bluuebunny/crossref_metadata_2025 huggingface dataset:
Increasing the meal frequency does not promote exercise‐induced muscle hypertrophy in rats
Authors: Tanaka Chihiro , Fujii Takako , Nakashima Ayumi , Xu Minjun , Sonou Tomohiro , Okamura Koji
Published: June 2021

Skeletal muscle protein synthesis has been reported to be maximized after ingesting 20 g of protein, and protein consumption of 2.0 g/kg BW/day is the upper limit to promote exercise‐induced muscle synthesis in humans. Therefore, 100 g of protein maximizes the muscle synthesis in a 50 kg human, and consuming 20 g of protein 5 times a day may promote muscle hypertrophy. To examine the effect of meal frequency on exercise‐induced muscle hypertrophy, male rats performed climbing exercise (19:00 – 20:00) three times a week were divided into a group fed 2 meals (08:00 – 09:00 and 20:00 – 21:00; 2M) or a group fed 5 meals (every three hours between 08:00–21:00; 5M). The amount of protein given to both groups was equal. The rats were killed pre‐and post‐exercise on the last day of the 8‐week study. The flexor hallucis longus muscle weight tended to be higher in 2M than 5M, while the weights of other muscles did not differ. The plasma urea level was significantly higher in 5M than 2M. The urinary urea nitrogen excretion did not differ significantly but it was higher in 5M than 2M by 19%. The pre‐exercise plasma branched‐chain amino acid level tended to be higher in 5M than 2M, while it was similar post‐exercise. The plasma levels of other amino acids did not differ between the groups pre‐or post‐exercise. Therefore, increasing the meal frequency may increase amino acid catabolism, and does not promote exercise‐induced muscle hypertrophy.
Removal of glutamate from diet suppresses fat oxidation and promotes fatty acid synthesis in rats
Authors: Nakamura Hidehiro , Kawamata Yasuko , Kuwahara Tomomi , Uneyama Hisayuki , Sakai Ryosei
Published: June 2021

Glutamate (Glu) is the most abundant amino acid (AA) in foods. Our previous study has shown that removal of Glu from diet increased body fat mass and hepatic triglyceride. This study investigated the underlying mechanism of the fat increase caused by Glu‐free diet. Male SD rats of 3 weeks old were fed fixed amount of diets containing either balanced AA mixture (4% Glu), Glu‐free mixture or low AA mixture (isonitrogenous to the Glu‐free). Fat oxidation was estimated by monitoring 13CO2 in the exhaled gas after ingestion of experimental diets containing [1,1,1‐13C3]tripalmitin. De novo lipogenesis was estimated from deuterium incorporation of given D2O into hepatic fatty acids. Feeding of Glu‐free diet suppressed the oxidation of dietary tripalmitin but promoted de novo lipogenesis in the liver. The mRNA expression involved in fatty acid synthesis (FAS and Elovl6) was also up‐regulated. Three weeks ingestion of Glu‐free diet resulted in remarkable increase in contents of fatty acids such as palmitate, stearate and oleate in hepatic triglyceride. These findings suggest that dietary Glu has the inhibitory effect on fat accumulation by both enhancement of fat oxidation and suppression of de novo lipogenesis.
Warfarin‐induced vitamin K deficiency is associated with alterations in sphingolipid status in rats
Authors: Ferland Guylaine , Tamadon‐nejad Sahar , Ouliass Bouchra
Published: June 2021

Vitamin K (VK) is involved in sphingolipid metabolism and menaquinone‐4 (MK‐4), the main K vitamer in brain, is strongly correlated to cerebral sulfatides, sphingomyelin and gangliosides. Warfarin (W), a widely used oral anticoagulant, acts by blocking the VK cycle. In a previous report, we observed W‐induced VK deficiency (model of Price et al. 1982) to result in cognitive deficits, an observation associated with a drastic decrease in brain MK‐4. Here we report that in these rats, W‐tx is associated with an alteration in sphingolipid status. Male Wistar rats were treated with 15 mg W/kg/d (in drinking water) and subcutaneous VK (85 mg/kg), 3X/wk, for 10 wks; control rats were treated with normal water and injected with saline. Sphingolipids (gangliosides, ceramides, cerebrosides, sphingomyelin and sulfatides) were determined by solid phase chomatography and the ganglioside subtypes were further assessed by HPTLC. Compared to the control condition, W‐tx altered ceramides, sphingomyelin, sulfatides and gangliosides in specific brain regions (p<0.05) and resulted in a loss of correlation between sphingolipids and brain MK‐4. W‐tx also resulted in significant changes in gangliosides GD1a and GT1b in key brain regions (p<0.05). In conclusion, W‐induced VK deficiency alters sphingolipid status in brain, a finding that could contribute to the detrimental effects of W on cognition. (Funded by CIHR.)
Synergistic effects of polyphenols with leucine and β‐hydroxy‐ β‐methylbutyrate (HMB) on energy metabolism
Authors: Bruckbauer Antje , Baggett Brooke , Zemel Michael B
Published: June 2021

We recently demonstrated synergy between low dose resveratrol (200 nM) with leucine or its metabolite HMB on energy metabolism. We have now assessed potential synergy of other polyphenols with leucine and HMB. Dose‐response curves for fatty acid oxidation (FAO) were constructed and the highest non‐effective dose (typically 1 – 10 nM) was combined with either leucine (0.5 mM) or HMB (5 μM) to treat adipocytes and myotubes for 24h. Significant synergy was detected for the stilbenes (resveratrol, piceatannol) and a stilbene‐containing grape seed extract, resulting in stimulation of FAO in adipocytes (60–70%, p<0.05) and myotubes (>;2000%, p<0.01) and stimulation of AMPK (~50%, p<0.03) and Sirt1 (~65%, p<0.001) activities. Hydroxycinnamic acids, including chlorogenic and cinnamic acids and their derivatives, exhibited robust synergy with leucine and HMB, increasing FAO (300–1300% p<0.01), AMPK activity (~50%–150%, p<0.01), Sirt1 activity (~70%, p<0.001) and insulin sensitivity/glucose utilization (~50 – 150%, p<0.05). In contrast, more complex polyphenol structures (ellagic acid, epigallocatechin gallate) required higher concentrations (>;1 μM) and exerted little or no synergy with leucine or HMB. These data indicate synergy of several naturally occurring polyphenols with leucine or HMB in concentrations which produce no independent effects and are readily achievable via dietary supplementation.
Bioaccessibility of carotenoids from transgenic provitamin A biofortified sorghum
Authors: Lipkie Tristan E , De Moura Fabiana F , Zhao Zuo‐Yu , Albertsen Marc C , Che Ping , Glassman Kimberly , Ferruzzi Mario G
Published: June 2021

