bluuebunny/crossref_metadata_2025 huggingface dataset:
Dynamic alterations of chemical component profiles during Cordyceps sinensis maturation and comparisons with those for Hirsutella sinensis and Paecilomyces hepiali mycelia
Authors: Zhu Jia‐Shi , Wu Zimei , Qi Ying , Lu Jihong , Zhang Li , Zhang Lijuan
Published: June 2021

Cordyceps sinensis (Cs) is a precious Chinese herb traditionally used for anti‐aging and rehabilitation from diseases. Co‐existence of Paecilomyces hepiali (Ph) and Hirsutella sinensis (Hs) in Cs has been reported (FESAB 19:A1033, 2005; 21:A1079, 2007). We examined maturational alterations of component proteins and small organic chemicals in fresh Cs (180 pieces) and compared the profiles with those for Ph and Hs. Cs of 2 maturation stages were used, less mature Cs (T1) with a plump caterpillar body (CB) and a short stroma (Stm), and mature Cs (T2) with a less plump CB and a long Stm. Cs (CB and Stm) and freeze‐dried mycelia of Ph and Hs were individually grinded in a mortar into powder in liquid nitrogen and dissolved in water or methanol before subjected to the profiling analysis. SELDI‐TOF MS was used for protein profiling and HPLC with an ODS‐C18 column for small molecule profiling. Different profiles were noted for Cs (CB, Stm) in 2 maturational stages. The profiles for Ph and Hs did not match completely to those for Cs. But Ph mycelia appears to show closer profile patterns to Cs, than Hs mycelia does. In conclusion, Cs maturation associates with dynamic changes in profiles of component chemicals, supporting its superior function profiles. As compared to Hs, the profiles for Ph are closer to those for Cs.
Tracing Cell Lineage in Mammalian Cardiovascular Development
Authors: Sucov Henry M.
Published: June 2021

As is true for most developing tissues and organs, cardiovascular development involves interactions between cells of different lineage origins, which in many cases migrate to their ultimate positions from distant locations. We have utilized Cre/lox approaches for mapping cell lineage contributions to the cardiovascular outflow tract during normal mouse development, and have combined these approaches with conventional and conditional mutant backgrounds to explore the roles of retinoic acid and TGFb receptors during these processes.
Biotin Requirement and Its Biosynthesis Blockage in the Free‐living Nematode, Caenorhabditis elegans
Authors: Liu Chun , Lu Nancy
Published: June 2021

The free living C. elegans were cultivated in the chemically defined C. elegans Maintenance Medium (CeMM) supplemented with 0, 0.00600, 0.0300, 0.150, 0.750, 3.75, 18.8, 93.8 and 469 μg/ml biotin. No population growth was observed when biotin was omitted from the CeMM (0 μg/ml). A significantly decreased population growth (p ≤.05) was observed at 0.00600 μg/ml biotin. Optimal population growth occurred at biotin levels between 0.0300 and 18.8 μg/ml. However, the nematodes appeared to move sluggishly at 0.0300 and 0.150 μg/ml biotin. These results, therefore, suggested that 0.75 to 18.8 μg/ml were the optimal range to support a maximal growth of C. elegans as well as to prevent sluggish movement resulted from a biotin deficiency. Significant reductions of population growth (p ≤.05) were observed at 93.8 μg/ml and 469 μg/ml biotin. In the blockage experiment, dethiobiotin, the last‐step precursor in the biotin biosynthesis pathway, was used to investigate if it supported the population growth of C. elegans. Three levels of dethiobiotin (0.0263, 0.132, and 3.29 μg/ml) were used to replace biotin at the equal molar concentrations, respectively, in the CeMM. At all three levels of dethiobiotin, no population growth of C. elegans was observed. These finings suggested that C. elegans might have lost the ability to produce the enzyme, biotin synthase. Therefore, biotin became a dietary essential nutrient for C. elegans. (Supported by San Jose State University Foundation Grant and Circle of Friends Research Award of Department of Nutrition & Food Science, San Jose State University, CA)
The role of β‐carotene‐15,15′‐oxygenase (CMO1) during mammalian embryonic development
Authors: Kim Y‐K. , Wassef L. , Wyss A. , Blaner W.S. , Quadro L.
Published: June 2021

β‐carotene is the best‐known vitamin A precursor. By the action of β‐carotene‐15,15′‐oxygenase (CMO1), one molecule of β‐carotene can be cleaved symmetrically, ultimately resulting in the formation of two molecules of retinol. Vitamin A is essential for embryonic development. In order to meet vitamin A requirements, the mammalian embryo relies on maternal circulating retinoids (vitamin A and its derivatives). We previously showed that, upon maternal dietary vitamin A deprivation, normal embryonic development is impaired in mice lacking retinol‐binding protein (RBP), the sole specific carrier for retinol in the bloodstream. To investigate the relationship between carotenoid and retinoid metabolism during development, we generated a mouse strain that lacks both CMO1 and RBP (CMO1−/−RBP−/−). We show that CMO1−/−RBP−/− mice are viable and fertile when maintained on a vitamin A‐sufficient diet. However, E14.5 embryos from CMO1−/−RBP−/− dams bred on a vitamin A‐deficient diet are malformed, and the severity of malformations is greater than those seen in RBP−/− embryos. No differences in circulating retinoids levels were observed between CMO1−/−RBP−/− and RBP−/− dams. Also, embryonic development in CMO1−/− mice is normal. We are now investigating the reasons for the embryonic phenotype of the CMO1−/−RBP−/− mice to elucidate the physiological role played by CMO1 and β‐carotene during development.
Bioactive compounds with high antioxidant potential in biofortified carrots do not influence provitamin A carotenoid bioefficacy in gerbils
Authors: Mills Jordan P , Simon Philipp W , Tanumihardjo Sherry A
Published: June 2021

