bluuebunny/crossref_metadata_2025 huggingface dataset:
Challenges in the Separation of Purified Cardiac Mitochondrial Membranes
Authors: Rodríguez Graciani Keishla M , Chapa‐Dubocq Xavier R , Javadov Sabzali
Published: June 2021

Background

Mitochondria are the bioenergetic engines of the cell, thus maintaining mitochondrial health is essential for meeting energy demands. Mitochondrial function is achieved upon the balance of structural processes that involve mitochondrial biogenesis, fusion and fission. Structural and functional integrity of the mitochondrial membranes, the outer mitochondrial membrane (OMM) and the inner mitochondrial membrane (IMM) is important for the maintenance of the mitochondrial quality control and mitochondrial metabolism. Therefore, elucidation of the individual contribution of each membrane to mitochondrial metabolism and function, as well as mitochondria‐mediated cell survival pathways and cell death is important. In this study, we attempted to set up the methods for isolation of mitoplasts (mitochondria without OMM) and the OMM from the rat heart.

Methods

Cardiac mitochondria were isolated from Sprague Dawley rats (250–300g) by differential centrifugation and purified via Percoll gradient (continuous layers of 19%, 30% and 60% concentration). Isolation of mitoplasts was performed by incubation in different hypotonic and hypertonic buffers, sonication (20% output/power), and separation of membranes by ultracentrifugation using sucrose continuous gradients (20–80%). Mitochondrial swelling assay was made to confirm the swelling/shrinking method used. Mitoplast formation was validated by monoamine oxidase (MAO) activity, oxygen consumption in the presence and absence of cytochrome‐c and Western Blotting against specific inner and outer mitochondrial membrane proteins.

Results

Western Blot analysis showed pure isolated mitochondria, validated by the absence of GAPDH (cytosolic marker) upon Percoll gradient. In addition, more than 30% decrease in TOMM20 (OMM specific protein) expression in isolated mitoplasts in comparison to mitochondria. Mitochondrial respiratory rates and MAO activity suggests changes in mitochondrial membrane distribution. Mitochondrial swelling assay showed that cardiac mitochondria were resistant to swelling in hypotonic solution, therefore other methods involving the addition of Ca+2 or EGTA were used to induced separation of mitochondrial membranes.

Conclusion

Despite application of different preparative approaches, we were not able to isolate purified OMM, and only one method was proved to make a slight reduction of OMM contamination in mitoplasts.

Support or Funding Information

Supported by the NIGMS NIH grant SC1GM128210

This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Synthetic Biology Approaches To New Chemistry
Authors: Chang Michelle
Published: June 2021

Living systems have evolved the capacity to carry out many chemical transformations of interest to synthetic chemistry if they could be redesigned for targeted purposes. However, our ability to mix and match enzymes to construct de novo pathways for the cellular production of small molecule targets is limited by insufficient understanding how chemistry works inside a living cell. Our group is interested in using synthetic biology as a platform to study how enzymes function in vivo and to use this understanding to build new synthetic pathways for the production of pharmaceuticals, nanomaterials, and fuels using living cells.

This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Direct and Natural Killer cell‐mediated anti‐tumor effects of low‐dose Bortezomib in Hepatocellular Carcinoma
Authors: Krusch Matthias , Armeanu Sorin , Baltz Katrin M. , Lauer Ulrich M. , Steinle Alexander , Bitzer Michael , Salih Helmut R.
Published: June 2021

Hepatocellular carcinoma (HCC) displays a particular resistance to conventional cytostatic agents. Therefore, alternative treatment strategies focus on novel substances exhibiting anti‐neoplastic and/or immunomodulatory activity enhancing e.g. NK cell reactivity. However, the tumor‐associated ligands engaging activating NK cell receptors are largely unknown. Exceptions are the NKG2D ligands (NKG2DL) of the MIC and ULBP families. We here studied the consequences of proteasome inhibition with regard to direct and NK cell‐mediated effects against primary human hepatocytes (PHH) from different donors and hepatoma cells. Bortezomib treatment inhibited hepatoma cell growth with IC50 values between 2.4 and 7.7 nM. These low doses increased MICA/B mRNA levels and protein expression in hepatoma cells, which stimulated cytotoxicity and IFN‐γ production of cocultured NK cells. Importantly, while IFN‐γ production of NK cells was concentration‐dependently reduced, low‐dose Bortezomib neither induced NKG2DL expression or cell death in PHH nor altered NK cell cytotoxicity. Thus, low‐dose Bortezomib mediates a specific dual anti‐tumor effect in HCC by inhibiting proliferation and by priming hepatoma cells for NK cell anti‐tumor reactivity. Our data suggest the clinical evaluation of Bortezomib treatment in HCC, especially in combination with immunotherapeutic approaches like adoptive NK cell transfer.
Processed meat intake, doneness preference and risk of colorectal cancer in Northeastern Pennsylvania
Authors: Miller Paige Elizabeth , Lesko Samuel M , Muscat Joshua E , Lazarus Philip , Hartman Terryl J
Published: June 2021

