bluuebunny/crossref_metadata_2025 huggingface dataset:
Zinc regulation of Runx2 expression through BMP‐2 signaling in osteoblastic MC3T3‐E1 cells
Authors: Cho Young‐Eun , Shin Hong‐In , Kwun In‐Sook
Published: June 2021

Runx2, a bone‐specific transcription factor, is a key regulator of osteoblastic differentiation. Runx2 expression is induced by BMP‐2 stimulation suggesting that Runx2 is a downstream transcription factor in BMP‐2 signaling. In the present study, we investigated whether Zn deficiency modulates BMP‐2 expression in osteoblastic MC3T3‐E1 cells via activation of Smad1 which is the molecule of BMP‐2 signaling. Two osteoblastic MC3T3‐E1 subclones, subclone 4 and subclone 24 were cultured in OSM (normal osteogenic medium), Zn‐ (1 µM ZnCl2) or Zn+ (15 µM ZnCl2) for 24 h. BMP‐2 mRNA and protein expression and extracellular BMP‐2 secretion decreased in Zn‐ at 24 h, compared to OSM or Zn+ (p<0.05). Downstream regulator of BMP‐2 signaling, Smad1 and p‐Smad1 expression was decreased in Zn‐. Again, the Runx2 mRNA and protein expression and Osterix, the downstream regulator of Runx2, was also decreased in Zn‐, which might be due to de‐creased p‐Smad1 since Runx2 is affected by BMP2 signaling. Bone marker gene expression (ALP, Pro COLI, OC and OPN) were decreased in Zn‐. The study results suggest that Zn deficiency decreases BMP‐2 signaling pathway via decreased Smad1 activation, and then decreased Runx2 expression. The decreased Runx2 expression may cause the decreased bone marker gene expression in osteoblastic MC3T3‐E1 cells. (Supported by KOSEF grant No. R01‐2006‐000‐10640‐0 and KRF grant No. KRF‐2007‐313‐C00810)
Flaxseed combined with ultra low‐dose estrogen therapy preserves bone mass in ovariectomized rats
Authors: Sacco Sandra Maria , Jiang Jessica Ming Yi , Reza‐Lopez Sandra , Ma David WL , Thompson Lilian U , Ward Wendy Elizabeth
Published: June 2021

Flaxseed, rich in phytoestrogens and alpha‐linolenic (ALA) acid, that may modulate bone metabolism, is commonly consumed by postmenopausal women in combination with pharmaceuticals such as estrogen replacement therapy. We determined if a 10% FS diet modulates the effect of ultra‐low dose estrogen therapy (ULD) on bone mineral density (BMD) and biomechanical bone strength, a surrogate measure of fracture risk, using the ovariectomized rat model of postmenopausal osteoporosis. Ovariectomized rats (n=48) were randomized to: i. basal diet (BD, AIN93M); ii. BD+ULD implant; or iii. BD containing 10% FS+ ULD for 12 weeks. A sham‐operated control group was fed BD. BMD and strength properties of the lumbar vertebrae (LV), femurs and tibias were analyzed by dual energy x‐ray absorptiometry and biomechanical strength testing, respectively. Bone fatty acid composition was determined by gas chromatography. Unlike ULD, FS+ULD resulted in greater (p<0.05) LV BMD and strength compared to ovariectomy alone, and higher (p<0.05) n‐3 fatty acid levels and lower n‐6 fatty acids levels in LV and tibias compared to all groups. ALA (r=0.52, p=0.03) and total n‐3 fatty acid (r=0.52, p=0.03) levels in LV were positively correlated with LV strength. In conclusion, FS enhances the effect of ULD at the lumbar spine and these benefits are associated with changes in fatty acid composition.

Grant Funding Source

Supported by NSERC Discovery Grant (W. Ward), NSERC Postgraduate Scholarship (S. Sacco), CONACyT Scholarship (S. Reza‐Lopez).RC
Canine Adipose Tissue Transcriptome Changes Following Four Weeks of Rapid Weight Gain
Authors: Grant Ryan W. , Vester Brittany M. , Ridge Tonya K. , Graves Thomas K. , Swanson Kelly S.
Published: June 2021

Changes that occur in organs and tissues during the early stages of weight gain prior to the onset of clinical obesity remain unclear. The purpose of this study was to characterize adipose tissue transcriptome changes occurring after 4 wk of rapid weight gain and relate these changes to circulating metabolites. Intact female beagles were fed a commercially available high‐fat diet and randomized to either ad libitum feeding (n=5) or weight maintenance (n=4). Adipose tissue biopsies and blood samples were collected at baseline and at 4 wk. Transcriptome changes were assessed using Affymetrix Canine 2.0 microarrays and analyzed using the Limma package with an FDR = 0.05. After 4 wk, BW, body fat mass, and blood leptin and fasting glucose concentrations were elevated (P<0.05) in dogs fed ad libitum, but blood NEFA, triglyceride, insulin, adiponectin, CRP, and TNF‐α concentrations were unchanged. 139 gene transcripts were differentially expressed in adipose tissue following rapid weight gain. The majority of these changes were observed in 5 functional classes: metabolism, apoptosis/cell cycle regulation, cell adhesion, redox state, and transport. These results indicate that major changes associated with cell growth, metabolism, and redox status occur during rapid weight gain and adipose tissue expansion in the absence of systemic low‐grade inflammation. Funding: Illinois CFAR # CF071‐003‐SEN

Grant Funding Source

Illinois CFAR # CF071‐003‐SEN
Botanical extracts from St. John's Wort inhibit adipogenesis and induce insulin resistance
Authors: Amini Zhaleh , Doucet Julie , Ribnicky David M , Stephens Jacqueline M
Published: June 2021