Dietary Guidelines for Americans recommend a diet rich in fruits and vegetables of a variety of colors. Colored carrots contain several groups of phytochemicals. Provitamin A carotenoid bioefficacy was measured in four carrot varieties (purple/orange, purple/orange/red, orange/red and orange) using gerbils. Freeze‐dried carrot powders were mixed into diets to achieve 6 nmol beta‐carotene equivalents/g diet and fed to 4 groups (n = 11/group) for 4 wk. Diets were equalized for carrot powder using carotenoid‐free white carrot powder. Diets containing carrot varieties rich in lycopene and/or anthocyanins were formulated to have equal concentrations of these compounds. Antioxidant capacities of the carrot powder, diets, serum and livers were determined with the ABTS+ free radical scavenging decolorization assay. All four colored carrot‐fed groups had higher liver retinol stores (0.230 + 0.029 to 0.259 + 0.047 μmol/g) compared to the white carrot‐fed control group (0.124 + 0.036 μmol/g), but did not differ from each other. Hydrophilic antioxidant capacities of the anthocyanin‐rich purple varieties were 5 and 10‐fold higher than white carrot. Lipophilic antioxidant capacities were 10 to 20‐fold higher in the carotenoid‐rich colored carrots compared to white carrot. Provitamin A carotenoids in biofortified carrots maintained vitamin A status with no discernable influence of lycopene or anthocyanins on bioefficacy at the levels fed in this study. Supported by Hatch WIS04975; Standard Process, Inc., Palmyra, WI; and IEDR UW‐Graduate School 118–0617.
Lifetime consumption of a low vitamin K diet leads to learning impairment in aged rats: potential links with sphingolipid metabolism
Authors: Ferland Guylaine , Carrié Isabelle , Bélanger Élisabeth , Rochford Joseph
Published: June 2021

In a recent report, we showed VK to preferentially accumulate in brain regions rich in white matter and to positively correlate with certain sphingolipids. In rodents, pharmacological VK deficiency has resulted in behavioral perturbations. Whether a more physiological deficiency leads to similar effects has not yet been determined. To gain insight on this issue, we investigated learning abilities (Morris water maze) in 6, 12 and 20 month‐old female SD rats which had been fed diets containing either very low (VL: 80), low (L: 500) or moderately high (H: 2000) levels of K1 (μg/Kg diet; n=5–10/group) since weaning. In 20‐months rats, cerebroside, sulfatide, sphingomyelin, ceramide and gangliosides were also assessed in cerebellum, midbrain, pons medulla, striatum and hippocampus (Bodennec et al. Meth. Enzymol. 312:101–14, 2000). Lifetime consumption of a low VK diet led to cognitive deficits in old age, rats from VL group showing longer latencies to reach the platform than those from the L and H groups (p<0.05), a finding associated with higher concentrations of ceramides in the hippocampus and lower gangliosides in the pons medulla and midbrain. Learning abilities at 6 or 12 months were not affected by diet. In conclusion, we show for the first time that lifetime consumption of a low VK diet leads to learning impairments in old age and that this may be linked to disturbances in sphingolipid metabolism. Supported by NSERC.
Regulatory effect of crude extract of medicinal herbs on atopic allergic reaction
Authors: Park Kimoon , Yang Heejin , Lee Kyungjin , Cho Soomuk , Chun Hyekyung
Published: June 2021

The atopic dermatitis syndrome(ADS) is one of most common chronic inflammatory skin illness and characterized by eczematous skin lesion and IgE hyperproduction. This study was carried out to investigate effect of crude extract of medicinal herbs on atopic dermatitis syndrome in vitro. The crude extract of medicinal herbs significantly inhibited compound 48/80‐induced beta‐hexosaminidase release from HMC‐1 cells(human mast cell line). It also inhibited tumor necrosis factor‐alpha, interleukin‐6 and interleukin‐8 secretion from the phorbol 12‐myristate 13‐acetate(PMA) and A23187‐induced HMC‐1 cells. And when the cytokine mRNA expression was examined by RT‐PCR, the crude extract of medicinal herbs significantly inhibited tumor necrosis factor‐alpha, interleukin‐6 and interleukin‐8 mRNA expression. These results suggest that crude extract of medicinal herbs has an inhibitory effect on the atopic allergic reaction and thus may be useful in the treatment of atopic dermatitis.
Protective Effect of Ginseng and Several Medicinal Herbs on Heat Stress by IFIT1 and IFIT2 Gene Expression
Authors: Yoon Kye Yoon , Kim Kui‐Jin , Lee Hee‐Hyun , Hong Hee‐Do , Lee Boo‐Yong
Published: June 2021

Ginseng and several medicinal herbs are known to enhance stamina and immune function, it has been suggested to have pharmacological activities in the cardiovascular, endocrine, immune, and central nervous systems. Heat stress can trigger a cellular stress response characterized by induction to against heat environment. IFIT1 and IFIT2, which are bound to the translation initiation factor eIF3, inhibit translation of cellular stress response. Rats divided to 6 groups and were adapted at room temperature for four weeks. One group was raised at room temperature condition, another group was raised at heat stress condition for two weeks (38°C, 5hr/day), the others were administrated with ginseng and medicinal herbs on heat stress condition. To investigate protective effect of ginseng and medicinal herbs on heat stress, expression level of IFIT1 and IFIT2 mRNAs of rat liver examined by RT‐PCR and real‐time PCR. Compared to unstressed controls, the expression level of genes was generally increased in the heat stressed rats. But the expression levels of IFIT1 and IFIT2 of groups, which were administrated with ginseng and medicinal herbs, was decreased more than those of group which was only stressed by heat. In conclusion, ginseng and several medicinal herbs are effective on protecting rat against heat stress.
Effects of Saw Palmetto Extracted Oil Supplementation on Human Benign Prostatic Hyperplasia.
Authors: Hong Heeok , Kim Chun‐Soo , Lee Jung Sug , Maeng Won Jai
Published: June 2021

