directly test this hypothesis by measuring unlinking rates of single left‐ and right‐handed DNA crossings. Combining these measurements with Monte Carlo simulations of the DNA crossings provides a measure of the preferred crossing angle for Topo IV. Surprisingly, the preferred crossing angle is larger than previous estimates and can not account for the asymmetric supercoil relaxation rates. Single‐molecule relaxation measurements reveal that the asymmetry results from processivity differences of Topo IV in relaxing positively and negatively supercoiled DNA.
Interactome Networks
Authors: Vidal Marc
Published: June 2021

For over half a century it has been conjectured that macromolecules form complex networks of functionally interacting components, and that the molecular mechanisms underlying most biological processes correspond to particular steady states adopted by such cellular networks. However, until recently, systems‐level theoretical conjectures remained largely unappreciated, mainly because of lack of supporting experimental data.

To generate the information necessary to eventually address how complex cellular networks relate to biology, we initiated, at the scale of the whole proteome, an integrated approach for modeling protein‐protein interaction or “interactome” networks. Our main questions are: How are interactome networks organized at the scale of the whole cell? How can we uncover local and global features underlying this organization, and how are interactome networks modified in human disease, such as cancer?
Regulation and function of the TSC‐mTOR pathway in cell growth
Authors: Guan Kun‐Liang
Published: June 2021

The mammalian target of rapamycin (mTOR) plays a central role in the regulation of cell growth. mTOR exists in two distinct complexes, mTORC1 and mTORC2. mTORC1 is known to receive inputs from multiple signaling pathways, including growth factors, nutrients, and cellular energy levels to stimulate protein synthesis by phosphorylating key translation regulators such as ribosomal S6 kinase (S6K) and eukaryote initiation factor 4E binding protein 1 (4EBP1). In contrast, mTORC2 regulates cell morphology and also phosphorylates AKT. Recent studies have established that dysregulated mTOR activity is associated with several hamartoma syndromes and metabolic disorders. The mechanism of mTOR regulation by numerous intracellular signaling pathways and the function of mTOR in physiological regulation will be discussed.
Integrative genetics and genomics study of the hypertriglyceridemia in a polygenic type 2 diabetes mouse model
Authors: Kim Jung Han , Kim Hyoung Yon , Stewart Taryn , Saxton Arnold
Published: June 2021

Hypertriglyceridemia (HTG) is the dominant dyslipidemia in type 2 diabetes (T2D). The aim of this study was to elucidate genetic networks underlying the HTG in a polygenic model for T2D, TALLYHO/JngJ (TH) mice. We conducted a large‐scale microarray gene expression analysis using 16 F2 male mice from a (C57BL/6J x TH) F1 intercross; 8 each from the upper and lower tails for plasma triglyceride distribution of 400 F2 mice. At 24 wk, mice were killed, and liver, muscle, and pancreas were collected and the total RNA isolated and hybridized on the mouse genome 430 2.0 array (Affymetrix). Mice were also genotyped with 68 genetic markers spaced across the whole genome. Individual probe data were extracted using Bioconductor, and the gcRMA normalization was used to produce a signal measure for each gene. Statistical analysis was performed using SAS software, and a mixed ANOVA model was run on the normalized data, fitting genotype and tissue effects, and using array variation as the experimental error. Genes with significant (p<0.05) ANOVA interaction, and significant pair‐wise False Discovery Rate were considered differentially expressed, identifying 1377 in pancreas, 494 in muscle, and 1094 genes in liver. Examining differentially expressed genes using known pathway networks revealed the insulin signaling pathway as the most commonly enriched category in the three tissues tested. American Diabetes Association 7‐04‐RA‐52
The Effects of Escalating Quantities of Salvia hispanica L. (Salba) on Postprandial Glycemia and Appetite in Healthy Individuals
Authors: Vuksan Vladimir , Dias Andre , Chiavaroli Laura , Lee Amy
Published: June 2021

Objective: To assess the effects of escalating quantities of the whole grain Salvia hispanica L. (Salba) on postprandial glycemia and appetite.

Methods: Using an acute randomized controlled design, 12 healthy individuals (7M;5F;BMI 22.3±2.8 kg/m2) received either 0, 7, 15 or 24 g of Salba baked into white bread. The control (0g) was repeated twice. All meals contained 50 g of available carbohydrates. Thus, subjects consumed 5 different meals with at least 3 days in between visits. Fingerprick blood samples and ratings of satiety scores on a 100 mm visual analog scale were taken at 0, 15, 30, 45, 60, 90 and 120 minutes post‐consumption.

Results: There was a dose‐response reduction of postprandial glycemia with the highest and intermediate quantities of Salba resulting in an IAUC blood glucose reduction of 43% (p=0.0013) and 33% (p=0.0115), respectively. Only the intermediate dose resulted in a significant reduction in combined satiety score, with an average reduction of 47% compared to the two other experimental meals and controls at every time point after 15 minutes.

Conclusions: Addition of the whole grain Salba to white bread lowers postprandial glycemia and appetite, and may be potentially cardioprotective. Thus, further research is warranted to assess the effects of Salba on long‐term reduction of cardiovascular risk factors, carbohydrate metabolism and control of body weight.