Biofortified sorghum lines are being developed to target vitamin A deficient populations in Sub‐Saharan African. The purpose of this study was to screen diverse samples of transgenic sorghum for the content and delivery of provitamin A carotenoids using an in vitro digestion model. The germplasm background and transgenic sorghum contained 1.0–1.5 and 3.3–14.0 μg/g β‐carotene equivalents on a dry weight basis (DW), respectively. Test porridges made from milled sorghum contained 16–250 μg/100g β‐carotene equivalents on a fresh weight basis. Micellarization efficiency of all‐E‐β‐carotene was lower (p<0.05) from transgenic sorghum (0.9–4.9%) than from null/non‐transgenic sorghum (6.3–11.1%). ABS 188 sample #11 contained the greatest bioaccessible β‐carotene content (0.67±0.12μg/g DW) in relation to null/non‐transgenic samples (0.08–0.20 μg/g DW). While the bioavailability and bioconversion of provitamin A carotenoids from these grains must be confirmed in vivo, these data support the notion that biofortification can enhance the total and bioaccessible concentrations of β‐carotene in sorghum. Support provided by HarvestPlus, DuPont Pioneer, and a grant from the USDA National Needs Program.
Effects of acute Pils‐beer consumption on alcohol‐induced liver steatosis in a mouse model
Authors: Landmann Marianne , Kanuri Giridhar , Bergheim Ina
Published: June 2021

Chronic alcohol abuse is still among the leading causes of liver‐related diseases worldwide. Studies suggest that not only the amount but also the type of alcoholic beverage may be critical in the development of alcoholic liver diseases. In the present study, the acute effects of Pils‐beer and plain ethanol intake on the liver were determined.

C57Bl/6J mice either received a bolus ethanol, beer (6 g/kg BW, isocaloric) or maltodextrin solution. Fat accumulation, markers of lipogenesis and mRNA expression of MyD88 as well as concentration of 4‐hydroxynoneal protein adducts (4‐HNE), protein levels of the inducible NO synthase (iNOS) and ApoB were analyzed in livers 6h after the acute alcohol exposure.

Despite similarly elevated plasma alcohol levels in both alcohol exposed groups, acute ethanol exposure caused a ~5‐fold increase (p<0.05) in hepatic lipid accumulation while the ingestion of beer only lead to a ~2‐fold increase (n.s.) in comparison to controls. The less harmful effects of the beer were associated with a protection against an elevation of MyD88, SREBP‐1c and FAS mRNA expression as well as the increase of iNOS protein levels and 4‐HNE protein adducts in the liver. Protein levels of hepatic ApoB were significantly induced of Pils‐beer but not ethanol‐treated mice.

Taken together, these data suggest that the acute ingestion of Pils‐beer may be less harmful on the liver than ethanol in mice.
Oral administration of nutrients recovers intestinal functions in total parenteral nutrition (TPN) model of rats
Authors: Suzuki Takuji , Kasahara Ayako , Keta Ayumi , Sato Ayaka , Mayanagi Yuki
Published: June 2021

It has been considered that influx stimulation of nutrients in intestinal tract is important for keeping the intestinal functions. Therefore, we investigated whether the influx stimulation of nutrients through the intestinal tract recover the intestinal function using intestinal dysfunction model of rats. We performed total parenteral nutrition (TPN) against 8 weeks old rats for 7 days (TPN7). After 7days, rats were administered respective nutrients orally, i.e. carbohydrates, amino acids and lipids, for 3 days. We collected jejunum, and analyzed expression levels of digestion/absorption of nutrients related genes by qRT‐PCR and measured disaccharidase activity. Although the expression of digestion/absorption of nutrient related genes markedly increased by lipids and carbohydrates admin, the enzyme activities did not change. These results suggested the expression levels of nutrients digestion/absorption related gene were recovered by nutrients for 3 days, but it needed more time to express the intestinal function by nutrient admin orally. In conclusion, our data suggest that influx stimulation of nutrients to intestinal tract is important to keep on the intestinal functions, and fructose and medium‐chain triglyceride may be important for recovery of intestinal function.
Zinc supplementation increases expression of transcription factors PU.1, KLF4, and Nrf2 in the lungs of alcohol‐fed rats
Authors: Joshi Pratibha C , Fan Xian , Koval Michael , Guidot David M
Published: June 2021

Our group has previously shown in a rat model of alcohol ingestion that (1) cultured type II alveolar epithelial cells form a leaky barrier, (2) there is a decrease in transcription factors Nrf2 and KLF4 in these cells, (3) GM‐CSF signaling is dampened, and (4) either in vitro or in vivo zinc treatment increases tight junction proteins and tightens the epithelial barrier. In this study, we examined the expression of Nrf2, KLF4, and PU.1, a master transcription factor for GM‐CSF, by real time PCR in primary alveolar epithelial cells isolated from rats fed the Lieber‐DeCarli liquid diet with or without alcohol and alcohol plus zinc acetate (10, 100, 500 mg/L) for 8 weeks. Zinc supplementation of the alcohol diet significantly increased (p<.02) expression of PU.1, KLF4, and Nrf2 in a dose‐dependent manner as compared to the alcohol group and reached the control levels with 10, 100 and 500 mg/L zinc in the diet, respectively. The increase in Nrf2 expression by zinc treatment may explain why zinc is purported to have antioxidant properties whereas the higher expression of KLF4 may be due to its interaction with PU.1. In fact, KLF4 is a PU.1 target gene and we have shown before that zinc restores GM‐CSF signaling in the lungs of alcohol‐fed rats. Collectively, these data suggest a delicate interplay of transcription factors in the lungs of alcohol‐fed rats and their differential modulation by the bioavailability of zinc.
Identification of miR‐26b and miR‐203 gene targets in colon cancer cells
Authors: Shah Manasvi Shailesh , Davidson Laurie A , Chapkin Robert
Published: June 2021