The purpose of this research is the possibility of treatment of benign prostatic hyperplasia (BPH) by saw palmetto extracted oil. This study was performed for 6 months with total 33 BPH patients aged 51 years old. Measured parameters were international prostate symptom score(IPSS), quality of life (QoL), serum prostate specific antigen(PSA), prostate volume, maximum flow rate, serum total cholesterol, HDL‐cholesterol(HDL), LDL‐cholesterol(LDL), triglyceride(TG), and blood pressure. The materials used in this study were sweet potato starch(control group) and saw palmetto extracted oil(SP group) used 320mg per day, respectively. The IPSS, and QoL measured after 3 and 6 months treatment decreased significantly in SP group as compared with pre‐experimental level, respectively(p<0.05). But the PSA measured after 3 and 6 months decreased significantly in control group as compared with pre‐experimental level, respectively(p<0.05) but those of SP group weren't changed during treatment. Urine speed in all groups increased significantly after 6 months treatment(p<0.05). All groups were kept normal criteria range during treatment for total cholesterol, HDL, LDL and TG. In SP group, systolic blood pressure increased significantly after 6 months(p<0.05). Therefore, saw palmetto extracted oil supplementation is possible effectiveness of complementary for BPH patients.
Oral supplementation of gromwell (Lithospermum erythrorhizon) extract prevents the development of atopic dermatitis with reducing ceramide degradation in the epidermis of NC/Nga mice
Authors: Kim JungMin , Kim YoungRan , Seo DaeBang , Kim SungHan , Lee SangJun , Cho Yunhi
Published: June 2021

Abstract Gromwell (Lithospermum erythrorhizon) is widely used in the treatment of abnormal skin condition, but its systemic efficacy, especially in atopic dermatitis (AD), is not clear. To examine the systemic efficacy of gromwell on the clinical manifestation of AD‐like skin lesions, NC/Nga mice, a murine model of AD, were fed control diet (group CA: atopic control) or diet with 70% ethanol extracts from 5% gromwell (Group LE) for 10 wks. In group LE, the clinical manifestation of AD‐like skin lesion was prevented as the level of serum IgE, epidermal hyperproliferation, and the number and duration of scratching episode, which were greater in group CA, were significantly reduced to the similar level of normal control group of BALB/c mice. In addition, the level of ceramides, the major lipid maintaining epidermal barrier, in the epidermis of group LE was increased, which was inversely associated with a decreased protein level of ceramidase, an enzyme of ceramide degradation. However, mRNA and protein levels of serine palmitoyl transferase (enzyme for de novo ceramide synthesis) in groups C, CA and LE did not differ. We demonstrate that oral supplementation of gromwell extracts prevents the development of atopic dermatitis with reducing ceramide degradation coupled with a low expression of ceramidase protein.
Selective, Multi‐Target Protein Kinase Inhibitors, Rho iso‐alpha acids (RIAA) and Proanthocyanidins, Combined with Cholesterol Lowering Foods, Reduced Cardiovascular Risk Factors in Humans with Metabolic Syndrome
Authors: Tripp Matthew , Lerman Robert , Lamb Joseph , Minich Deanna , Darland Gary , Konda Veera , Emma Dennis , Carroll Brian , Babish John , Pacioretty Linda , Bland Jeffrey
Published: June 2021

Background: Metabolic Syndrome (MetS), insulin resistance and hypercholesterolemia are major cardiovascular disease (CVD) risk factors. Epidemiological studies suggest that a phytochemically poor diet may be implicated, however, the effect of specific dietary phytochemicals on MetS remains largely unexplored.

Objective: To identify phytochemicals which improve insulin sensitivity using in vitro models, and investigate their effects in animal models of insulin resistance, and on CVD risk factors in humans with MetS.

Results: RIAA and proanthocyanidins improved insulin signaling in an in vitro 3T3L1 adipocyte screen. In cell free assays they were found to be selective, multi‐target protein kinase inhibitors (e.g., GSK3α and β; PKCβ2; IKKβ), possibly explaining their mode of action. In combination they reduced insulin and glucose to a similar degree as metformin in obese db/db mice. Thus, a human clinical study was conducted. In this 2‐arm, open‐label, 12 wk study, volunteers with MetS and hypercholesterolemia were randomized to either a phytochemical rich Mediterranean‐style, low glycemic load diet, MED (n=19) or MED plus tailored phytochemicals (PED) (n=25) including soy protein, plant sterols, RIAA, and proanthocyanidins. Phytochemical supplementation (PED) led to a greater decrease in CVD risk compared with diet alone (P<0.001), a result independent of weight loss or caloric restriction.
Identification of gene differences between dogs with poor periodontal scores compared dogs with good periodontal scores
Authors: Frantz Nolan Z. , Coffman Lori , Stiers Cheryl , Gao Xiangming , Friesen Kim G. , Yamka Ryan M. , Al‐Murrani Samer
Published: June 2021

Fifty‐six (> 1 years of age) spayed/neutered beagles were identified for this study. Dogs were scored for periodontal lesions and fed commercially available adult foods. At the time around scoring, a blood sample was drawn into a PAXgene Blood RNA tube. RNA was extracted according to the procedures provided in the PAXgene Blood RNA Kit Handbook (Qiagen, Valencia, CA). RNA was hybridized to an Affymetrix GeneChip Canine‐2 Genome Arrays and normalized using Robust Multi‐Array Average. Transcripts having a P < 0.01 (following a false discovery rate adjustment value of 0.1) and a fold change range of at least 1.2 were considered different among the two groups. Analysis of the RNA found differences in 284 genes between the dogs with poor periodontal scores and dogs with good periodontal scores. Of the genes identified, 157 were up‐regulated and 127 down‐regulated in dogs with poor periodontal scores and dogs with good periodontal scores. Genes associated with inflammation process and pain response were increased, while cell adhesion and attachment, heavy metal binding, glycoprotein synthesis, and cytoskeleton structure support were decreased in dogs with poor periodontal scores compared to dogs with good periodontal scores.
Effect of Heparin and Low Molecular Weight Heparin on the Regulation of Microparticle in Atrial Fibrillation
Authors: Samikkannu Michael , Hoppensteadt Debra , Adiguzel Cafer , Cunanan Josephine , Iqbal Omer , Tobu Mahmut , Fareed Jawed
Published: June 2021