Adipocytes are insulin sensitive cells that play a major role in energy homeostasis. Obesity is the primary disease of fat cells and a major risk factor for the development of Type II diabetes, cardiovascular disease, and metabolic syndrome. Obesity and its related disorders results in dysregulation of the mechanisms that control adipocyte gene expression and function. To identify potential novel therapeutic modulators of adipocytes, we screened 425 botanical extracts for their ability to modulate adipogenesis and insulin sensitivity. We observed that less than 2% of the extracts had substantial effects on adipocyte differentiation. Two of the positive hit botanicals we screened that inhibited adipogenesis were extracts from St. John's Wort (SJW). Our studies revealed that two different SJW extracts inhibited adipogenesis as judged by lipid staining and marker gene expression. We also examined the effects of these SJW extracts on insulin sensitivity in mature adipocytes. One of the SJW extracts substantially inhibited insulin sensitive glucose uptake in a time and dose dependent manner. SJW is commonly used in the treatment of depression. However, our studies have revealed that SJW may have a negative impact on adipocyte related diseases by limiting adipocyte differentiation of preadipocytes and significantly inducing insulin resistance in mature fat cells.
Effects of hamburger patties with bamboo leaf (Sasa borealis) extract or sea tangle (Laminaria japonica) powder on plasma glucose and lipid profiles
Authors: OH HYUN KYUNG , Lim Hyeon‐Sook
Published: June 2021

This study was performed to determine the effects of hamburger patties with water extract of bamboo leaf (Sasa borealis) or powder of dried sea tangle (Laminaria japonica) on plasma glucose and lipid profiles. Three kinds of patties were prepared; one was a control patty (C), S was the patty with 2.5% of the bamboo leaf extract substituted for the meat, and L was the patty with sea tangle substituted for 2.5% of the meat. A total of ten healthy adult women voluntarily participated in the study. Plasma glucose concentrations and lipid profiles were measured at 0, 30, 60, 90, 120, 180, and 240 minutes after consuming each patty. Δ‐AUCs (Change in area under the curve) of plasma glucose concentrations when consuming S or L were significantly lower than that when consuming C. Δ‐AUCs of plasma concentrations of triglyceride and total cholesterol consuming L were significantly less than those when consuming C or S, respectively. However, Δ‐AUCs of plasma concentrations of HDL‐ and LDL‐cholesterol were not significantly different among the three patties. These results indicate that the patties with 2.5% of the meat with water extract of the bamboo leaf or powder of dried sea tangle may improve blood glucose and, in addition, the patty with sea tangle powder may also improve plasma lipid profiles.
Increased Anaphylatoxin (C5a) and Bradykinin Levels in Patients with End‐Stage Renal Disease on Maintenance Hemodialysis: Potential Relevance to Heparin Mediated Hemodynamic Responses
Authors: Adiguzal Cafer , Bansal Vinod , Cunanan Josephine , Litinas Evangelos , Hoppensteadt Debra , Fareed Jawed
Published: June 2021

In addition to upregulation of inflammatory mediators, patients on hemodialysis due to end stage renal disease (ESRD) are periodically exposed to heparin and contact activation due to procedural settings. Recently the presence of oversulfated chondroitin sulfate in heparin was linked with adverse reactions and deaths in these patients. We measured C5a anaphylatoxin and bradykinin levels in ESRD patients prior to and after maintenance hemodialysis and in a control group of 40 normal healthy individuals. C5a and bradykinin levels were elevated in pre‐dialysis samples (C5a: 14.2± 4.6 vs. 3.2±0.6 ng/ml, bradykinin: 9.3±2.4 vs. 4±1.8 ng/ml). Dialysis itself increased both C5a and bradykinin levels. The postdialysis samples were further increased, suggesting that dialysis and heparinization result in the up‐regulation of these mediators. Supplementation of heparin, contaminated heparin or isolated contaminant to the plasma also resulted in the generation of C5a and bradykinin. The plasma samples included in these studies were obtained from patients who were not treated with contaminated heparin. These results suggest that C5a and bradykinin are up‐regulated in ESRD patients and this level can be further augmented by dialysis and heparinization. Therefore, additional factors may have contributed to the complex adverse reaction profiles and deaths in patients administrated contaminated heparin.
Interferon‐gamma inhibits hypoxia‐inducible factor (HIF) in intestinal epithelial cells through transcriptional repression of HIF‐1 beta
Authors: Glover Louise Elaine , Irizarry Karina , MacManus Christopher , Scully Melanie , Colgan Sean
Published: June 2021

Hypoxia and inflammation occur coincidentally in mucosal diseases such as inflammatory bowel disease (IBD). Hypoxia inducible factor (HIF) has been implicated as a regulatory pathway under such conditions. HIF is a transcription factor critical to adaptation to low oxygen states and several studies have indicated that intestinal epithelial HIF provides protection in murine models of colitis. We therefore hypothesized that inflammation‐relevant mediators may influence intestinal epithelial expression and function of HIF.

Guided by an initial screen of relevant inflammatory mediators, we analyzed the influence of IFN‐γ on HIF expression and transcriptional activity in intestinal epithelial cells. Epithelial exposure to IFN‐γ resulted in a concentration‐dependent repression of HIF activity (p<0.025) that was reversible in the presence of transcriptional inhibition. A real‐time PCR screen revealed prominent inhibition of HIF‐1β, but not HIF‐1α expression, by IFN‐γ. Analysis of the human HIF‐1β promoter confirmed the inhibitory influence of IFN‐γ on HIF‐1β activity. Taken together, these studies demonstrate that HIF activity and expression in intestinal epithelial cells is specifically repressed by IFN‐γ through a mechanism involving transcriptional repression of the HIF‐1β promoter. These observations may be relevant to the pathophysiology of colitis. (NIH, USA)
HSV‐2 infection induces NF‐kB‐dependent cytokines in human cervical epithelial cells
Authors: Li Hui , Li Xiaoling , Wei Yun , Tan Yun , Wu Xinxing
Published: June 2021