We recently demonstrated that the unique combination of chemoprotective dietary fish oil (containing eicosapentaenoic acid and docosahexaenoic acid) plus pectin (fermented to butyrate in the colon) upregulates a subset of rat mucosal miRNAs, miR‐19b, miR‐26b and miR‐203, and downregulates their predicted target genes PTK2B, PDE4B and TCF4 as compared to a corn oil cellulose diet in the presence of carcinogen. To further elucidate the biological effects of diet and carcinogen on miRNAs and their targets and to validate the predicted miRNA:mRNA associations, we manipulated miRNA levels by conducting loss and gain of function analyses in the human colon cancer cell line HCT116 and measured changes in protein expression of the targets. Furthermore, using luciferase reporter assays, we demonstrated that PDE4B and TCF4 are direct targets of miR‐26b and miR‐203, while PTK2B is an indirect target of miR‐19b. In addition, the physiological function of these miRNAs was assessed by examining effects on apoptosis and cell proliferation, showing that these miRNAs increased apoptosis. This is the first report to show the chemoprotective effects of combined fish oil and pectin feeding antagonize the oncogenic effects of carcinogen, in part, by modulating the expression of colonic miR‐19b, miR‐26b and miR‐203 and their targets.

Grant Funding Source: CPRIT grant RP100473 and NIH grants CA168312, CA129444 and CA59034.
Effects of frequent sweetener intake on murine splenic T lymphocyte surface marker expression
Authors: Sánchez‐Delgado Marcela , Estrada José Antonio , Serrano‐Váldes Jessica , Váldes‐Ramos Roxana , Contreras Irazú
Published: June 2021

Metabolic and immunological alterations are common in chronic conditions, such as diabetes, which promotes low‐level chronic inflammation. The use of non‐nutritive sweeteners has increased not only in patients suffering from this illness, but in the general population. The effects of these compounds on the immune system are not well known. The objective of this study was to determine the effects of the frequent consumption of sucrose and stevia on T lymphocyte surface marker expression. 14 week‐old BALB/c mice were orally administered either sucrose or stevia in drinking water for 6 weeks. Subsequently, splenic T cells were purified and analyzed by flow cytometry using the following surface markers: CD3, CD4, CD8, CD25 and CD69. Our results showed an increase in CD4+ T cells of the sucrose group, compared to control and stevia groups. We observed an increase in CD8+ T cells in the sucrose and stevia groups. In contrast, we found no change in CD4+/CD25+ T cells among groups, but we observed a decrease in the percentage of CD4+/CD69+ T cells in the sucrose and stevia groups. Our data suggests that the frequent use of sweeteners modifies the frequency and phenotype of T cell subsets in the spleen.
SELECTIVE INHIBITION OF GLIADIN IMMUNE REACTIVITY BY TRANSAMIDATION OF WHEAT FLOUR WITH MICROBIAL TRANSGLUTAMINASE
Authors: Iaquinto Gaetano , Lombardi Emanuela , Bergamo Paolo , Maurano Francesco , Bozzella Giuseppina , Luongo Diomira , Mazzarella Giuseppe , Aufiero Vera Rotondi , Rossi Mauro
Published: June 2021

Celiac disease (CD) is an immune‐mediated enteropathy caused by the ingestion of wheat gluten. The modification of gluten by intestinal tissue transglutaminase (tTGase) plays a crucial role in CD pathogenesis. In this study, we observed that extensive transamidation of wheat flour with lysine ethyl ester (K‐C2H5) by microbial TGase (mTGase) yielded water‐soluble (spf) and insoluble transamidated gliadin (K‐gliadins) fractions. Using DQ8 transgenic (tg) mice as a model of gluten sensitivity, we observed a dramatic reduction in IFNγ production in gliadin‐specific spleen cells challenged with spf and K‐gliadins in vitro (N=12; median values: 813 vs. 29 and 99; control vs. spf and K‐gliadins, P=0.012 for spf, P=0.003 for K‐gliadins). We also observed an increase in the IL‐10/IFNγ protein ratio (N=12; median values: 0.3 vs. 4.7, control vs. spf, P=0.005). In intestinal biopsies from untreated CD patients challenged in vitro with gliadins (N=10), K‐gliadins dramatically reduced the levels of antigen‐specific IFNγ mRNA in all specimens responsive to native gliadins (4/10; P <0.05). In addition, the analysis of glutathione‐S transferase (GST) and caspase‐3 activities in the enterocytic Caco‐2 cells showed that neither activities were modified by flour transamidation.