Recent evidence suggests higher intakes of processed meat and greater degrees of cooking meat are associated with increased risk of colorectal cancer (CRC). We conducted a pilot case‐control study in Northeastern PA, consisting of 92 incident cases and 156 healthy controls. Data were obtained by a 150 item food frequency questionnaire and preferences for degree of doneness were recorded with the aid of colored visuals during an in‐person interview. Multivariate logistic regression models (adjusted for age, gender and total energy intake) were used to examine associations between intakes of meat products and risk of CRC. Results suggest that higher intakes of specific processed meats and well‐cooked meats were positively associated with CRC risk. The OR for the highest versus the lowest quartile of bacon intake was 1.44 (95% CI 0.71–2.93; p‐trend = 0.132). Compared to individuals who did not consume well‐cooked bacon, the OR for those in the highest quartile of well‐cooked intake was 1.39 (95% CI 0.62–3.10; p‐trend = 0.225). ORs across quartiles of medium‐well to well‐cooked hamburger intake were 1.70 (95% CI 0.75–3.84), 2.69 (95% CI 1.13–6.37) and 1.06 (0.52–2.25; p‐trend = .199) compared to those consuming rare/medium‐cooked hamburgers. Our preliminary findings indicate that a larger sample and a more specific instrument capable of capturing meat consumption in greater detail are warranted. Funding by PSCI.
Calcium intake and adiposity in adolescents 12–16 years from Guadalajara, México
Authors: Romero‐Velarde Enrique , Vargas‐Hernández Gabriela , Vásquez‐Garibay Edgar M. , Vizmanos‐Lamotte Bárbara , Troyo‐Sanromán Rogelio
Published: June 2021

With the purpose to explore the relationship between calcium intake and adiposity in a group of adolescents a cross‐sectional study was carried out with 125 adolescents 12 to 16 years old; 76 girls (60.8%) and 49 boys (39.2%). Calcium intake was assessed by 24 hour dietary recalls and a food frequency questionnaire and adiposity was measured through anthropometric measurements; fat mass and percentage of body fat by bioelectrical impedance (BIA). Pearson's correlation coefficients were used to evaluate the relation between calcium intake (mg/d) and adiposity measurements. Multivariate models were performed with body mass index (z‐score), fat mass and percentage of body fat as dependent variables. The mean age was 13.5 ± 0.8 years and a negative correlation was found between calcium intake (mg/d) and body weight, tricipital, sub scapular and suprailiac skin fold thickness, body mass index, fat mass and percentage of body fat (p < 0.05). Multivariate analysis showed a negatively relationship between adiposity measures and calcium intake, adjusted by sexual maturity and gender (r2 −0.09 to −0.20; p < 0.05). In conclusion, it was found a negative relationship between calcium intake and adiposity in adolescents.
Maternal overweight predicts infant caloric intake from complimentary foods and weight‐for‐length at age 6 months
Authors: Gilchrist Janet M , Andres Aline , Smith Stacee A , Badger Thomas M
Published: June 2021