Microparticles (MPs) represent fragments released from the plasma membranes of either stimulated cells or epithelial cells. Although flow cytometry methods have been used in the detection of MPs, only very limited data is available on the functional measurement of these particles and ischemic stroke. More recently, a functional method for the measurement of MP procoagulant activity in plasma (Zymuphen MP‐Activity) has become available. This assay measures the ability of MPs to mediate the generation of thrombin. We hypothesized that patients with atrial fibrillation may have an up regulation of MPs. In order to validate this hypothesis, we measured functional MP in a sub‐study in conjunction with a recently published clinical trial [Assessment of Cardioversion Using Transoesophageal Echocardiography (ACUTE) II a randomized multicentre study]. We analyzed 50 patients plasma and measured the MP levels. The patients’ plasma samples were obtained from two separate groups of patients (n=25), each group was treated with either heparin (UFH) or a low molecular weight heparin (Enoxaparin) (E). In comparison to the normal controls (n=50) the baseline samples from the patient groups exhibited a much higher level of MPs (5.8±3.1 versus 17.1±5.8 nm). Both UFH and E treatment resulted in a decrease of the functional MP levels. These results suggest that in atrial fibrillation MPs are upregulated and may potentially mediate the thrombogenic responses.
Differential Interaction of Anti‐heparin Platelet Factor 4 Antibodies with Branded and Generic Versions of Low Molecular Weight Heparins. Pathophysiologic and Bioequivalence Implications.
Authors: Fareed Jawed , Hoppensteadt Debra , Iqbal Omer , Adiguzel Cafer , Messmore Harry , Walenga Jenaine
Published: June 2021

Low molecular weight heparins (LMWHs) such as enoxaparin (E) and dalteparin (D) are now widely used for the management of thrombotic and cardiovascular complications. Several generic versions of these drugs have recently become available. Due to the compositional differences in the branded agents, such as E and D, their interactions with platelet factor 4 (PF4) differ and potentially have impact on immunogenic responses. Similarly, because of the apparent differences in the source material and oligosaccharide composition, obvious differences in PF4 titration have been noted. To compare the potential interactions of D and one of it's generic versions daltehep (DH) and E and two of its generic versions, namely, lupenox (LU) and loparan (LO), each of these agents were tested in an in vitro heparin induced platelet aggregation (HIPA) system, where pooled serum from clinically diagnosed heparin induced thrombocytopenia (HIT) patients was used as an antibody source. Significant differences in the aggregation responses were noted between D and E. There was no difference in D and DH (30.8 ± 7.8% vs. 34.7 ± 8.1%). However, significant differences between E (18.2 ± 4.2%), LO (38.1 ± 9.0%) and LU (68.1 ± 9.1%) were noted. These results underscore the observation that HIT antibodies interact differentially between the branded and generic LMWHs, owing to their oligosaccharide composition.
Comparative Anticoagulant Effects of Suledoxide and Enoxaparin in Citrated Whole Blood
Authors: Adiguzel Cafer , Iqbal Omer , Hoppensteadt Debra , Cunanan Josephine , Fareed Jawed
Published: June 2021

Suledoxide represent a novel antithrombotic agent with multiple sides of actions on blood coagulation process. This drug has been used widely in various vascular indications. Suledoxide has been recently shown to produce therapeutic effects in diabetic nephropathy. Unlike LMWH, this agent can also be used orally. The purpose of this study was to compare the anticoagulant effects of Suledoxide with a LMWH Enoxaparin which is use mainly via subcutaneous route. Suledoxide represent of mixture of fast mobilizing heparin 80% and 20% Dermatan Sulfate. Within average molecular weight of 20.8 kDa. Suledoxide may be effective in subcutaneous indications. In order to investigate the anticoagulant effect of Suledoxide this agent was supplemented to citrated whole blood at concentration of 0–25 ug/ml such blood test ha PT, aPTT, Heptest, and Thrombin time were measured. This agent was compared Enoxaparin at equigravimetric concentrations.

In comparison to Enoxaparin and Suledoxide produced a stronger anticoagulant effect in the PT assay. Similarly Suledoxide produce a much stronger anticoagulant effect in the aPTT assay. In the Heptest assay, Suledoxide produced anticoagulant effects. Thrombin Time results is also much stronger with Suledoxide in comparison to Enoxaparin.

These results highly suggest that Suledoxide is a relatively stronger anticoagulant than Enoxaparin. Suledoxide may also exhibit subcutaneous bioavailability. These results warrant further investigation on Suledoxide in Pharmacologic studies.
Development of siRNA systems to knock‐down arginase I‐specific or iNOS‐specific gene expression
Authors: Guo Zhong , Bucher Brian T , Shao Lifang , Geller David A
Published: June 2021

The L‐arginine/iNOS pathway has been recognized to play critical roles during infection, inflammation, organ injury, and transplant rejection. The development of vector‐based siRNAs provides a powerful genetic approach to study the pathophysiology in vitro and in vivo by suppressing the endogenous arginase I or iNOS expression. Here, we have screened and identified the siRNA sequences specific for arginase I or iNOS gene, which are both conserved among human, rat and mouse species. Transfection of RAW cells with arginase I‐specific siRNA significantly reduces arginase‐I protein expression and arginase activity after stimulation with cytokines. Moreover, Transfection of DLD −1 cells with iNOS‐specific siRNA also significantly decreased iNOS protein expression and nitrite production after stimulation with cytokines. Non‐specific siRNA served as control, and did not knock‐down either gene expression. These siRNAs should provide useful tools to study the effects of iNOS and arginase I expression in biologic systems.
Expression of C5a receptor in osteoblasts
Authors: Jiang Tao , Gao Hongwei
Published: June 2021