Herpes Simplex Virus type 2 (HSV‐2) is one of the most common sexually transmitted pathogen and can establish lifelong latency infection. Our previously studies have shown that HSV‐2 infection up‐regulates TLRs expression and activates NF‐kB signaling in human cervical epithelia (HCE) cells. In this study, we examined the cytokines production of HCE cells in response to HSV‐2. HCE cells were infected with HSV‐2 at 3 MOI and cultured for the certain period of times. The result showed that the secretion of IL‐6 and IFN‐beta increased dramatically after HSV‐2 infection and maintained high levels during the course of experiment . We asked whether or not the induction of cytokines requires NF‐¿B activity. To this end, we treated cells with Isohelenin and harvested the supernatants for ELISA assay. The presence of NF‐¿B inhibitor, Isohelenin significantly blocked the production of both IL‐6 and IFN‐beta in response to HSV‐2 compared to the untreated control. Thus, our data provide the direct evidence that the production of both IL‐6 and IFN‐beta induced by HSV‐2 in HCE cells was NF‐¿B dependent suggesting a critical role of NF‐¿B as a key regulator of cytokine production in response to HSV‐2 infection in its natural host cells.

This work was supported by grant from the National Natural Science Foundation of China (No. 30500465, No. 30810103052)
Gender Differences in Estrogen Regulation of Intrahepatic Biliary Epithelial Cell IL‐6 Expression
Authors: Isse Kumiko , Kang Liang‐I , Specht Susan M , Lunz John G , Mizuguchi Yoshiaki , Demetris Anthony Jake
Published: June 2021

Females are significantly more susceptible to primary biliary cirrhosis and debilitating consequences of adult polycystic liver disease than males. Also, biliary strictures contribute to poor outcome of female‐to‐male liver transplant compared to other donor‐recipient sex combinations. Since IL‐6 is critically important in biliary epithelial barrier function and wound repair and estrogens regulate IL‐6 expression, we tested the hypothesis that estrogens differentially regulate biliary epithelial cell (BEC) IL‐6 production in males and females. Results show that estradiol treatment either inhibited or had no effect on male mBEC IL‐6 expression but increased female IL‐6 expression. Male mBEC expressed less estrogen receptor‐alpha (ERα) protein than female mBEC but had similar levels of estrogen receptor‐beta (ER β). Female mBEC were also more dependent on estradiol for robust survival. Male mBEC, however, in an estrogen‐rich environment, upregulated ERα and then became estrogen‐responsive. In vivo studies show that biliary epithelia from female mice in estrus expressed IL‐6, but anestrus female mice did not. Experiments in two male human cholangiocarcinoma cell lines showed that signaling through ERα, and not the underlying sex, was responsible for the differential regulation of IL‐6 by estrogen. In conclusion, female BEC are estrogen responsive and dependent because of higher levels of ERα. Male BEC can adapt to an estrogen‐rich environment by upregulating ERα. Since estrogens can influence BEC barrier functions and wound healing both directly and indirectly via IL‐6 signalling, the differential regulation of BEC IL‐6 in males and females likely contribute significantly to sex differences seen in biliary diseases and cross‐sex liver transplants.
Cardiac ischemic/reperfusion injury in diabetic rats treated with a superoxide dismutase mimetic
Authors: Schwartz Dean D , Carson Melissa , Rogers Colin B , Judd Robert L , Plaisance Eric P
Published: June 2021

The diabetic heart is at an increased risk for myocardial infarction. The aim of this study was to examine the effect of the superoxide dismutase mimetic EUK‐134 on the susceptibility of the diabetic heart to ischemic/reperfusion injury. Rats were injected with streptozotocin (STZ) or its vehicle (Control) and implanted with an osmotic minipump containing the SOD mimetic EUK‐134 or its vehicle. After 4 weeks, in vivo left ventricular (LV) pressure and contractility were similar in all four groups. Hearts were then isolated and perfused in the Langendorf mode for the measurement of LV pressure and contractility before and after a 30 min global ischemia. Baseline LV pressure was similar in all groups. Following 30 min of ischemia, LV pressure was significantly reduced in the control rats treated with vehicle whereas hearts from STZ rats treated with vehicle or EUK‐134 and control rats treated with EUK‐134 all recovered more quickly and regained normal mechanical function following ischemia/reperfusion. Heart lipid peroxidation was also increased in the vehicle‐treated control rats compared to the other groups. These results suggest that hyperglycemia associated with STZ diabetes and/or treatment with the SOD mimetic EUK‐134 reduce cardiac lipid peroxidation and enhance cardiac mechanical activity after ischemia/reperfusion.
RhoA links PI3K/Akt/mTOR signaling to p38 MAPK/GATA‐4 activation in leptin–induced cardiomyocyte hypertrophy
Authors: Zeidan Asad , Hunter Craig , Javadov Sabzali , Karmazyn Morris
Published: June 2021