We assessed the role of maternal overweight on infant dietary intake and body size during the first 6 months of life. Participants were from the Beginnings study, an on‐going, longitudinal cohort. Trained research assistants measured infant weight and length; weight‐for‐length percentiles (WL%) and Z‐scores (WLZ) were calculated with the CDC EpiInfo program. Mothers provided 3‐day records of formula and complimentary foods consumed by infants, which were analyzed using NDSR software. Matched data were available for 47 formula‐fed (FF) infants at 3‐ and 6‐months of age. At 3 months, all infants were exclusively FF; at 6‐months, all but one also ate complimentary foods. Groups were divided based on maternal BMI: normal weight (<25kg/m2, n = 21) vs. overweight (≥25 kg/m2, n = 26). At 3 months, infants did not differ in total caloric intake, WL%, or WLZ (p > 0.05). At 6 months, although total calories did not differ between groups, infants of overweight mothers had a greater caloric intake from complimentary foods (120 vs. 99 kcals/day, n.s.), higher WL% (76 vs. 56, p = 0.007), and WLZ (0.99 vs. 0.21, p = 0.004). Review of food records suggests that overweight mothers were more likely to offer “Stage 2 or 3” foods, such as meats, mixed dishes, and desserts. The transition from FF to table foods may be an important period for future study regarding the development of childhood obesity. Funded by USDA/ARS CRIS# 6251‐51000‐005‐03S.
Biological activities and utilizations of red pigments in Lithospermum erythrorhizon
Authors: Ma Seung‐Jin , Song Jung‐Hee , Cheon Gil‐Yong , Kang Seong‐Gook , Park Yang‐Kyun , Lee Jung‐Ju , Heo Kyung‐Chel , Kim Ki‐Man , Kim Sung‐Ho , Jung Soon‐Teck
Published: June 2021

The root of Lithospermum erythrorhizon has been used as a traditional folk remedy for treatment of blood circulation, injuries and burns in Korea. Especially its red pigments suggested to be main biological constituents in the root were used in Korean traditional liquor. Six kinds of shikonin derivative were isolated as red pigments from the ethanol extract of the root and investigated their biological activities. The isolated compounds showed strong biological activities such as antioxidant, antimicrobial and cytotoxicity. Although shikonin derivatives have potent biological activities, they were used in the limited food application because of the hydrophobic property. In the derivatives, shikonin is relatively bright red color and has low hydrophobicity and cytotoxicity. However, shikonin was a minor compound to be consisted in below 5% among its derivatives. So, the enzymatic method to liberate their derivatives was investigated to produce shikonin from the extracts for increasing the utilization. Enzymatic properties of commercial enzymes that are related to the hydrolysis of an acyl group in triglyceride were investigated. We found esterase originated from pig liver showed very strong activity for producing shikonin (up to 90%) from the crude extract including its derivatives. The application of bioactive red pigments as an antioxidant and antimicrobial agents were also investigated in this study.
HECA‐452 is a Non‐blocking Antibody for SLe‐x Mediated Adhesion to Endothelial Expressed E‐selectin
Authors: Kummitha China Malakondaiah , Burdick Monica M , Goetz Douglas J
Published: June 2021

Sialyl Lewis x (sLex) and its related glycans are found on the surface of leukocytes and are known to be ligands for endothelial expressed E‐selectin. HECA‐452 is a rat IgM mAb which recognizes sLex and its related glycans. It has been shown that pre‐treatment of HECA‐452 positive T‐lymphoblasts with HECA‐452 does not inhibit adhesion to E‐selectin under flow conditions. This lack of inhibition could be due to (a) HECA‐452 not binding to the exact set of glycoproteins/glycolipids that bind to E‐selectin (b) HECA‐452 binding to the exact set of glycoproteins/glycolipids that bind to E‐selectin but not at the glycans that binds to E‐selectin or (c) HECA‐452 binding to the involved glycans but not at the same epitope as E‐selectin. It is difficult to discern amongst the various scenarios with whole cells. However, if one has a particle that only has sLex, one can investigate scenario (c). Therefore we sought to determine if HECA‐452 is a blocking mAb for sLex adhesion to E‐selectin. We conjugated leukocyte‐sized microspheres with sLex. Flow cytometric analysis revealed that HECA‐452 recognized and bound avidly to the sLex microspheres. We next tested the adhesion of the sLex microspheres to 4 hr. interleukin‐1β (IL‐1β‐ an inducer of E‐selectin expression) activated human umbilical vein endothelial cells (HUVEC). sLex microspheres exhibited significant adhesion to IL‐1β activated HUVEC but did not bind to unactivated HUVEC. Pre‐treatment of activated HUVEC with anti‐E‐selectin mAb inhibited the adhesion of sLex microspheres. Pre‐treatment of the sLex microspheres with HECA‐452 had no effect on sLex microsphere adhesion to IL‐1β activated HUVEC. Combined, these results suggest that HECA‐452 is a non‐blocking mAb for sLex mediated adhesion to endothelial expressed E‐selectin.
Role of transient receptor potential vanilloid‐1 (TRPV1) and cannabinoid CB1 receptors in paracetamol induced antinociception and hypothermia in mice
Authors: Hamza May
Published: June 2021