Anaphylatoxin C5a signaling plays an important role in inflammation‐associated bone diseases such as rheumatoid arthritis. However, expression and function of C5a receptor (C5aR) in the osteoblasts are largely unknown. The objective of this study is to determine C5aR expression and its function in the osteoblasts. Using a human osteoblast‐derived osteosarcoma cells, MG‐63, we show that osteoblasts express C5aR by RT‐PCR and immunostaing. Unexpectedly, we found that C5a down‐regulates LPS‐induced IL‐6 release from the MG‐63 cells. We further show that C5a causes a decreased gene transcription for IL‐6 induced by LPS, suggesting a transcriptional regulation is involved. Interestingly, we found that in the presence of LPS, there is a C5a‐dependent up‐regulation of C5aR expression, suggesting that its up‐regulation in a C5a dependent manner brings into play a mechanism whereby inflammatory mediator production such as IL‐6 from osteoblasts is contained. In conclusion, C5aR is expressed in osteoblasts, and may play an important regulatory role in inflammatory bone diseases. This work is supported by North Dakota Experimental Program to Stimulate Competitive Research (NSF EPSCoR).
Wound Repair by Activated Heart Valve Interstitial Cells is Regulated by Transforming Growth Factor‐β
Authors: Liu Amber Chang , Chen Yingming Amy , Gotlieb Avrum I
Published: June 2021

Valve interstitial cells (VICs) undergo phenotypic changes in response to valve injury and disease, expressing α‐smooth muscle actin (α‐SMA), a marker of activation. We tested the hypothesis that TGF‐β is an important regulator of VIC activation and repair. We created in vitro experimental wounds by mechanical denudation of a confluent monolayer cultured in M199 containing 10% fetal bovine serum 2% penicillin/streptomycin/amphotericin. In TGF‐β neutralizing antibody non‐treated and treated wounds, we characterized VIC activation, TGF‐β expression and apoptosis by α‐SMA, TGF‐β, activated caspase‐3 and annexin V immunofluorescence respectively. We also quantified proliferation by BrdU labeling and repair by measuring wound closure. VICs at the wound edge are activated showing prominent α‐SMA staining which decreases over 24–96 hours post‐wounding. Upon wound closure, α‐SMA staining becomes similar to that of a quiescent nonwounded monolayer. TGF‐β and pSmad2/3 staining are more prominent and apoptosis is observed more frequently at the wound edge at 24 hours post‐wounding compared to the nonwounded monolayer. Addition of TGF‐β neutralizing antibody decreases VIC activation, proliferation and wound closure in a dose‐dependent manner. In conclusion, wounding activates VICs which upregulate TGF‐β. TGF‐β maintains VIC activation and in turn regulates proliferation and wound repair.
Methods for investigating integrin‐matrix interactions as a function of matrix mechanics and composition
Authors: Sazonova Olga , Nugent Matthew A , Wong Joyce Y
Published: June 2021

To investigate the role of mechanics in cell signaling, we have engineered polyacrylamide (PAAM) gels with precisely tunable stiffness and fibronectin (FN) content. Varying the acrylamide / bis ratio alters PAAM stiffness, as determined with tensile testing and atomic force microscopy (AFM). To promote cell attachment, FN is covalently linked throughout the gel, and can then be quantified with an ELISA. Analyzing AFM data with the Hertz and Oliver‐Pharr models, we calculated gel elastic moduli ranging from 11 – 344 kPa and 7.5 – 95 kPa. We also found that stiffer gels incorporate more FN than softer gels, and that adding FN reduces the modulus of softer gels by ~10%. Controlling for these factors allowed for precise and independent changes in gel mechanics and FN content.

To study integrin‐FN interactions on PAAM gels, we adapted published methods for quantifying integrin binding. After vascular smooth muscle cells (VSMCs) were grown on gels or FN‐coated plastic, bound FN‐alpha5 integrin units were cross‐linked and assayed with an ELISA. Preliminary data suggest that integrin activity per cell is reduced with decreasing stiffness or higher cell density. Future studies using the methods presented herein will provide insight into how VSMCs recognize and respond to changes in ECM composition and stiffness, aiding the rational design of new treatment for vascular disease. Supported by NIH grants HL56200 and HL72900.
Defective capacity of CD8+ memory T cells developed from absence of IL‐12 priming for secondary expansion
Authors: ye zhenmin
Published: June 2021

In vivo CD8+ T cell responses consist of three main phases, namely: (i) a proliferation phase consisting of effector T cell clonal expansion, (ii) a contraction (transition) phase leading to the transition from the large population of effector T cells to a smaller population of long‐lived memory T (Tm) cells, and (iii) a maintenance phase involving Tm cell survival in the host. Recently, it has also been shown that IL‐12 priming during in vitro Ag stimulation changes properties of CD8+ T cells such as downregulation of CD69, alteration in the distribution of lipid rafts, a decrease in T cell receptor structural avidity and cytolytic activity. However, the potent functional property of CD8+ Tm cells without IL‐12 priming for secondary expansion upon Ag reencounter is still unclear.In this study, we attempted to clarify the role of IL‐12 by transferring the CD8+ effector T cells into wild‐type C57BL/6 and IL‐12 gene KO mice, respectively. Our findings showed that IL‐12 priming is important not only in CD8+ T cell clonal expansion in the proliferation phase, but also in generation of CD8+ Tm cells with the capacity of secondary expansion upon antigen re‐encounter. However, IL‐12 signaling is neither involved in CD8+ effector T cell survival in the transition phase nor in the CD8+ Tm cell's secondary expansion in recall responses.
ET‐1 induces cyclooxygenase‐2 expression through MAPKs and NF‐ κB in Osteoblast MC‐3T3 Cells
Authors: Lai Szu‐Chia , Yang Chuen‐Mao
Published: June 2021