Sponsor: Salba Inc, Toronto
Including rapeseed protein in a high‐fat meal prevents postprandial vascular endothelial dysfunction in rats
Authors: Magné Joëlle , Huneau Jean‐François , Tomé Daniel , Mariotti François
Published: June 2021

High saturated fat/ high sucrose meals induce oxidative stress associated with vascular endothelial dysfunction, an early hallmark in atherogenesis, but the impact of meal protein on vascular homeostasis remains misunderstood. Here, we investigated the potential of rapeseed protein, an emergent cysteine‐rich protein, to modulate the adverse effects of a high‐fat meal (HFM).

In a cross‐over design, 12 healthy rats received 3 isoenergetic HFM (saturated fat: 60%; sucrose: 20%; proteins: 20% of total energy) with the protein source being either total milk protein (C), rapeseed protein (R), or milk protein supplemented with cysteine and arginine up to the same level as in R (AA). Endothelium‐related vascular reactivity, measured as acetylcholine‐induced transient fall in blood pressure, and plasma triglycerides, hydroperoxides and cGMP were measured before and 2, 4 and 6 h after meal.

While plasma triglycerides rose similarly in all meals, the decrease in endothelial function after C was attenuated after AA and steadily blunted after R. The postmeal decrease in plasma cGMP was lessened 2h after R, as compared to after meal C. Plasma hydroperoxydes increased after the 3 meals, but were lower 6h after meals AA and R than after C.

These results show that rapeseed protein specifically suppresses the acute deleterious effects of HFM on endothelial function, and this may be mediated in part by cysteine and arginine.
Studies in mutant mice: vitamin A and liver fibrosis
Authors: Wongsiriroj Nuttaporn , Piantedosi Rosean , Blaner William S
Published: June 2021

Hepatic stellate cells (HSC) are the major site of hepatic vitamin A storage as well as hepatic extracellular matrix synthesis. In liver injury, HSCs become activated, lose their vitamin A‐containing lipid droplets, and aberrantly produce extracellular matrix proteins giving rise to fibrosis. We have shown that lecithin:retinol acyltransferase‐deficient mice (LRAT−/−) lack lipid droplet in HSCs. Our in vivo studies indicated that diacylglycerol acyltransferase 1 (DGAT1), an enzyme expressed in liver, catalyzes acyl‐CoA‐dependent retinyl ester formation. Surprisingly, about 30% of 3 month‐old double mutant mice lacking LRAT and DGAT1 (LRAT−/−/DGAT1−/−) fed a chow diet develop spontaneously liver fibrosis as evidenced by trichrome staining. No evidence for fibrosis was seen in age‐ and gender‐matched wild type, LRAT−/− or DGAT1−/− littermates. Expression levels of CYP26A1 (a retinoic acid (RA)‐metabolizing enzyme) in LRAT−/− and LRAT−/−/DGAT1−/− mice were significant elevated over those of wild type and DGAT1−/− mice (p<0.0001). Strikingly, CYP26A1 expression of LRAT−/−/DGAT1−/− was significant higher than LRAT−/− (p = 0.002). Since CYP26A1 is a marker for RA levels, our data suggest that higher RA levels in LRAT−/−/DGAT1−/− may play a role in the development of liver disease. The mechanisms underlying the relationship between hepatic vitamin A and fibrosis development are being explored.
Effects of Dietary Components on Cadmium‐Induced Bone Loss in An Ovariectomized Rat Model of Osteoporosis
Authors: Soliman Amani F. , Stoecker Barbara J. , Soung Do Y. , Arjmandi Bahram H.
Published: June 2021

This study examined dietary supplementation effects of potassium phosphate (KPhos) and/or dried plum (DP) on Cd‐induced bone damage. Fifty, 90 day‐old ovariectomized Sprague‐Dawley rats were assigned to five treatments (n=10): 1) control, 2) 50 mg Cd/kg diet, 3) 50 mg Cd plus 12 g KPhos/kg, 4) 200 mg Cd/kg, and 5) 200 mg Cd plus 12 g KPhos/kg. After 45 days of treatment, half the rats in each group had 15% DP added to their diets for an additional 3 months. At necropsy, L4 vertebrae and femora were scanned using microcomputed tomography (uCT) to assess microarchitecture of the trabecular and cortical bone at the midshaft respectively. Bone strength was evaluated using finite element analysis. Bone volume fraction (BV/TV) was lowered by Cd and by KPhos (p< 0.0001). Trabecular number (TbN) was significantly decreased while trabecular separation (TbSp) was significantly increased by Cd and KPhos. DP was beneficial in rats fed 50 ppm Cd but not in those fed 200 ppm Cd with KPhos. Cortical thickness was decreased by Cd and by KPhos (p < 0.0001) but increased by DP (p = 0.002). Force to compress L4 trabecular bone was significantly reduced by Cd and KPhos but increased by DP. Bone ash was reduced by Cd (p<0.0001) and KPhos (p<0.0005) but was enhanced (<0.0105) by DP. Our results indicate that Cd and KPhos impaired bone microarchitecture and strength. DP increased cortical thickness but DP effects on L4 depended on dietary Cd and KPhos.