Paracetamol is one of the most popular and widely used analgesic antipyretics; however, its mode of action has not been clear. It was recently shown that paracetamol is metabolized in the brain and the spinal cord to form the potent TRPV1 agonist N‐acylphenolamine (AM‐404). AM‐404 is also an inhibitor of the cellular uptake of the endogenous cannabinoid, anandamide. This study was designed to study the contribution of TRPV1 and CB1 receptors in the antinociceptive and hypothermic effects of paracetamol in mice. Capsazepine, a TRPV1 antagonist, significantly reversed the antinociceptive effect of paracetamol in the formalin test, when administered i.p. at a dose of 10 mg/kg. A higher dose (30 mg/kg), however, failed to reverse the paracetamol induced antinociceptive effect. In contrast, the higher dose partially blocked paracetamol induced hypothermia but not the lower dose. Furthermore, AM‐251, a cannabinoid CB1 receptor antagonist, when administered i.p. at a dose of 10 mg/kg, reversed the antinociceptive effect of paracetamol in the formalin test and blocked paracetamol induced hypothermia after 2 hours of paracetamol injection. In conclusion, both TRPV1 and CB1 receptors are involved in paracetamol induced antinociception in mice. Hypothermia, however, might involve other mediators and further studies are needed to investigate this point.
Selective inhibition of voltage‐activated Ca2+ channels by mu‐opioid receptor agonist in the primary sensory neurons devoid of T‐type Ca2+ channels: Mechanisms of action
Authors: Wu Zi‐Zhen , Cai You‐Qing , Pan Hui‐Lin
Published: June 2021

Opioids have a unique effect on nociception with little effect on other sensory modalities. However, the cellular mechanisms for this preferential effect are not clear. We have found recently that the μ‐opioid agonists inhibited high voltage‐activated Ca2+ channels (HVACC) only when the low voltage‐activated (T‐type) Ca2+ channels are not present in dorsal root ganglion (DRG) neurons (15 – 30μm). In this study, we determined the potential mechanisms for the differential effect of the μ‐opioid agonists on HVACC in DRG neurons with and without T‐type Ca2+ channel currents. Whole‐cell voltage clamp recordings were performed in acutely isolated DRG neurons in adult rats. In DRG neurons with T‐type Ca2+ channel currents, the μ‐opioid agonist DAMGO had no significant effect on both T‐type and HVACC currents. This occurred despite the fact that a large ω‐conotoxin GVIA‐sensitive (N‐type) Ca2+ current was present in the cells with T‐type currents. DAMGO still had no effect on HVACC currents when T‐type Ca2+ currents were blocked by amiloride or Ni2+ in these neurons. However, intracellular dialysis of GTP‐γ‐S to activate G proteins significantly attenuated HVACC currents, and the strong depolarization partially relieved GTP‐γ‐S‐induced inhibition. In the neonatal DRG neurons with T‐type Ca2+ currents, the GABAB receptor agonist baclofen but not DAMGO potently inhibited HVACC. Importantly, single‐cell RT‐PCR analysis showed that none of the DRG neurons with T‐type Ca2+ currents had μ‐opioid receptor mRNA. Thus, this study provides novel information about the heterogenous effects of μ‐opioid receptor agonists on HVACC in different populations of sensory neurons. This finding provides an intrinsic cellular mechanism for the selective effect of μ‐opioids on pain transmission.
In vivo receptor occupancy method that simultaneously quantifies four tracers: raclopride, pirenzepine, MDL‐100907, and SB‐221284
Authors: Schmelzer Kara , Craig Darren , Bonhaus Doug W , Tabatabaei Ali
Published: June 2021