Mature bone homeostasis is maintained by bone remodeling cycle involved in osteoblast bone formation and osteoclast bone resorption. Induction of COX‐2 and PGE2 expression through various intracellular signaling pathways by endothelin‐1 (ET‐1) may play an important role in inflammation and directly stimulates osteoblastic activity. Here, the machanisms of MAPKs, AP‐1, and NF‐κB pathways for ET‐1‐induced COX‐2 expression were investigated in MC3T3 osteoblast cells. ET‐1‐induced expression of COX‐2 protein and mRNA was attenuated by the inhibitors of MEK1/2 (PD98059), p38 MAPK (SB202190), JNK (SP600125), NF‐κB (PDTC), and AP‐1 (curcumin). Consistently, ET‐1‐stimulated phosphorylation of p42/p44 MAPK, p38 MAPK, and JNK was also attenuated by PD98059, SB202190 and SP600125, respectively. Pretreatment with BQ123 (an inhibitor of ETA receptor) attenuated ET‐1‐induced COX‐2 expression, indicating the involvement of ETA receptors. The implication of NF‐κB in ET‐1‐mediated responses was supported by translocation of NF‐κB into the nucleus and degradation of IκB‐α stimulated by ET‐1. Taken together, these results suggest that in MC3T3 osteoblast cells, activation of MAPKs and NF‐κB translocation is essential for ET‐1‐induced COX‐2 gene expression. These results provide new insights into the mechanisms by which ET‐1 may promote inflammatory responses and will create chances for the development new therapeutic strategies for bone diseases.
Automated quantification of fibrosis in myocardium
Authors: Mouchaers Koen Thijs Bert , Hadi Awal M. , van der Laarse Willem J. , Jaspers Richard T. , Vonk‐Noordegraaf Anton , Belien Jeroen A. M.
Published: June 2021

Background

Fibrosis can affect many organs and is associated with a variety of pathologies. Current quantification methods are time consuming. We developed a new automated quantification technique for fibrosis in myocardium, and validated this in a rat model for Pulmonary Hypertension (PH).

Methods

PH was induced in Wistar rats by monocrotaline (40 mg/kg). Cryosections of myocardial tissue (5 μm) of PH rats (n=8) and controls (n=5) were stained using Picrosirius red and scanned with a MIRAX slide scanner (Zeiss). From these sections 21 images were taken randomly of each heart. We developed a macro for ImageJ, to automatically determine the amount of fibrosis. As comparison, fibrosis was determined in the same areas using polarisation filters. Stereology served as golden standard to validate both methods.

Results

Fibrosis significantly increased in PH hearts vs. control (0.97±0.14 vs 3.30±0.42 % of total area, p=0.0002). Polarisation quantification confirmed this increase (1.14±0.09 vs 3.06±0.16%, p<0.0001). Both automated and polarisation analysis correlated linearly with stereology (p<0.0001, r2=0.77; p<0.0001, r2=0.69, respectively). A non‐linear relation between automated analysis and polarisation was found (rs=0.76), were polarisation has less discriminative power at higher percentage fibrosis.

Conclusion

Automated quantification of fibrosis is feasible, objective, and time‐efficient.
Stabilization of [beta]‐catenin by cPLA2[alpha]‐mediated activation of PPAR[delta]
Authors: Han Chang , Lim Kyu , Xu Lihong , Li Guiying , Wu Tong
Published: June 2021

Cytosolic phospholipase A2α (cPLA2α) is a rate‐limiting key enzyme that releases arachidonic acid (AA) from membrane phospholipids. This study shows a novel role of cPLA2α for activation of peroxisome proliferator‐activated receptor‐δ (PPARδ) and β‐catenin in the nuclei. Overexpression of cPLA2α in human cholangiocarcinoma cells induced the binding of PPARδ to β‐catenin and increased their association with the TCF/LEF response element. These effects are inhibited by the cPLA2α siRNA and inhibitors as well as by siRNA knockdown of PPARδ. Overexpression of PPARδ or treatment with the selective PPARδ ligand, GW501516, also increased β‐catenin binding to TCF/LEF response element and increased its reporter activity. Addition of AA and GW501516 to nuclear extracts induced a comparable degree of β‐catenin binding to TCF/LEF response element. Furthermore, cPLA2α protein is present in the PPARδ and β‐catenin binding complex, suggesting that PPARδ may direct the import of cPLA2α into the nucleus. Thus the close proximity between cPLA2α and PPARδ provides a unique advantage for their efficient functional coupling in the nucleus, where AA produced by cPLA2α becomes immediately available for PPARδ binding and subsequent β‐catenin activation. These results depict a novel interaction linking cPLA2α, PPARδ and Wnt/β‐catenin signaling pathways in human cells.
Aging and the anti‐allodynic efficacy of combination treatment with opiates and antioxidants
Authors: Crisp Terriann , Giles Jennifer R. , Egli Shannon D. , Henry Matthew K. , Finnerty Edward P.
Published: June 2021

Pharmacological studies were undertaken to investigate age‐related differences in the efficacy of co‐administration of morphine and antioxidants (á‐lipoic acid) to reverse the allodynic symptoms of peripheral nerve injury. The L‐5 spinal nerve was ligated to induce peripheral nerve injury in young and aged rats (2–4 or 26–30 months old, respectively). Calibrated von Frey filaments were used to measure changes in paw withdrawal threshold. Dose‐response curves were generated for morphine sulfate (1, 3 or 10 mg/kg, i.p.) or á‐lipoic acid (30, 100 or 300 mg/kg, i.p.) in young and aged rats prior to and following L‐5 spinal nerve ligation. In subsequent experiments, combination subanalgesic doses of morphine and á‐lipoic acid were administered and the antiallodynic efficacy of these agents was assessed. Results revealed that, in young animals, doses of morphine (1 mg/kg i.p.) or á‐lipoic acid (100 mg/kg i.p.) that were ineffective at reversing tactile‐evoked allodynia when administered alone were highly efficacious at inhibiting the allodynic symptoms of peripheral nerve injury when co‐administered. In the aged cohort of rats, the results were similar. These data support a mediatory role of oxidative metabolites in persistent pain, and reinforce the potential therapeutic usefulness of combination opiate/antioxidant therapy for treating humans with persistent pain of neuropathic origin.
Study on Vasorelaxant Mechanism induced by Extract of Zanthoxylium piperitum Leaves
Authors: Li Xiang , Kang Dae Gill , Lee Jun Kyoung , Choi Deok Ho , Lee An Sook , Lee Ho Sub
Published: June 2021