Obesity is associated with increased leptin production which may contribute to cardiac hypertrophy. However, the mechanism of leptin‐induced cardiac hypertrophy remains incompletely understood. The Rho family and mTOR have recently emerged as important regulators of cell growth. In this study we explored the roles of phosphatidylinositol 3‐kinase (PI3K), mTOR and the Rho family in the regulation of actin polymerization in leptin‐induced hypertrophy in cultured neonatal rat cardiomyocytes treated with 3.1 nM leptin. Leptin treatment resulted in activation of RhoA and Rac1 (by 330% and 160%, respectively; P<0.05) but not cdc42, 5 min after treatment, as determined by Western blotting. The hypertrophic effect of leptin was associated with an increase in phosphorylation of p70S6K by 110% (P<0.05). The specific mTOR inhibitor, rapamycin (10 nM) attenuated leptin‐induced RhoA and Rac1 activation. Furthermore, the decrease in the G/F actin ratio, a measure of actin polymerization, was blunted by rapamycin. Leptin produced activation of the transcriptional factor GATA‐4 which was attenuated by the RhoA inhibitor C3, the p38 MAPK inhibitor SB203580 (10 µM) or rapamycin. Our results demonstrate that leptin‐induced cardiomyocyte hypertrophy is associated with GATA‐4 activation which is dependent on RhoA, PI3K/mTOR/p70S6K and p38 MAPK pathways. Supported by the Canadian Institutes of Health Research.
Metabolic and structural remodeling of heart‐derived H9c2 cells by AMPK activation
Authors: Dai Jiazhen Minnie , Saeedi Ramesh , Sharma Vijay , Saran Varun , Parsons Hannah , Dyck Jason , Allard Michael
Published: June 2021

AMP‐activated protein kinase (AMPK), a kinase involved in control of cellular metabolism, inhibits pathologic cardiac hypertrophy development but also plays a role in skeletal muscle adaptation to exercise. We tested the hypothesis that AMPK activation per se results in structural and metabolic remodeling of heart muscle cells characteristic of that seen in physiologic cardiac hypertrophy. Adenoviral‐mediated over expression of a constitutively active form of AMPK (CA‐AMPK) was used to increase AMPK activity in H9c2 cells, a cell line from embryonic rat heart ventricle. Then, indices of cellular hypertrophy and substrate use were measured 48 hrs later. AMPK activity was ~40‐50% higher in CA‐AMPK treated cells than in untreated cells. This was accompanied by a ~20‐25% increase in total protein content and phenylalanine incorporation without a measurable increase in atrial natriuretc peptide expression. CA‐AMPK stimulated fatty acid oxidation and glycolysis, but did not significantly alter glucose oxidation. Thus, AMPK activation causes structural and metabolic remodeling in heart muscle cells different from that seen in pathologic settings, indicating AMPK activation alone leads to a physiologic form of hypertrophy in heart muscle cells and may alter the reciprocal relationship between fatty acid and glucose utilization.

This research project is funded by Canadian Institute of Health Research.
Evaluation of Medicinal Herbs for Antidiabetic Activity
Authors: Obih Patience Ogechi , Bennett Lunawati L , Burnett Derrick D , Thomas Rodney B , Onor Ifeanyi O
Published: June 2021

Medicinal plants and natural products have contributed immensely to the development of treatment strategies for various types of diseases. The study reported here is the screening of local herbs for antidiabetic activity. The incidence of diabetes mellitus is increasing in alarming rate around the world. Diabetes mellitus is a major cause of morbidity and mortality. There is dire need for production of more potent drugs for the treatment of diabetes. This project is therefore directed to study the effect of extracts of medicinal herbs on hyperglycemia. Water Extracts of medicinal herbs Vernona amygdalina, Allium sativum, and Thymus vulgaries were administered to streptozotocin‐induced diabetic rats (STZ‐D). Sprague Dawley rats weighing 250‐300 gm were made diabetic by injecting them with 55 mg/kg streptozotocin intraperitoneally. Animals with blood glucose level of 250 mg/dL and above, received extracts of these herbs via the oral route. At intervals of 1 hr, 2hr, 3 hr after extract administration, measurement of blood glucose was repeated using glucometer. The water extract of Allium sativum did not reduce the rat hyperglycemia significantly. Thymus vulgaries appears to slightly increase hyperglycemia 1‐2 hr after administration. Vernon amygdalina showed some activity. Further work will test the alcoholic and chloroform extracts. This work will not only boost research in diabetes but will also provide valuable information needed for effective management of diabetes mellitus.
Effect and mechanism of auricularia auriculajudae polysaccharide in hippocampus of streptozotocin‐induced diabetic cognitive impairment rats
Authors: Fei Jiaqian , Fan Ying , Zhang Xiaoming , Ye Tingmei , Chen Yingying , Shen Yueliang , Wang Linlin
Published: June 2021

Aim

To investigate the effect and mechanism of auricularia auriculajudae polysaccharide(APP) in the hippocampus of streptozotocin‐induced diabetic cognitive impairment rats.

Methods

APP group was treated with intragastric administration of APP (150mg/day) for 4 weeks after intraperitoneal streptozotocin(STZ) injection (60 mg/kg). Their capacity for learning and memory were detected by the Morris water maze. Microstructural and ultrastructural changes were also analysed by transmission electron microscope (TEM). Thrombospondin‐1 (TSP‐1) expression in the CA1 and dentate gyrus (DG) hippocampal area was examined by immunohistochemistry and RT‐PCR.

Results

Four weeks after STZ induction, the diabetic rats showed decreased capacity for learning and memory. However, the cognitive impairment was significantly less severe in APP group as indicated by decrease in the time spent in the target quadrant (P < 0.05) and the number of crossings of the previous location of the platform (P < 0.05). TEM also revealed that APP group had less swollen of mitochondria, decreased heterochromatin accumulation and increased synaptic contacts when compared with the diabetic group. Morever, TSP‐1 expressions in the CA1 and DG were significantly lower in APP group than in diabetic group.