The atypical antipsychotics share the common attributes of working on serotonin and dopamine receptors. However, a comprehensive mechanism of action of these agents is unknown. In order to better elucidate the mechanism by which these drugs produce their therapeutic and the undesired effects, a robust method for measuring in vivo receptor binding is needed. The authors developed a liquid chromatography/triple quad mass spectral detectors (LC/MS/MS) method to measure the brain distribution of receptor occupancy using tracers targeting dopamine D2, serotonin 5‐HT2A, 5‐HT2C and muscarinic M1 receptors. The tracers are raclopride, MDL‐100907, SB‐221284 and pirenzepine, respectively. All four non‐radiolabeled tracers were detectable in discrete rat brain areas after intravenous administration. These tracers demonstrated a differential brain distribution corresponding to the regional differences in their respective receptor densities. Oral pretreatment of various antipsychotic agents that occupy these receptors decreased this differential distribution in a dose‐dependent manner. Our results demonstrate the utility of LC/MS/MS to quantify the in vivo receptor occupancy of antipsychotic and other agents acting in the CNS.
HIV‐1 protease inhibitors suppress insulin secretion in pancreatic β cells : role of oxidative stress and endoplasmic reticulum stress and protection by thymoquinone (TQ)
Authors: Chandra Surabhi , Mondal Debasis , Agrawal Krishna C
Published: June 2021

The advent of protease inhibitors (PIs) in highly active anti‐retroviral therapy (HAART) has considerably reduced the mortality in HIV‐1 positive patients; however, HAART is associated with cardiovascular dysfunction, lipodystrophy, and insulin resistance syndrome (IRS). The molecular mechanism(s) of IRS remain largely unknown. We determined the role of PI‐induced oxidative and endoplasmic reticulum (ER) stress in decreasing insulin secretion in rat pancreatic β‐cells (INS‐1). Exposure to the PIs, nelfinavir (5–10 μM) and atazanavir (8–20 μM), but not saquinavir, significantly decreased pre‐pro‐insulin gene expression and insulin secretion (50%) within 24 h. There was a significant increase (50%) in ROS levels and ER stress molecules (Grp78 and phospho eIF2α‐2–2.5 fold) in cells treated with nelfinavir (10 μM) but not with atazanavir. Nelfinavir (10 μM) suppressed (40%) superoxide dismutase (Cu/Zn SOD) and glutathione levels. Simultaneous treatment with TQ (2.5 μM), an active ingredient of black seed oil, significantly inhibited ROS production and ER stress in cells exposed to nelfinavir and increased glucose mediated insulin secretion to normal levels. These results suggest a role of ROS and ER stress in nelfinavir‐induced pancreatic β‐cell dysfunction and demonstrate TQ as a potential therapeutic agent for ameliorating IRS in HAART treated patients. (Supported by NHLBI grant # 1R01 HL73691)
Pharmacogenomic evaluation of a pediatric dextromethorphan fatality following cold medication therapy: the role of cytochrome P450 2D6 polymorphisms
Authors: Tobin Ann A , Little Colleen , Schneider Randal J , Thompson Victoria M , Jannetto Paul J , Wong Steven H , Gummin David D
Published: June 2021

Cold medications are commonly used to alleviate respiratory viral symptoms in children under two years of age, but pose a risk of adverse effects and overdose. Though side effects may be enhanced by immature drug metabolizing enzymes, they may even become lethal when combined with mutations that compromise enzyme function. We identify the role of pharmacogenomic variants of cytochrome P450 (CYP) in a pediatric cold medication fatality. Postmortem toxicological analysis of the infant's blood showed elevated levels of the cough suppressant, dextromethorphan (29.2 ng/ml; adult therapeutic range: 10–40 ng/ml). Genotyping was used to identify mutations in CYP enzymes that metabolize dextromethorphan to dextrorphan (CYP2D6 *2, 3, 4, 5, 6, 7, 8) and 3‐methoxymorphinan (CYP3A4 *1B and CYP3A5 *3) using pyrosequencing. As a confirmatory test, 27 allelic variations of CYP2D6 were genotyped using a Roche CYP450 microarray. The decedent's genotype was CYP2D6 *1/*4 and CYP2D6 *4/*9, respectively. Overall, genotyping identified the child as an intermediate metabolizer of dextromethorphan, a probable cause of the high drug level and death. Genotyping may improve respiratory infection therapy in the neonate by predicting efficacious cold medication doses while minimizing potentially lethal side effects. Supported by grants from the Medical College of Wisconsin, Roche Diagnostics, Abbott Diagnostics and GE Healthcare.
Inhibitive effect of EGCG on MCF‐7 cell migration and invasion
Authors: Fan Biao , Wei Lei , Zhang Yimin , Han Guoge , Dong Huimin , Wang Yongping , Zhou Xuan
Published: June 2021