A methanol extract of Zanthoxylium piperitum (MZP) Leaves relaxed vascular smooth muscle in rats. Therefore, the aim of the present study was to define the mechanism of the MZP‐induced vascular relaxation in rat thoracic aorta. Among the four solvent partitions of MZP, n‐butanol (BZP) and water extract (WZP) of Zanthoxylium piperitum Leaves induced relaxation of the phenylephrine‐precontracted rat aorta in a dose‐dependent manner, respectively, and its effects were both abolished by the removal of functional endothelium. Pretreatment of the endothelium‐intact aortic tissues with NG‐nitro‐L‐arginine methyl ester (L‐NAME) and 1H‐[1,2,4]‐oxadiazole‐[4,3‐α]‐quinoxalin‐1‐one (ODQ) inhibited the relaxation induced by BZP and WZP, respectively. On the other hand, vascular relaxation induced by BZP or WZP were not blocked by several inhibitors including indomethacin, atropine, propranolol, tetraethylammonium (TEA), glibenclamide, verapamil, and diltiazem, respectively. In addition, phenylephrine or epinephrine‐induced aortic contraction was also blocked by treated with BZP and WZP, respectively. These results suggest that BZP and WZP dilate vascular smooth muscle via endothelium‐dependent nitric oxide/cGMP signaling pathway.
Gender and estrogen specificity of the renal vasodilatory effect of nicotine in rats
Authors: El‐Mas Mahmoud M , El‐gowilly Sahar M , Goher Eman Y , Ghazal Abdel‐Rheem M
Published: June 2021

This study investigated whether the renal vasodilatory response to acute nicotine exposure is sexually dimorphic and the role of estrogen in modulating the nicotine effect. Nicotine vasodilation was evaluated in phenylephrine‐preconstricted perfused kidneys of male, proestrus female, ovariectomized (OVX) and estrogen‐replaced OVX (OVXE2) rats. Nicotine infusion (5×10−4 M) produced significantly greater decreases in the renal perfusion pressure (RPP) in proestrus female compared with male kidneys. Inhibition of NOS by NG‐nitro‐L‐arginine abolished nicotine vasodilation in the two genders and brought the RPP of male and female kidneys to similar levels. Surgical (OVX) or physiological (diestrus phase) estrogen depletion almost abolished nicotine vasodilation. Further, estrogen or L‐arginine supplementation of OVX rats largely restored the renal vasodilatory response to nicotine. Blockade of estrogen receptors by tamoxifen attenuated the increase in nicotine vasodilation demonstrated in OVXE2 rats. Together, these findings suggest that the renal vasodilator activity of nicotine is entirely dependent on NOS activation and is more pronounced in female than in male kidneys. Also, estrogen mediates, at least partly, the renal vasodilatory effect of nicotine probably via facilitation of NOS‐NO signaling.

Supported by Faculty of Pharmacy, Univ. of Alexandria, Egypt.
Characterization of cardiac H3 receptor expression and function
Authors: Milicic Ivan , Yao Betty , Polakowski Jim , Campbell Thomas , Fryer Ryan , McDermott Jeff , Martin Ruth , Gintat Gary , Brioni Jorge , Esbenshade Timothy
Published: June 2021

Histamine H3 receptors (H3R) modulate the release of neurotransmitters and are hypothesized to play a role in cognition, sleep, and weight regulation thus providing an attractive drug target. Although expressed primarily in the CNS, it is known that this receptor can modulate neurotransmitter release at peripheral tissues including the heart. Using quantitative RT‐PCR, we demonstrate that H3R mRNA is expressed in canine Purkinje fibers although at a lower level than seen in brain. No H3R mRNA was detected in canine ventricle. Exposing isolated canine Purkinje fibers to increasing concentrations (0.01–1.0μM) of the H3R agonist R‐α‐methylhistamine (RAMH) did not affect the action potential duration determined from electrophysiological recordings of stimulated fibers (2 sec basic cycle length). In in vivo rat studies, i.v. infusion of RAMH (10 mg/kg) produced little changes in mean arterial pressure (MAP), heart rate (HR), cardiac contractility (dP/dt), and peripheral vascular resistance whereas 30 mg/kg RAMH caused large increases in MAP (10%), HR (36%), and dP/dt (62%) relative to vehicle treated rats. In a rat myocardial ischemia/reperfusion model involving occlusion of the main coronary artery, RAMH (3 mg/kg, i.v.) failed to reduce infarct size when infused 10 min prior to and during the duration (3 hr) of reperfusion. These results suggest that although H3R mRNA is present in Purkinje fibers, H3R agonists such as RAMH have no direct modulatory effects on Purkinje fiber function. However, RAMH can induce marked changes in cardiac function at high doses whereas lower doses do not appear to protect against reperfusion injury. Additional studies utilizing more potent and selective H3R agonists may be warranted to further elucidate the role of H3Rs in cardiac function.
Amino acid residue 24 of the human equilibrative nucleoside transporter 1 (ENT1) is important for transport activity
Authors: Zimmerman Eric , Leisewitz Andrea , Huang Min , Graves Lee
Published: June 2021