Conclusion

APP could alleviate the impairment of memory and cognition in diabetic rat, which may be mediated by decreasing the expression of TSP‐1 in the hippocampus.
Influence of the state of shosphorylation on the interaction of anthracene‐9‐carboxylic acid with Ca2+‐activated Cl‐ channels in pulmonary artery myocytes
Authors: Wiwchar Michael , Huebner Marissa , Greenwood Ian A. , Leblanc Normand
Published: June 2021

Ca2+‐dependent Cl− currents (IClCa) are down regulated by phosphorylation in arterial smooth muscle cells. We recently showed that niflumic acid (NFA), an inhibitor of IClCa, is less efficacious at blocking the current in conditions promoting phosphorylation. This study aimed to assess whether another Cl− channel blocker, anthracene‐9‐carboxylic acid (A9C), is also affected by channel phosphorylation. A9C blocks IClCa at positive potentials but paradoxically stimulates the inward IClCa tail after repolarization to negative potentials. IClCa was evoked by pipette solutions containing 500 nM free Ca2+ with or without 5 mM ATP to alter the state of phosphorylation. A9C (10‐500 μM) produced voltage‐dependent block of IClCa that was more potent in cells dialyzed with ATP vs. 0 ATP. A9C enhanced IClCa tail at ‐80 mV by causing a large negative shift in voltage‐dependence in both cell groups. Finally, phosphorylation greatly attenuated the rate of recovery of IClCa at +80 mV upon washout of A9C, but not IClCa tail at ‐80 mV. As for NFA, the complex actions of A9C with IClCa are profoundly influenced by the state of channel phosphorylation and we propose the existence of multiple binding sites for A9C.
PACS‐1 Mediates Phosphorylation‐Dependent Ciliary Trafficking of the CNG Channel in Olfactory Sensory Neurons
Authors: Jenkins Paul Michael , Zhang Lian , Thomas Gary , Martens Jeffrey R.
Published: June 2021

Ciliopathies are an emerging class of human disorders that involve defects in ciliary protein trafficking or assembly. Although impaired ciliary protein transport in olfactory sensory neurons (OSNs) leads to anosmia, surprisingly little is known regarding the mechanisms controlling this transport. Here, we show a role for phosphofurin acidic cluster sorting protein 1 (PACS‐1) in the ciliary trafficking of the olfactory CNG channel. PACS‐1 is an intracellular sorting protein that mediates its effects through the binding of acidic clusters on cargo protein. This interaction is dependent on CK2 phosphorylation of both PACS‐1 and its cargo. Sequence analysis reveals that CNGB1b contains two putative PACS‐1 binding sites, while in vitro kinase reactions confirm that CNGB1b is a CK2 substrate. Additionally, we show that PACS‐1 is expressed in OSNs and interact in complex with the CNG channel. In a model system, we demonstrate that alterations in PACS‐1 function results in deficits in CNG channel ciliary trafficking. Importantly, inhibition of CK2 in native OSNs causes a loss of CNG channel from cilia and subsequent olfactory dysfunction. These results provide a mechanism for the subunit‐dependent ciliary trafficking of the CNG channel and provide further insight into the mechanisms of ciliary transport. This work supported by NIH grants DC009606 (JRM), DK37274, AI49793 (GT), GM007767, DC00011, and DC009524 (PMJ).
differential regulation of P2X7R activation by extracellular NAD in murine macrophages and T cells
Authors: Hong Shiyuan , Brass Anette , Haag Friedrich , Koch‐Nolte Friedrich , Dubyak George
Published: June 2021

The ATP‐gated P2X7 receptor (P2X7R) plays important roles in pro‐inflammatory responses. Recently, extracellular NAD has been shown to induce activation of P2X7R in murine T cells via ADP‐ribosylation involving an ADP‐ribosyltransferase ART2.2 that is constitutively expressed on murine T‐cell surfaces. We have recently reported that ART2.1, functionally and structurally similar to ART2.2, is markedly up‐regulated by pro‐inflammatory activation of murine macrophages which constitutively express high levels of the P2X7R. Here, we showed that extracellular NAD acts to covalently modify the P2X7R in both macrophages and T cells but with distinct functional consequences on the gating of channel activity. We monitored P2X7R channel activity by fura2‐Ca2+ influx assays and P2X7‐mediated pore formation by ethidium influx assays. Co‐expression of P2X7R with ART2.1 or ART2.2 in HEK293 cells verified that the P2X7R is a substrate for ART2.1, as well as ART2.2. In contrast with T cells, the ART2‐mediated ADP‐ribosylation of P2X7R in macrophages (or HEK293 cells) is not sufficient to gate the P2X7R. However, this allosteric modification of the macrophage P2X7R potentiates the ability of ATP to gate the channel as indicated by a left‐shift in the ATP dose‐response relationship. This supports a different role for extracellular NAD in the regulation of P2X7R‐dependent inflammatory responses of different cell types.
Posttranscriptional regulation of kappa opioid receptor by RNA‐binding protein Grb7
Authors: Tsai Nien‐Pei , Wei Li‐Na
Published: June 2021

The expression of opioid receptor is responsible for the binding of opioid drugs or endogenous ligands. While studies of opioid receptor gene transcription have been conducted extensively, much less is known about the regulation of receptor protein expression at posttranscriptional level. In this study, we identified a novel RNA‐binding protein, growth factor receptor bound‐protein 7 (Grb7), that binds to a specific stem loop of 5' untranslated region (5'UTR) of kappa opioid receptor (KOR) transcript. Upon binding to the KOR transcript, Grb7 stimulates its nuclear export through the Exportin‐1 pathway that requires an adaptor protein Hu antigen R (HuR). Grb7 represses kor translation by blocking its mRNA 5'end recognition by the pre‐initiation complex subunit eIF4E. Upon stimulation by growth factor or cytokine, such as neuron axon guidance factor netrin‐1 or epidermal growth factor EGF, Grb7 is phosphorylated at tyrosine residues 483 and 495 located in its carboxyl terminus by focal adhesion kinase (FAK). This release Grb7 from kor mRNA and the Grb7‐free KOR mRNA can then be actively translated. Our findings reveal a novel growth factor signaling pathway that stimulates rapid expression of KOR protein, mediated by the action of a newly identified RNA‐binding protein Grb7. This work was supported by DA11190, DA11806, DK54733, DK60521, K02‐DA13926 to L.‐N. W.
Induced deficit in hippocampal‐dependent spatial memory development of pups from lithium treated pregnant rats
Authors: Dyer Rayon Martino , Gossell‐Williams Maxine , Young Lauriann
Published: June 2021