Epidemiological studies have described the beneficial effects of dietary green tea on the reduction of the risk of breast cancer. We surveyed the role of green tea catechin epigallocatechin gallate (EGCG) in the metastasis of MCF‐7 cell and analyzed the possible mechanism of EGCG's ability to inhibit breast cancer metastasis. To determine the effect of EGCG on the migration of MCF‐7 cells, we used would‐healing assay. The results show that the cell migration velocity was reduced by EGCG at 50μg/mL (P<0.05). In transwell invasion assay, the EGCG group showed lower invasion capacity than the control group significantly (P<0.05). The activity of Rac1 pathway decreased after treating with EGCG, which was estimated by GST pull‐down assay. On the contrary, the expression level of VASP was incresaed by Rac activator fMLP, while migration velocity of MCF‐7 cells was increased significantly. Furthermore, the result revealed that knockdown of Rac1 by siRNA decreased the expression of VASP. Thus, our study demonstrated the inhibitive effect of EGCG on MCF‐7 cell migration and invasion, which may be produced by down regulated of VASP expression level via Rac1 pathway. (This work was supported by National Natural Sciences Foundation of China under Grant No.30770966)
Preventive effect of α‐lipoic acid and ebselen on rat intestine against ischemia/reperfusion injury
Authors: Tunc Turan , Guven Ahmet , Topal Turgut , Kul Mustafa , Korkmaz Ahmet , Gundogdu Gokhan , Onguru Onder , Ozturk Haluk
Published: June 2021

Purpose: Reactive oxygen species (ROS) and reactive nitrogen species(RNS) generated during reperfusion of the tissue are characteristic of ischemia and reperfusion (I/R) injury. The present study was designed to evaluate whether a‐Lipoic Acid (a‐LA) and Ebselen have protective effect in intestinal I/R injury.

Methods: Fourty Sprague‐Dawley rats were divided into five groups equally: Group‐1 Sham‐operated; Group‐2 I/R; Group‐3 I/R+a‐LA; Group‐4 I/R+Ebselen; Group‐5 I/R+a‐LA+Ebselen. Intestinal ischemia for 45 min and reperfusion for 3 days were carried out. Ileal specimens were obtained to determine the tissue levels of Malondialdehide (MDA), Protein Carbonyl (PC) content, Superoxide dismutase (SOD) and Glutation Peroxidase (GPx) and histologic changes.

Results: There was a statistically significant decrease in SOD and GPx levels, with an increase in MDA and PC content and intestinal mucosal injury in intestinal I/R group (p<0.05). a‐LA and Ebselen led to a significantly increase in SOD and GPx level, with a decrease in MDA and PC content and intestinal injury when compared with I/R group, (p<0.05). Although shortness of villous and epithelial lifting was seen in the rats subject to I/R, there was slightly injury in mucosa in treatment groups.

Conclusion: a‐LA and Ebselen played significant protective role in attenuating I/R injury of the intestine by scavenging ROS and RNS.
Imaging of synaptically‐evoked blood volume and blood oxygenation optical changes in rat somatosensory cortex
Authors: Winn Richard H. , Meno Joseph , Hochman Daryl
Published: June 2021

The objective of these studies was to determine the extent to which optical imaging can dissociate changes due to either blood volume or blood oxygenation during synaptic activation of the cortex. Towards that aim, a closed cranial‐window rat preparation was used that allowed for optical imaging of somatosensory cortex during stimulation of a sciatic nerve. High‐magnification imaging using 550 nm light showed that optical changes were restricted to the dilating pial arterioles (thus representing blood volume changes), whereas the changes occurring under 660 nm light were restricted to the venous compartments (thus representing changes in blood oxygenation). A simple linear model revealed that blood volume optical changes acquired with 550 nm light were tightly correlated to the diameters of the pial arterioles (p < 0.001). Low‐magnification imaging showed that blood volume changes were closely localized to cortical regions undergoing increased neuronal activity. In contrast, blood oxygenation changes were maximal in the larger veins at sites distant to the areas of increased neuronal activity. In conclusion, these data demonstrate that optical imaging can dissociate changes due to either blood volume or blood oxygenation by selecting the appropriate optical wavelength. Further, optical images of blood volume provide a more accurate localization of neuronal activity than do those of blood oxygenation.
Perinatal taurine exposure alters renal potassium excretion in adult conscious rats
Authors: Roysommuti Sanya , Malila Pisamai , Lerdweeraphon Wichaporn , Jirakulsomchok Dusit , Wyss J. Michael
Published: June 2021