Nucleoside analogs such as cytarabine (Ara‐C) and gemcitabine are commonly used to treat cancer (leukemia and breast cancer among others). It has been previously shown that a correlation between drug resistance and reduced uptake of these agents is due to decreased expression of a major equilibrative nucleoside transporter, ENT1. [3H]‐Uridine uptake and [3H]‐NBMPR binding revealed that the CEM‐AraC/8C cells, a leukemia cell line resistant to Ara‐C, are devoid of functional ENTs. Sequence analysis for ENT1 gene from CEM‐AraC/8C revealed a missense point mutation in the ENT1 gene sequence that confers a glycine to arginine substitution at residue 24. To determine the importance of G24 to ENT1 transport activity G24R, G24E, and G24A ENT1 mutants were expressed in PK15 cells, an ENT‐deficient cell line. Expression of wt ENT1 produced a time‐dependent increase in [3H]‐uridine uptake whereas cells that expressed G24R ENT1 were devoid of [3H]‐uridine uptake. Expression of G24E and G24A ENT1 led to only partial [3H]‐uridine incorporation, compared to wt ENT1. Furthermore, the expression of wt ENT1 in PK15 cells increased nucleoside analog‐induced cell death whereas a reduced effect was observed during the expression of the ENT1 mutants. This study reveals that CEM‐AraC/8C resistance to Ara‐C may be attributed to the G24R mutation in ENT1 and that this single amino acid is important for ENT1 nucleoside transport activity.
Molecular determinants for substrate selectivity of OATP1B3
Authors: Gui Chunshan , Hagenbuch Bruno
Published: June 2021

Human organic anion transporting polypeptides (OATP) 1B1 and 1B3 are liver‐specific transporters located at the basolateral membrane of hepatocytes and mediate uptake of amphipathic organic compounds from blood into hepatocytes. The amino acid sequence of OATP1B3 is 80% identical to that of OATP1B1 and the two transporters have common as well as distinct substrates. At low concentrations, estrone‐3‐sulfate is preferentially transported by OATP1B1, while cholecystokinin octapeptide (CCK‐8) is a specific substrate for OATP1B3. To determine which domains are important for the substrate selectivity of OATP1B3 we constructed a series of chimeric proteins between OATP1B1 and 1B3. These chimeras were transiently expressed in human embryonic kidney cells (HEK293), uptake of estrone‐3‐sulfate and CCK‐8 was measured and surface expression was determined. Most of the chimeras could be expressed and detected at the cell surface. A first set of chimeras revealed that the substrate recognition/transport site is within the second half of the protein. Using additional chimeras, we found that when OATP1B3 contained transmembrane domain 10 and extracellular loop 5 of OATP1B1 it was not able to efficiently transport CCK‐8. Thus, we conclude that the region around transmembrane domain 10 is crucial for recognition and/or transport of the OATP1B3‐specific substrate CCK‐8.
Ischemia Reperfusion‐Induced microRNAs are Blunted by Ischemic Preconditioning
Authors: Tsutsumi Yasuo M , Panneerselvam Mathivadhani , Roth David M , Patel Hemal H
Published: June 2021

Introduction: MicroRNAs (miRNAs) are endogenously expressed, noncoding, small RNA molecules that regulate protein synthesis via degradation of mRNA or inhibition of translation to modulate cellular physiology. Little is known regarding the expression of miRNAs in the heart in response to ischemia/reperfusion (I/R) injury and how this expression is impacted by an ischemic preconditioning (IPC) stimulus. We hypothesized that a distinct expression of miRNA characterizes cardiac injury in response to I/R and IPC is able to normalize this expression towards basal. Methods: miRNA was isolated from hearts of four groups of C57BL/6 mice (n=4 mice/group): control; 30 min ischemia/24 hr reperfusion; 3–5 IPC‐30minischemia/24 hr reperfusion; 3–5 IPC 24 hr reperfusion. The miRNA was hybridized to the NCode miRNA microarray (Invitrogen) and expression analysis performed. miRNA were ranked in order of most highly induced in MI relative to control. Results: Analysis of miRNA expression showed an induction (>50‐fold) of 33 distinct miRNA in hearts exposed to I/R injury relative to control hearts. An IPC stimulus prior to ischemia/reperfusion resulted in a blunted expression (>40%) of 14 of the miRNA induced by I/R alone. IPC prior to I/R resulted in the induction of 14 miRNAs, 50 % of which were not induced in the I/R injury alone group.

Conclusion: The results suggest that miRNAs are involved in ischemia/reperfusion injury and IPC limits deleterious miRNA expression. Targeted suppression of miRNAs induced by ischemia/reperfusion injury may represent a novel therapeutic target to mimic IPC.
Transcriptional regulation of the human neutral ceramidase promoter
Authors: O’Neill Sean Michael , Kester Mark
Published: June 2021

Promitogenic and proinflammatory metabolites of ceramide, including sphingosine‐1‐phosphate (S‐1‐P), contribute to atherogenic plaque formation. Ceramidases play a critical role in generating S‐1‐P by hydrolyzing ceramide into sphingosine, a substrate for sphingosine kinase. The objective of the present study is to elucidate the transcriptional regulation of human neutral ceramidases (hnCDase) in vascular smooth muscle (VSM) cells. Using human genomic DNA, we cloned a 3000 base pair (bp) region upstream of the translational start site of the hnCDase gene. Luciferase reporter analyses identified a 200 bp essential promoter region with multiple candidate transcriptional response elements (TRE). Electrophoretic mobility shift assays identified several growth factor regulated transcription factors, including AP‐1, AP‐2, and NF‐Y. Mutagenic analyses of the TRE revealed that these sites regulated promoter activity. We demonstrated that PDGFββ, TGFβ, as well as serum induced hnCDase expression in human coronary artery smooth muscle cells, which correlated with elevation of the mass of hnCDase‐dependent metabolites, sphingosine and S‐1‐P. Taken together, our studies indicate that hnCDase is transcriptionally regulated by growth factors that contribute to VSM cell proliferation or inflammation, both hallmarks of atherosclerotic arterial injury. These studies were funded by NIH RO1 HL076789 to MK.