In conclusion, we found that K‐C2H5 cross‐linking via mTGase specifically affected gliadin immunogenicity, reversing the inducible inflammatory response in models of gluten sensitivity without influencing the cytotoxic properties of gliadins.
Matrix Metalloproteinase‐9 Inhibition Attenuates Wall Thinning but Increases Neutrophil Infiltration Post‐Myocardial Infarction in Mice
Authors: IYER RUGMANI PADMANABHAN , PATTERSON NICOLLE LYANNE , FIELDS GREGG B , LINDSEY MERRY L
Published: June 2021

Targeted deletion of the matrix metalloproteinase‐9 (MMP‐9) gene attenuates cardiac remodeling post‐myocardial infarction (MI), but whether MMP‐9 inhibition post‐MI is effective as a therapeutic strategy has not been evaluated. Adult male C57BL/6 mice (3–6 months old, n=80) were subjected to left coronary artery ligation. Osmotic pumps with saline or MMP‐9 inhibitor (MMP‐9i; 0.03 μg/day) were inserted subcutaneously 3h post‐MI, and the mice were sacrificed at day 7 post‐MI. Infarct area was similar in the saline (57±2%) and MMP‐9i (55±1%, p=0.22) groups, indicating similar initial injury. Survival rates were 33% (12/36) for saline and 27% for the MMP‐9i (12/44; p=0.54). Infarct wall thinning was lower in the MMP‐9i group (0.95±0.10 mm) vs. saline (0.66±0.08 mm; p=0.04). Out of 84 inflammatory genes examined in the infarct region, 13 pro‐inflammatory and 2 anti‐inflammatory genes were increased in the MMP‐9i group, indicating an overall increase in inflammation. One upregulated cytokine was Ccr3, the eotaxin receptor that regulates neutrophil influx. Eotaxin was also 1.5 fold increased in the plasma of the MMP‐9i mice. Further, neutrophil numbers were 10× higher in the MMP‐9i group at day 7 post‐MI, indicating prolonged neutrophil presence in the infarct region. In conclusion, MMP‐9 inhibition post‐MI attenuated LV wall thinning and facilitated neutrophil infiltration through increased eotaxin levels.
The mechanism of CD99 and CD99L2 in leukocyte transendothelial migration
Authors: Watson Richard L , Mikhailov Alexei , Muller William A
Published: June 2021

Transendothelial migration (TEM) is the process by which leukocytes move out of the vasculature into the interstitium and is one the most important steps of the inflammatory response. PECAM and CD99 are two critical proteins that function sequentially to regulate TEM (PECAM upstream of CD99). These proteins partially reside in a novel, intracellular endothelial cell compartment, known as the Lateral Border Recycling Compartment (LBRC), which supplies the migrating leukocyte with unligated proteins to facilitate its movement through the endothelium, in a process called targeted recycling (TR). Blocking PECAM function has been shown to inhibit TR and subsequently prevent TEM. Here we show that TR is able to be initiated in the face of a CD99 block. We hypothesized that CD99 regulates further rounds of LBRC recruitment. Stimuli that also lead to intracellular membrane recruitment such as histamine (and in preliminary experiments, VEGF) selectively overcome CD99 blockade (but not PECAM blockade), in a calcium‐independent manner. A novel paralog of CD99, CD99L2, has been shown to control inflammation in mice. We show that human CD99L2 functions at the same step as CD99. We are currently investigating if CD99L2 also resides in the LBRC and the mechanism behind its function.

This research was supported by R21 HL102519, R01 HL046849, and R37 HL064774 to WAM.
Ozonation as an anti‐microbial technique in dental treatment units
Authors: Jacinto Roberta Bassi Soares , Mergulhao Eliane , do Carmo Fornel Adriana , Navarro Ricardo , Zangaro Renato Amaro
Published: June 2021

Dental units are characterized by systems that allow the use of various techniques for dental treatments in patients, part of this structure is made up of a water‐based hydraulic system, which allows cleaning and irrigation during the dental clinical procedure in patients, the fluid dynamic circuit is characterized by a system of compressed air pressure from a water reservoir, with valves and piping the output to hoses for liquid injection in the oral cavity of patients. Requirements with respect to water quality, oral fluid reflux problems during the dental clinical procedure, inter alia, promote the microbiological proliferation in hydraulic system of the unit, this allows the occurrence of biofilm formation throughout the circuit of internal walls making up the system of water, thus demonstrating the great potential of transmitting diseases among these the cystic fibrosis. Traditional techniques of cleaning the system, such as the peroxidation, have permitted the microbiological control and inhibition of biofilm formation. The ozone gas presents the characteristic of being more reactive compared to chlorine, demonstrating great potential for elimination of microorganisms, a new Protocol was developed with the use of ozone water to promote cleaning the fluid system hydraulic circuit, various dental units were adopted, in which microbiological tests were focused on qualitative and quantitative conditions. Preliminary results showed a significant reduction of microorganisms highlighting also the removal of the biofilm, this research project aims to demonstrate the feasibility of the use of ozone in the hydraulic system for cleaning and microbial control of dental treatment units.
EPAC1 controls vascular endothelial cell permeability and the adaptation of these cells to differential fluid shear stress
Authors: Rampersad Sarah N , Kaczmarek Milosz , Maurice Donald H
Published: June 2021

The permeability of a monolayer of vascular endothelial cells (VECs) to the paracellular transit of macromolecules is largely dependent on their ability to form vascular endothelial cadherin (VEcad)‐based contacts between neighbouring VECs and on the strength of the linkages between these VECad‐based structures and the actin cytoskeleton. We reported previously that cAMP‐elevating agents reduce VEC permeability largely through an exchange protein activated by cAMP‐1 (EPAC1)‐mediated strengthening of these inter‐VEC adhesions. Specifically, we showed that integration of EPAC1 into VECad‐based macromolecular signaling complexes allowed localized EPAC1‐dependent activation of Rap‐1 at regions of VEC intercellular contacts and reduced permeability. Since high VEC permeability occur in vivo in areas of the vasculature that experience low fluid shear‐stresses and these areas are more likely to develop atherosclerotic lesions, we studied the impact of EPAC1 activation on VEC permeability under conditions of high or low laminar shear stress. Our results indicated that while VECs aligned poorly when subjected to low fluid shear‐stresses, EPAC1 activators markedly promoted alignment under these conditions.
The Role of TLRs in Cardiac Myocyte Apoptosis
Authors: Heiserman James P. , Chen Le , Kim Bum‐Soo , Kim Se‐Chan , Tran Alice , Siebenborn Nina , Knowlton Anne A.
Published: June 2021

Recently, Toll‐like receptors(TLRs) have been investigated for their role in apoptosis. Previously we have shown that low endotoxin extracellular(ex) HSP60 causes cardiac myocyte apoptosis, and we hypothesized that this would be blocked in cardiomyocytes that have nonfunctioning TLR4.