Perinatal taurine exposure alters renal hemodynamics and sodium excretion in adult rats. This study further tests the effect of perinatal taurine on renal potassium excretion in adult conscious rats. Female Sprague‐Dawley rats were fed normal rat chow and given water alone (C), 3% beta‐alanine in water (taurine depletion, TD) or 3% taurine in water (taurine supplementation, TS), either from conception until delivery (fetal period; TDF or TSF) or from delivery until weaning (lactation period; TDL or TSL). In Experiment 1, after weaning, male offspring were fed normal rat chow and tap water. In Experiment 2, female pups were treated similarly, but from conception until weaning. At 7–8 weeks of age, renal potassium excretion at rest and after an acute saline load (5% of body weight) was studied in conscious, restrained rats. All male groups displayed similar renal potassium excretion. TSF increased fractional potassium excretion at rest but not in response to saline load, whereas TDF did the opposite. Plasma potassium levels were lower only at rest in TSF but at rest and during the challenge in TDF. Their glomerular filtration rates did not significantly different among groups. In female offspring, perinatal taurine supplementation decreased renal potassium excretion, glomerular filtration rate, and plasma potassium concentration at rest and after the saline load. It is unlikely that increased tubular reabsorption caused these effects, since the fractional potassium excretion significantly increased, especially at rest. Perinatal taurine depletion had no significant effect on renal potassium excretion in the female offspring. The present data suggest that perinatal taurine exposure influences renal potassium excretion in the adult rats.
Skeletal muscle fiber type switching and atrophy in diabetes mellitus
Authors: Donaldson Sue K , Price S Russ , Li Haiyan , Eaton Eric , Roberts Tiffany K
Published: June 2021

This study demonstrates that switching of mammalian skeletal muscle fibers, identified as to Myosin Heavy Chain (MHC) type, from MHCI to MHCII occurs along with selective MHCII fiber atrophy in Diabetes Mellitus (DM). DM characterized by reduced plasma insulin level was induced by streptozotocin (40 mg/kg body wt STZ in buffer) injection of 6 wk old male Sprague‐Dawley rats; no exogenous insulin was administered. Rats, matched as to birth date and weight, were randomly assigned to test STZ‐DM or Control (injected with buffer only) group; after 3 wks the STZ‐DM (2) and Control rats (2) were sacrificed and their white gastrocnemius (GAST) and soleus (SOL) muscles excised. A 3–5 mm transverse section of each excised muscle was immediately cut and flash frozen (−80°C). The frozen tissue was cut into 12 μm cross‐sections in a cryostat; fibers in cross‐section were stained and identified as to type using 3 different MHC antibodies in serial cross‐sections. For each SOL and GAST cross‐section, 100 contiguous fibers were MHC typed. The proportions and CSA of combined MHCIIa, x/d,b and MHCI (nonhybrid) fibers were analyzed for GAST and SOL, comparing STZ‐DM to Control. MHCI to MHCII type switching in STZ‐DM was significant (p<0.05) only for SOL, although it was evident in GAST. CSA of only the STZ‐DM MHCII fibers was significantly (p<0.05) decreased in both SOL and GAST. Supported by Emory University (SKD) and R01 DK50740‐09 (SRP).
Angiotensin receptor binding in rostral ventrolateral medulla (RVLM) is reduced by destruction of the C1 neurons
Authors: Bourassa Erick A. , Stedenfeld Kristen A. , Li Xia , Speth Robert C. , Sved Alan F.
Published: June 2021