Lithium carbonate is routinely used in the treatment of patients suffering from bipolar disorder, but can decrease the availability of phosphatidylcholine and choline. Both nutrients are important during pregnancy and are required for foetal brain development. This study aims to assess the effect on memory development of pups from lithium treated dams (LTD). Pregnant Sprague‐Dawley rats were administered water (control) or lithium carbonate 30mg/kg/day by oral gavage throughout the course of pregnancy until birth and access to food and water ad libitum. Pups were given access to food and water ad libitum. After 4 months, offspring were tested in the cued and spatial version of the Morris water maze task, and their rate of acquisition and retention were compared. Pups from the LTD exhibited a cognitive deficit in hippocampal‐dependent spatial memory, however no significant difference was seen in the rate of acquisition of cued task. It can therefore be concluded that lithium therapy during pregnancy can impair the memory development in offspring, an effect that may be related to choline and phosphatidlycholine supply.

Source of research support: Graduate Studies & Research U.W.I Mona.
Chronic constriction sciatic nerve injury in mice delays acquisition, impairs retention, and alters motivation in autoshaping and progressive ratio tasks
Authors: Foley John J , Ward Sara Jane , Gordiienko Oleg , Radzievsky Alexander , Walker Ellen A
Published: June 2021

Persistent pain can produce long‐lasting changes in both physical and psychological functioning in patients. In the present study, we assessed the impact of a prior history of neuropathic pain on subsequent learning and reward processing in mice to further understand the role of past pain states in normal functioning. Under anesthesia, mice (N=19) received two ligatures tied loosely around the right common sciatic nerve and were allowed approximately 20‐30 days to recover. The neuropathic (N=19) and control (N=19) mice learned to respond for a 50:50 (v:v) water:Ensure® solution predicted by an audible tone during two consecutive daily sessions in an autoshaping procedure. After the autoshaping procedure, the mice were trained in a progressive ratio procedure and tested with various concentration of Ensure solution (50, 10, 3, and 0%) over a 4‐8 week period. Neuropathic mice responded with longer latencies and more errors than control mice although the rates of responding were not different between the two groups in the autoshaping procedure. Despite delayed acquisition and impaired retention in the autoshaping procedure, the neuropathic mice responded with higher breakpoint values than control mice for 10% and 3% Ensure solutions, indicating an increase in the reinforcing efficacy of Ensure in the neuropathic mice. Taken together, these data suggest neuropathic pain can modulate learning and reinforcement processes for weeks after surgery.
Effects of aripiprazole and (‐)NPA, dopamine D2‐like receptor agonists of varying intrinsic efficacy, on cocaine vs. food choice in monkeys
Authors: Czoty Paul W. , Coller Michael , Icenhower Michelle , Nader Michael A.
Published: June 2021

Partial agonists of dopamine D2‐like receptors have been suggested as treatments for substance abuse. We examined effects of D2‐like receptor agonists on the relative reinforcing strength of cocaine using a food‐drug choice procedure in male cynomolgus monkeys. In daily self‐administration sessions, monkeys chose between food pellets and injections of cocaine (0.003‐0.1 mg/kg). The available cocaine dose increased during the session, so that a complete dose‐effect curve was generated each day. The high‐efficacy D2‐like agonist (‐)NPA (0.001‐0.0056 mg/kg) had biphasic effects on cocaine choice; a lower dose shifted the curve down whereas a higher dose shifted it leftward. In contrast, the low‐efficacy D2‐like partial agonist aripiprazole (ARI; 0.01‐0.1 mg/kg) increased choice of low cocaine doses and decreased choice of higher cocaine doses, flattening the dose‐effect curve. Moreover, decreases in cocaine intake were larger after ARI treatment compared to (‐)NPA. Effects were observed below doses that significantly decreased the number of food pellets and total reinforcers earned. The results suggest that ARI and (‐)NPA, whose intrinsic efficacies differ in vitro, produce qualitatively different effects on the reinforcing strength of cocaine, and support the view that low‐efficacy D2‐like receptor partial agonists may be effective pharmacotherapies for cocaine abuse. Support: DA12460, DA10584.
The role of injection cues in the effects of morphine preexposure on morphine‐induced conditioned taste aversions (CTAs)
Authors: Davis Catherine Marie , de Brugada Isabel , Riley Anthony L
Published: June 2021

Given that the attenuating effects of LiCl preexposure on LiCl‐induced taste aversions have been reported to be controlled associatively, via injection‐related cues, the present experiment examined the effects of morphine exposure on morphine‐induced taste aversions to assess the generality of this control. Specifically, in Experiment 1 rats were exposed to morphine every other day for five exposures prior to aversion conditioning in which saccharin was paired with morphine (5 mg/kg) for five trials. Between preexposure and conditioning, rats were either injected with saline for eight consecutive days (extinction) or received no treatment. Morphine exposure attenuated the morphine CTA, an attenuation unaffected by saline injections (i.e., extinction of the morphine‐associated cues). Experiment 2 showed that increasing the number of extinction injections to 16 had no effect on morphine preexposure, although it was somewhat weakened in all preexposed groups due to the increased time between preexposure and conditioning. Unlike with LiCl, the attenuating effects of morphine preexposure in aversion learning are most likely controlled by nonassociative mechanisms, such as drug tolerance. Supported by a grant from the Mellon Foundation to ALR.
Dynamics of structural adaptation after vascular occlusion.
Authors: Styp‐Rekowska Beata , Secomb Timothy W. , Pries Axel R.
Published: June 2021