Methods

We studied adult cardiac myocytes from C3H/HeJ mice, which have a mutant, nonfunctional TLR4, and compared the results with parallel studies in adult cardiac myocytes from wild type (WT) mice. We also looked at the response of WT mouse cardiomyocytes to hypoxia/reoxygenation(H/R) in the presence of TLR2 and TLR4 blocking antibodies. LDH release and NADPH Oxidase 2 (NOX2) activity were endpoints.

Results

We found exHSP60 caused apoptosis in WT, but not TLR4 mutant cardiomyocytes, based on increased DNA fragmentation and caspase3 activation. In experiments with WT myocytes treated with TLR2 and 4 blocking antibodies with H/R, LDH release was decreased by either blocking antibody. NOX2 activity increased in control cells with H/R and was decrease in WT cardiomyocytes treated with TLR2 and 4 blocking antibodies, suggesting TLR2/4 mediate increased NOX2 activity in H/R injury.
Effect of Pretreatment with Polyalthia longifolia var (Annonaceae) on Isoproterenol Induced Cardiotoxicity and Cardiac Hypertrophy in Rats
Authors: Ramesh Alluri , Rajashekar Perusomula
Published: June 2021

The Objective of present study was to investigate the effect of pretreatment with Polyalthia longifolia var PL on Isoproterenol induced cardiotoxicity and cardiac hypertrophy in Rats.

Method

Wistar rats 220–250 g were randomly divided into 5 Groups.

Control (olive oil 2 mL/kg orally for 18 days & water IP from days 9–18);

ISO group (olive oil 2 mL/kg orally 18 days & ISO 1 mg/kg IP from days 9–18);

(PL 300 mg/kg p.o 18 days + ISO);

(PL 600 mg/kg p.o 18 days + ISO);

Group V (CoQ10 100 mg/kg p.o 18 days + ISO).

ISO 1 mg/kg IP was administered in group Groups II, III, IV, V from days 9–18). Twenty‐four hours after the last dose of water or ISO, the rats were anesthetized and an ECG was recorded. Blood was withdrawn by retro‐orbital puncture for estimation of serum creatine kinase‐MB CK‐MB, lactate dehydrogenase LDH levels, and aspartate aminotransferase activities. The animals were euthanized using an overdose of ether. The hearts of 4 animals from each group were used for estimation of superoxide dismutase SOD activity, reduced glutathione GSH concentration, lipid peroxidation LPO, malondialdehyde MDA, and total protein concentration. Histopathology of the 2 remaining hearts in each group was carried out by a blinded technician.

Results

ISO‐treated rats had a significant change in the endogenous antioxidants significantly higher myocardial MDA concentration significantly lower myocardial GSH concentration and SOD activity and significantly higher serum activities of marker enzymes CK‐MB and LDH. Compared with the ISO group, the PL 600 mg/kg + ISO group had a significant changes in the endogenous antioxidants higher myocardial GSH concentration and SOD activity; significantly lower serum activities of marker enzymes CK‐MB and LDH.

Conclusion

Pretreatment with PL 600 mg/kg for 18 days was associated with moderate protection against ISO‐induced cardiac hypertrophy.
Isoform Selectivity of Adenylyl Cyclase Inhibitors and Identification of Novel Compounds
Authors: Brand Cameron S. , Hocker Harrison J. , Gorfe Alemayehu A. , Cavasotto Claudio N. , Dessauer Carmen W.
Published: June 2021

Nine membrane‐bound adenylyl cyclase (AC) isoforms produce the 2nd messenger cyclic AMP (cAMP) in response to a wide‐range of stimuli. Reduction of AC activity has well documented benefits, including for heart disease and pain. These roles have inspired development of isoform selective AC inhibitors, a lack of which currently limits exploration and/or treatment of dysfunctions where cAMP plays a role. Known AC inhibitors were flexibly docked to the catalytic site of two AC structure models. Predicted conformations of selective inhibitors suggested flexible catalytic site subregions which confer potency and selectivity. To identify and characterize novel AC inhibitors, a structure‐based virtual screen of 35,000 small drug‐like molecules produced 100 high scoring molecules tested for AC inhibition in vitro. Multiple candidates showed AC inhibition, including isoform selective AC inhibitors. Most known inhibitors are nucleotide analogs; our identified inhibitors show no significant similarity in chemical structure, demonstrating novel selective targeting of the AC catalytic site.

Research was funded by Keck Center training fellowship NIGMS T32GM089657 (CSB), and by NIH grants GM60419 and MH060397 (CWD).
Plasmodium falciparum cyclin G associated kinase as a drug target
Authors: Harmse Leonie , Coetzer Theresa
Published: June 2021

Kinases have become attractive drug targets in disease and several selective inhibitors are currently in clinical use. A P. falciparum kinase gene (PFL2280W) was identified in silico and investigated as a potential novel drug target. PFL2280W shows 57% similarity to mammalian cyclin G associated kinase (cGAK) which is essential for the survival of developing and mature mice.

The P. falciparum 3D7 strain was maintained in culture and DNA was isolated and used to amplify the catalytic domain and flanking region of the PfcGAK gene by PCR. The PCR product was cloned into a vector that yielded a fusion protein with a N‐terminal HA and a C‐terminal His (6X) tag respectively. Protein expression was induced in E. coli and isolated by affinity chromatography on Ni+ beads. The protein samples were analysed by SDS‐PAGE and Western blotting using antibodies against the 6xHis and HA tags. The function of the fusion proteins was assessed in a standard kinase assay using radiolabelled ATP and exogenous substrates.