Vascular occlusion results in substantial change in the hemodynamic stimuli. While flow is stopped in the occluded vessel, collateral vessels exhibit elevated flow. It is known that these changes lead to long term changes in vessel structure including vessel diameter and vessel wall thickness. To analyze the dynamics of these processes for vessels in the microcirculation, an intravital microscopy approach in the mouse skinfold chamber was used. Microvascular occlusion was carried out by laser coagulation, and vessels were observed at high magnification intermittently for 10 days. Flow velocity, intravascular oxygen saturation, luminal diameter and wall thickness were measured. The data obtained were analyzed using a previously described computer model (Pries et al., Hypertension 2005). This combined approach allows the investigation of time courses of the relevant stimuli and vascular reactions. It also shows that specific requirements with respect to vascular response characteristics exist in order to avoid unstable responses.
Microvascular NG2 expression patterns in response to aging, ischemic injury, and disease in mouse spinotrapezius muscle
Authors: Wheat Roy , Wapole Joseph , Mac Gabhann Feilim , Bailey Alexander M. , Glaw Jason , Murfee Walter L. , Peirce Shayn M.
Published: June 2021

In unstimulated mature microvascular networks, NG2 expression is limited to perivascular cells located in the arterial tree (e.g. arterial vascular smooth muscle cells) and along capillaries (e.g. pericytes). NG2 expression according to immunohistochemistry is notably absent in the perivascular cells present in the venule tree. During microvascular remodeling in response to an inflammatory or hypoxic stimulus, however, the NG2 proteoglycan is transiently expressed by perivascular cells (e.g. smooth muscle cells) in venules. Expression of NG2 by perivascular cells in venules spatially and temporally coincides with an abundance of angiogenic capillary sprouting from venules in the rat mesentery, for example. The objective of this study is to determine, using immunohistochemistry, the spatial and temporal expression profile of NG2 in the microvasculature of the murine spinotrapezius muscle, a thin stabilizing muscle that enables en face visualization of entire microvascular networks, in young vs. old mice, before and after ischemic insult, and in healthy vs. obese (ob/ob) and diabetic (db/db) mice. Results suggest that NG2 exhibits a differential expression pattern in the perivascular cells of arterial vs. venous trees in the quiescent spinotrapezius muscle, and ischemic insult induces NG2 expression by smooth muscle cells along remodeling capillaries. Support provided by: NIH‐HL082838‐02.
Biologic Nanoparticles Calcify and Form Bacteria‐like Biofilm In Vitro
Authors: Hunter Larry W , Lieske John C , Miller Virginia M
Published: June 2021

Biologic nanoparticles (NPs) isolated from human atheromas propagate and form an apatite shell when placed in cell‐culture conditions. These characteristics led us to hypothesize that NPs might derive from microorganisms. NPs isolated from human aortic aneurysms and maintained in DMEM, 10% fetal bovine serum at 37oC increased both in density (measured in nephelometric turbidity units, NTU) from 1.6 to 107.1 and in diameter (from < 0.2 μm to 0.5‐1 μm) over the course of 12 days. Crystals present on the surface of NPs were found by electron microscopy/elemental analysis to be composed of Ca2+ and Pi. NPs adhered to the surface of the culture flasks, forming a biofilm strikingly similar to that seen in microbial communities. Confocal laser scanning microscopy showed that the biofilm stained positively with concanavalin‐A, a probe for mannose and glucose, both major constituents of microbial biofilm matrix exopolysaccharides. Further, the biofilm stained positively by BacLight Green, a bacteria‐specific cell wall label, as well as the lipid membrane‐selective dye Vybrant CM‐DiI. These results reveal that calcifying NPs isolated from human atheromas share characteristics common to bacteria since they propagate when placed in cell culture conditions, form a biofilm containing polysaccharides, and stain with agents that recognize bacterial components. Supported by grants from the Fetzer Foundation and NIH HL88988.
Role of AT1‐receptors in hypercholesterolemia‐induced thrombosis.
Authors: Russell Janice M , Granger D. Neil
Published: June 2021

Hypercholesterolemia is associated with increased platelet aggregation and increased risk for thrombosis. Previous reports have described the ability of diet‐induced hypercholesterolemia (HC) to enhance thromboembolism in arterioles, but not venules, in rabbit mesentery (Broeders; ATBV 22:680, 2002). The objectives of this study were to determine whether: 1) HC promotes thrombus formation in microvessels of mouse cremaster muscle and 2) angiotensin II type‐1 receptors (AT1‐R) mediate the HC‐induced responses. Intravital videomicroscopy was used to monitor thrombus formation induced with a light/dye endothelial injury model in mice placed on either a normal diet (ND), 2 wks high‐cholesterol diet (HCD) or HCD mice treated with the AT1‐R antagonist, losartan (25mg/kg/day) in drinking water for 7 days. Arterioles in HCD mice exhibited an enhanced rate of thrombus formation compared to ND mice, decreasing the time of thrombus onset and the time to flow cessation. As previously reported, light/dye‐induced thrombosis was not altered in venules of HCD mice. The HCD‐enhanced thrombosis in arterioles was not altered by losartan treatment. These findings confirm the vulnerability of arterioles to HC‐induced thrombosis, and indicate that mechanisms other than AT1‐R activation underlie the increased risk for thrombosis during hypercholesterolemia. (Supported by HL26441).
Microthrombi and hydraulic conductivity increase induced by laser/dye treatment
Authors: Fu Bingmei , Liu Qin , Zeng Min
Published: June 2021