Recombinant PfcGAK showed kinase activity using casein as substrate. PfcGAK was shown to undergo autophosphorylation. PfcGAK has a typical serine/threonine catalytic domain except for the absence of the conserved glycine triad in subdomain 1. Bioinformatic analysis of PfcGAK revealed an unique repetitive nanomeric amino acid sequence of 81 amino acids (KLQND/ED/VSG) which occurs outside the catalytic domain. In one clone this repetitive sequence was deleted in frame, which enhanced the production of the recombinant protein. PfcGAK shows the presence of a conserved GOLD domain which indicates that it may function in the Golgi complex, protein sorting or protein‐protein interactions. The carboxyl terminus has a predicted prenylation site, implying that the protein may be anchored in a membrane. Further studies are required to elucidate the potential of PfcGAK as a drug target in P.falciparum. .
Influence of OPRM1 genotype on induction and maintenance of alcohol drinking in monkeys
Authors: Sawyer Eileen K , Sirbu Madelynn H , Vallender Eric J , Miller Gregory M , Platt Donna M
Published: June 2021

Studies in humans indicate that specific single nucleotide polymorphisms (SNPs) in the gene encoding the μ‐opioid receptor (OPRM1) may influence sensitivity to the abuse‐related effects of alcohol. Rhesus macaques express a functionally analogous SNP (C77G) and serve as a highly translational model of the human variant. We selected rhesus macaques (n=18) a priori such that the genotypes (G/G, C/G, C/C) were equally represented, and characterized alcohol pharmacokinetics and self‐administration behavior via established oral induction paradigms. Preliminary results suggest that G/G animals met drinking criteria sooner and more often during the induction period than C/C animals. We then evaluated their sensitivity to the reinforcing effects of alcohol by varying the concentration available (1–8%) for self‐administration during a 3‐hr daily access period. All animals maintained stable intakes with little variation in dose ingested across different alcohol concentrations. G‐allele carriers drank significantly higher doses of alcohol across the different concentrations. No significant pharmacokinetic differences between genotypes were observed. These data suggest that the G‐allele in macaques may increase alcohol drinking behavior, potentially modeling a pharmacogenetically‐determined vulnerability to alcohol use disorders in humans. Funding: AA016828 (DMP) and RR000168 (NEPRC; OD011103)
Effects of chronic treatment with the α2‐adrenergic agonist, lofexidine, on cocaine self‐administration
Authors: Kohut Stephen J. , Fivel Peter A. , Mello Nancy K.
Published: June 2021

Cocaine's abuse related effects have been attributed to its interaction with dopamine systems. Although adrenergic receptors are located in brain areas that modulate dopaminergic activity, adrenergic agents have received little attention as potential pharmacotherapies for cocaine addiction. Lofexidine, a a2‐adrenergic agonist, is being investigated as a treatment for reducing opioid withdrawal symptoms but little is known about its direct interactions with drugs of abuse. The present study assessed the effects of chronic treatment with lofexidine on cocaine selfadministration. Male rhesus monkeys were trained under a second order FR2(VR16) schedule of food and cocaine reinforcement. Chronic treatment (7 days) with lofexidine (0.1–0.32 mg/kg/hr, IV) potentiated the reinforcing effects of cocaine as evidenced by a leftward shift in the cocaine dose‐effect curve. Chronic lofexidine had no effect on food‐maintained responding and mild side effects during treatment. Fewer overt behavioral effects were also seen upon cessation of treatment compared to clonidine (0.032–0.1 mg/kg/hr, IV). Although clinical studies also find that lofexidine produces few unwanted side effects and has minimal withdrawal effects compared to clonidine, its potentiation of cocaine's reinforcing effects makes it unlikely that lofexidine will be effective for cocaine abuse treatment. Supported by NIDA DA02519 and DA026892
In vitro functional analysis of novel single nucleotide polymorphisms in OATP1B1
Authors: Yin Peter , Kim Richard B
Published: June 2021

The goal of our study is to functionally characterize six nonsynonymous single nucleotide polymorphisms (SNPs) in human organic anion transporting polypeptide 1B1 (OATP1B1) using in vitro transporter expression technologies. We hypothesize these SNPs will result in a decrease in transport activity compared to wild‐type. OATP1B1 cDNA packaged in pEF6/V5‐His TOPO was used as template, and 6 SNPs — 298G>;A (rs144508550), 419C>;T (rs147450830), 463C>;A (rs11045819), 1007C>;G (rs72559747), 1463G>;C (rs59502379), and 1738C>;T (rs71581941) — were introduced separately to wild‐type templates using QuikChange site‐directed mutagenesis. OATP1B1 variant cDNAs will then be transferred to pAd/CMV/V5‐DEST Gateway vector for further propagation as OATP1B1 variants expressed in adenovirus. We will determine the optimal titre of the OATP1B1‐ variant adenovirus for transporter expression using a monolayer cell lines such as HeLa. Subsequently, we will use prototypical substrates of OATP1B1 such as rosuvastatin to determine the relative affinity (Km) and capacity (Vmax) for uptake, followed by quantification of relative cell surface trafficking. Identification of new functional SNPs in OATP1B1 will give us further insights to genetic heterogeneity in OATP1B1 as a contributor to inter‐subject variation in response to OATP1B1 substrate drug therapy. Research is funded by the Canadian Institute for Health Research.
Lysophosphatidic Acid‐Induced Integrin Activation in Vascular Smooth Muscle Cells Requires Production of Reactive Oxygen Species
Authors: Staiculescu Marius Catalin , Hong Zhongkui , Sun Zhe , Ramirez‐Perez Francisco , Meininger Gerald A. , Martinez‐Lemus Luis A
Published: June 2021

Lysophosphatidic acid (LPA) stimulates vascular smooth muscle cell (VSMC) proliferation and migration, as well as, the production of reactive oxygen species (ROS) and the engagement of integrins with extracellular matrix proteins. We hypothesized that LPA‐induced integrin activation requires ROS. Using atomic force microscopy, we determined the effects of LPA on cytoskeletal stiffness and the binding activity of fibronectin (FN) with integrins on VSMC isolated from rat cremaster arterioles. Incubation of VSMC with LPA (2μM) significantly increased (P<0.05) cellular stiffness by 44% and the binding probability of FN to the cell surface by 18%. Pre‐incubation with antibodies vs. β1 and β3 integrins reduced the binding probability of FN to the cell surface, both, in control conditions and in the presence of LPA. This suggested that LPA induced the activation α5β1 and αvβ3 integrins. Suppression of ROS with the superoxide dismutase mimetic, tempol (250μM) or the NADPH oxidase inhibitor apocynin (300μM) blocked the increased binding probability of FN induced by LPA, but not the increased cytoskeletal stiffness. Our results indicate that ROS production by VSMC is required for LPA to induce integrin activation. This mechanism may be involved in the functional and structural changes of the vasculature that accompany increased circulating LPA such as inflammation. (NIH HL‐088105 to LAM and 1P01HL095486 to GAM)
High performance temperature and pressure telemetry for rodents
Authors: Clark Therese Marie , Millar Huntly , Lim Matthew , McCormick Daniel , Russell David , Guild Sarah‐Jane , Malpas Simon , Hu Patrick , Pauly Bob , Budgett David
Published: June 2021

We report on the ability to measure simultaneous temperature and pressure from a 2 Fr sensor (0.6 mm) located inside an artery of a rat. Over the temperature range from 25 to 45 degrees centigrade, the accuracy is better than 0.2 degrees. The pressure waveform is high fidelity with a 2 kHz sample rate. Performance data has been evaluated over a 30 day period and has illustrated very low drift and high precision. The sensor and accompanying telemeter are fully implanted. The telemeter is inductively powered and able to operate for the lifetime of the animal. The small size of the temperature sensor enables placement and the recording of temperature in select regions of the body.
Non‐invasive theranostic to predict and assess response to atherosclerotic drugs
Authors: Buckler Andrew John , Hamilton James Arthur , Madabhushi Anant , Keith James C.
Published: June 2021

Effective biomarkers are needed for assessing atherosclerotic plaque vulnerability for prognostic and longitudinal use. We report on a preliminary proof of concept using contrast MRI and textural kinetic analysis. Multivariate quantitative descriptors are developed using controlled outcome studies on a specialized model to identify candidate imaging markers that in composite are able to accurately distinguish vulnerable and stable plaque in both cross‐sectional and longitudinal applications. We hypothesize that characteristic patterns of lipid intermixed with extracellular matrix fibers, necrotic tissue, inflammatory cells and intra‐plaque hemorrhage are detectable via the use of steerable, multi‐scale Gabor filters, localized pixel statistics, and second order co‐occurrence textural features. These derived attributes are capable of discriminating functional microvascular networks without requiring resolution beyond that of standard DCE MRI. Neovascularization, presence of extracellular matrix and increased tissue permeability leading to increased distribution volume is evaluated by fitting cubic polynomials to the contrast media uptake signal to provide more effective separation between vulnerable and stable plaque than possible by merely relying on thresholding of time activity curves. Preliminary results demonstrate good discrimination between interventional study arms.
Open‐loop static characteristics of the carotid sinus baroreflex in rats fed a high‐fat diet
Authors: Abe Chikara , Iwata Chihiro , Morita Hironobu
Published: June 2021

It is still controversial whether high‐fat diet‐induced obesity attenuates the function of the carotid sinus baroreflex. To examine this, we employed open‐loop baroreflex analysis. Rats were fed a high‐fat diet from the 4th to 13th week, and then separated into 2 groups (Lean and Fat) by the body weight. The body weight in Fat group was significantly higher than that in Lean group (474 ± 8 vs 521 ± 4 g), while there was no difference in the baseline arterial pressure (AP) under free moving condition. In the open‐loop baroreflex analysis, the AP and renal sympathetic nerve activity (SNA) was measured in response to stepwise increment of isolated carotid sinus pressure (CSP) from 60 to 180 mmHg. The SNA was normalized by the maximal response induced by the ischemia. In the Fat group, CSP‐SNA relationship (neural arc) shifted to a higher SNA and slope of SNA‐AP relationship (peripheral arc) decreased compared with Lean group. Consequently, there was no difference in the operating point between Fat and Lean group (109 ± 4 vs 104 ± 6 mmHg). This result indicates that the sympathoexcitation‐induced AP increase was buffered by decrease in gain of peripheral arc in Fat group.
Caveolin‐1 overexpression repairs neuronal degradation in the setting of traumatic brain injury
Authors: Kellerhals Sarah Elizabeth , Niesman Ingrid , Schilling Jan , Kleschevnikov Alexander , Lysenko Larisa , Roth David , Patel Hemal , Patel Piyush , Head Brian
Published: June 2021

Each year, traumatic brain injury (TBI) contribute to a substantial number of deaths and cases of permanent disability. TBI causes a loss of pro‐growth signaling proteins found within neuronal membranes. Many of these pro‐growth components localize to membrane/lipid rafts (MLR), microdomains enriched in sphingolipids and cholesterol, and this localization is dependent upon the MLR protein caveolin‐1 (Cav‐1). MLR are essential for axonal growth, guidance and the development and stabilization of functional synapses. Our hypothesis is that neuron‐targeted Cav‐1 overexpression might reduce the extent of injury and enhance functional recovery after TBI. Our TBI model utilizes a controlled cortical impact (CCI) on mice, which leads to cortical and hippocampal injury ipsilateral and contralateral to CCI and reduces expression of MLR‐localized pro‐growth receptors and signaling components. Cav‐1 gene therapy to injured brain regions 24 hr prior and post CCI, preserves neurons and enhances neuronal growth in the hippocampus and enhances LTP, an indicator of memory formation. We are currently investigating anxiety, motor function, and learning and memory of mice treated with Cav‐1 post injury. Therefore, Cav‐1 may be a novel therapy to limit brain injury in the setting of TBI and potentially be an effective therapeutic intervention for other neurodegenerative diseases.
Norepinephrine mediated sympathetic over‐activity: the relationship to sodium homeostasis and blood pressure regulation.
Authors: Wainford Richard David , Kuwabara Jill Toshiko
Published: June 2021

Aim

Recent controversial studies in mice have suggested excess norepinephrine (NE) mediated sympathetic activity evokes impaired NCC mediated sodium homeostasis and salt‐sensitive hypertension. These studies examined the impact of excess NE on salt‐sensitivity and sodium homeostasis in conscious Sprague‐ Dawley (SD) rats.