To quantify microthrombi induced by photodynamic therapy, we measured thrombus initiation time, growth rate and microvessel occlusion time under laser/dye treatment. Rats (SD, 250‐300g) were anesthetized and kept warm on a heating pad. A midline incision (~2 inch) was made in the abdominal wall; the mesentery was gently taken out and arranged on a glass cover slip. Sodium fluorescein (NaF, 50 mg/kg in 1% BSA mammalian Ringer, ~0.5mg/ml in individual microvessels) was injected through the carotid artery simultaneously when the mesentery was exposed to a laser (495 nm, 0.38mW/mm2). Thrombi were initiated in microvessels (post‐capillary venules, n = 8) in 3.8 (±0.4 SE) min after laser/dye treatment; the mean thrombus growth rate was 0.038 vessel plane area/min; vessels were fully occluded in 25.5 (±2.2 SE) min. No flow was restored after 30 min laser off. We then tested the hypothesis that laser/NaF induced thrombosis was partially due to the increased hydraulic conductivity (Lp) by measuring Lp of a single microvessel under the same laser/dye treatment as for thrombosis. In ~5 min, Lp increased from a mean of 1.02 (±0.02 SE) to 2.12 (±0.25 SE, n = 13) × 10−7 cm/s/cmH2O (p = 0.002), no further increase after longer time exposure (up to 30 min). After laser was off for ~5min, Lp insignificantly decreased to 1.59 (±0.02 SE) ×10−7 cm/s/cmH2O (p = 0.07), no further decrease after 30 min off. The thrombosis is consistent with Lp increase.
Blockade of Pancreatic Digestive Proteases in Severe Hemorrhagic Shock Enhances Long‐term Survival Rate
Authors: DeLano Frank A , Schmid‐Schönbein Geert W
Published: June 2021

Our evidence suggests that the inflammation encountered in physiological shock may involve pancreatic digestive enzymes in the intestine. Once digestive enzymes breach the mucosal barrier they generate inflammation and organ dysfunction. Blockade of digestive enzymes in the intestine with protease inhibitors reduces inflammation. But no long‐term studies have tested the utility of pancreatic protease blockade as a measure to reduce multi‐organ failure and enhance survival. We exposed mature rats to acute hemorrhagic shock (30 mmHg mean arterial pressure for 120 min) by hemorrhage from the femoral artery followed by an observation period of two weeks until complete recovery and normal whole weight gain. Blockade of the digestive serine proteases after 60 min of hypotension in the lumen of the small intestine (tranexamic acid, 0.3 mM, in polyethylene glycol‐3350, 60 g/L) from the duodenum to the cecum by injection of equal aliquots directly into the intestinal lumen (~every 5cm with a total of 15 ml solution) lead to increase in survival rate (7/7) as compared without enzyme blockade (2/7; P<0.01). The treated but no untreated rat had low levels of tissue damage in several organs. These results showed a significant protection against multi‐organ failure by intraluminal blockade of digestive enzymes in the small intestine after severe hypotension.

(Supported by an unrestricted gift from Leading Ventures)
Impaired micro vascular function and prolongation of recovery time post‐ occlusion observed in diabetic non‐human primates
Authors: Tigno Xenia , Shamekh Rania , Hansen Barbara C
Published: June 2021

Microvascular dysfunction has been implicated as the underlying mechanism for many of the complications of diabetes. The post‐occlusive hyperemic response was studied in 24 monkeys (10 Normal (N), 7 Prediabetic (P) and 7 Diabetic (D)) after a standard occlusion of 2 minutes and measured using a Perimed Laser Doppler flowmeter. Mean fasting glucose levels were N: 65 ±14, P: 65 ± 17 and DM: 257± 17mg/dl. Mean HbA1C values were N: 4.35 ± 0.4, P: 4.60 ± 0.5 and DM: 9.4 ± 0.5%. Prediabetes in monkeys was defined as IGT, and the presence of hyperinsulinemia, hypertension, increased adiposity and dyslipidemia (3 of 5). In diabetic monkeys, blood flow remained elevated for a prolonged period after release of occlusion, whereas in control monkeys, the hyperemic response was very brief. Because of the prolonged recovery time, the area of hyperemia, a measure of the post‐occlusive hyperemic response, was significantly elevated among diabetic monkeys 3909 ±1184 (p=0.05) compared to normal monkeys ‐ 39 ± 990 unit/sec. Interestingly, the post occlusion hyperemic response was also significantly correlated with FPG (R = 0.57, p =.003) and HbA1C (R= 0.55, p = .006). The prolonged hyperemia in diabetic monkeys, reflective of a substantial loss in vascular tone, may be due to autonomic dysfunction, endothelial damage, local acidosis or circulating inflammatory cytokines. Supported by NIA HHSN2532008002C
A Selected History of Exercise Thermoregulation: A Century of Discovery 1910‐2010
Authors: Schneider Suzanne , Moseley Pope
Published: June 2021

Exercise is a tremendous challenge for homeostasis. It was known that body temperature increases with exercise (Haldane and Hunt, 1905), but not appreciated until 1938 that core temperature is controlled (M. Nielsen), and at a level proportional to relative exertion (Astrand 1960). A debate ensued whether core or peripheral temperature regulated thermoregulatory responses. This was settled when Benzinger (1959) demonstrated that intracranial temperature most closely corresponds to sweating and skin blood flow during exercise. Lind (1963) identified a "prescriptive zone", where core temperature is not affected by ambient conditions. The importance of heat acclimation on work capacity was described by Dresoti (1935) and applied to soldiers by Robinson (1943) and miners by Wyndham (1966). The effect of hydration was studied by Pitts (1940's) and the cardiovascular and body fluid effects were graphically illustrated during desert walking by Adolf (1947) and Dill (1972). Recent studies have challenged the concept of Hunt (1912) that water ingestion is the best method to restore fluid balance during work in the heat. A new fascinating area is to understand cellular responses during acute (Salo 1991, Ryan 1991) and chronic (Horowitz 1997, Yamada 2007, McClung 2008) heat exposure and the consequences for tolerance to other environmental and disease stressors.