Methods

Naïve male SD rats or rats receiving a s.c. NE infusion (600ng/min) were maintained on a normal 0.4% (NS) or high 8% NaCl (HS) diet for 14‐days, additional rats received s.c. hydrochlorothiazide (HCTZ 4mg/kg/day) and NE. On day‐14 MAP, 24h Na balance, FENa, MAP response to i.v. hexamethonium (30mg/kg), and peak natriuresis to i.v. HCTZ (2mg/kg) or amiliroide (2mg/kg) infusion was assessed (N=4/gp).

Results

NE increased MAP, FENa and vascular sympathetic tone. HS exacerbated NE induced hypertension, reduced FENa and prevented a salt stimulated reduction in HCTZ evoked natriuresis ‐ HCTZ co‐infusion abolished the salt‐sensitive component of NE stimulated hypertension.

Naive
s.c. NE
s.c. NE + HCTZ

NS
HS
NS
HS
HS

MAP (mmHg)
127±2
129±2
151±3φ
172±4* ≠
152±3 ≠ τ

24h Na+ balance (meq/day)
0.2±0.1
0.3±0.1
0.1±0.1
1.1±0.2* ≠
−0.7±0.3≠ τ

FENa (%)
0.60±0.1
0.93±0.1*
2.3±0.4φ
1.4±0.1* ≠
4.1±0.8 ≠ τ

Peak ΔHR to hexamethonium (mmHg)
−30±3
−32±4
−58±6φ
−52±8≠
−55±5≠

Peak ΔUNaV to Amiliroide (μeq/min)
7.7±0.7
4.2±0.9*
9.4±1.1
3.7±1.6*
7.1±1.1 ≠ τ

Peak ΔUNaV to HCTZ (μeq/min)
9.8±0.2
7.0±0.3*
11.1±1.1
10.8±0.4≠
0.4±0.2 ≠ τ

p<0.05 vs respective NS gp,

p<0.05 vs naïve NS gp,

p<0.05 vs naïve HS gp,

p<0.05 vs NE + HS gp.

Conclusion

These data support previous studies in mice and highlight an apparent opposing interaction between excess NE and high sodium intake on FENa and sodium homeostasis – which exacerbates NE‐induced hypertension via a mechanism independent of NE‐mediated vascular constriction that, as reported in mice, appears to involve altered function of the renal NCC channel ‐ HL107330.
Either sodium deprivation or sodium excess sensitizes angiotensin (ANG) II‐induced hypertension
Authors: Xue Baojian , Zhang Zhongming , Beltz Terry , Guo Fang , Johnson Alan Kim
Published: June 2021

Dietary sodium is known to regulate renin‐angiotensin‐aldosterone system (RAAS) activity, in which sodium deprivation activates the circulating RAAS, while sodium excess suppresses activity in that system. However, a recent study showed that the activity of RAAS was either not suppressed or, even augmented, after 4 weeks of salt loading despite high salt intake and increased blood pressure (BP). Our previous studies using an Induction‐Delay‐Expression (I‐D‐E) experimental design have demonstrated that one‐week ANG II pretreatment can sensitize the brain to produce an enhanced hypertensive response to subsequent ANG II. The present study tested the hypothesis that changes in dietary sodium (deficit or excess) can sensitize the pressor actions of ANG II. Three groups of male rats were implanted for telemetered BP recording and were maintained on 1) normal rat chow, 2) sodium‐deficient diet or 3) chow with 2% NaCl for two weeks, respectively. Then all groups were treated with subcutaneous ANG II (120 ng/kg/min) and normal chow for 2 weeks. Under both conditions where dietary sodium was manipulated, the animals responded with enhanced hypertension to ANG II (sodium deprivation 36.0±6.9 mmHg, high salt 34.8±4.1 mmHg, normal diet 19.6±3.8 mmHg). The results indicate that either sodium deprivation or sodium excess can sensitize the development of ANG II‐induced hypertension.
Increase in cyclooxygenase activity in the brain paraventricular nucleus mediates development of angiotensin II‐salt hypertension in the rat
Authors: Asirvatham‐Jeyaraj Ninitha , O'Brien Caitlin , Fink Gregory D
Published: June 2021

One in three adults in the US have chronic high blood pressure (hypertension; HTN) putting them at risk for cardiovascular diseases. Previous work from our laboratory suggests that inhibition of brain cyclooxygenases (COXs), which catalyze the rate limiting step in prostaglandin synthesis, prevents HTN development in rats treated with angiotensin II (AngII) and high salt (2% NaCl) diet. However, brain COX enzyme levels during HTN development were unaltered. So, we hypothesized that an early increase in brain COX activity in organum vasculosum lamina terminalis (OVLT), subfornical organ (SFO), paraventricular nucleus (PVN) and/or rostral ventrolateral medulla (RVLM) leads to AngII‐salt HTN development. Male Sprague‐ Dawley rats, fed a high‐salt diet were fitted with radio‐telemeters for continuous measurement of BP. After recording baseline BP, osmotic mini‐pumps were surgically implanted to deliver saline vehicle or AngII (150ng/kg/min, SC). Brains were collected after 4 days of infusion as rats became hypertensive. Above regions were punched and COX activity was measured using a commercially available kit. Hypertensive rats showed a significant increase in COX activity only in the PVN (p<0.05 vs. control rats)and not in other brain regions. Therefore, inhibition of COX signaling in specific regions of the CNS like PVN may represent a potential therapeutic target for patients with salt‐sensitive HTN.