The RVLM, a brainstem site critical in cardiovascular regulation, contains angiotensin Type 1 receptors (AT1R) and stimulation of these receptors increases arterial pressure. Elevated stimulation of these receptors may contribute to sympathoexcitation seen in some rat models of hypertension, as injection of AT1R antagonists into RVLM decreases arterial pressure in these hypertensive models. Within the RVLM, there are two groups of spinally‐projecting sympathoexcitatory neurons based on neurochemical phenotype: those that contain catecholamine biosynthetic enzymes, termed C1 cells, and those that do not. Although evidence supports the hypothesis that the C1 and the AT1R‐expressing populations of the RVLM overlap, this hypothesis has not been tested. Local injection of saporin toxin conjugated to a dopamine‐beta‐hydroxylase antibody(antiDBH‐Sap) selectively destroys C1 cells. Using this lesioning approach, we examined the effect of C1 cell depletion on density of AT1R binding sites in the RVLM. Autoradiographic localization of (125)‐I‐sarcosine(1), isoleucine(8) Ang II revealed high affinity AT1R binding in the RVLM. Selective unilateral lesions of C1 neurons in the RVLM reduced AT1R binding in the RVLM compared to the unlesioned side and the RVLM of rats injected with a control toxin. These data suggest that a large proportion of AT1R in the RVLM are located on C1 neurons. (NIH HL‐55687, HL‐76083)
On the reproducibility of oxygen uptake kinetics during heavy exercise
Authors: Cannon Daniel T. , Schenone Aaron , Kolkhorst Fred W
Published: June 2021

While the equations of Lamarra and colleagues remain the standard for a priori determination of adequate exercise transitions, if subject/patient populations do not allow for adequate laboratory visits, parameter estimation based on a single transition may be required. Thus, the purpose of this study was to examine the short‐term reproducibility of oxygen uptake kinetics during heavy intensity cycling. Fourteen amateur competitive cyclists (mean ± SE age = 30 ± 1.9; V̇O2max = 4.477 ± 0.280 l min−1) completed three constant workrate bouts in the heavy intensity domain. Breath‐by‐breath data were filtered (2.58 SD), combined, averaged, interpolated and characterized with least‐squares non‐linear regression modeling. No significant differences (P > 0.05) were observed across the 3 trials for the fundamental time constant (τ) (F(2,26) = 2.771), fundamental amplitude (F(2,26) = 0.013), or slow component magnitude (F(2,26) = 0.751). Intraclass correlation coefficient revealed good reliability for the fundamental τ (ICC(2,1) = 0.86), fundamental amplitude (ICC(2,1) = 0.98), and slow component (ICC(2,1) = 0.92). While confidence in parameter estimation may be greatly improved by combining multiple exercise transitions, these data indicated good reproducibility and the possibility of utilizing single exercise transitions. This research was not supported by external financial sources.
TRPV calcium‐permeable channels mediate mechanosensitive calcium reabsorption in renal cortical collecting duct cells
Authors: O'Neil Roger G , Wu Ling , Jin Min
Published: June 2021

The role of mechanosensitive TRPV channels in transepithelial calcium (Ca) reabsorption was evaluated in mouse cortical collecting duct (CCD) cells, M‐1. RT‐PCR and immunoblots revealed expression of TRPV2 and TRPV4, both swelling‐activated channels, in M‐1 cells while immunohistochemistry demonstrated TRPV2 and ‐4 expression at the plasma membrane. Immunofluorescence studies of mouse kidney cortical sections showed strong expression of TRPV4 at the luminal membrane (TRPV2 to be tested). Exposure of cells to hypotonic media (260 mOsm) induced a marked initial rise in intracellular Ca (fluorescence Ca imaging) that was inhibited by ruthenium red (RR, 1–10 μM), a TRPV channel blocker, while hypotonic exposure to cells grown on permeable supports induced a marked 3‐fold increase in net transepithelial Ca reabsorption (baseline = 7.8 +/− 0.8 nmol/cm2/hr) that was partially inhibited by RR. Knockdown of TRPV4 by siRNA techniques largely abolished the hypotonic‐induced rise in intracellular calcium, implicating a role for TRPV4 in calcium signaling and reabsorption. A potential supporting role of TRPV2 is being investigated. It is concluded that TRPV4 (and potentially TRPV2) is a key component of hypotonicity‐induced calcium signaling and appears to underlie hypotonicity‐induced calcium reabsorption in mouse CCD. (NIH grant DK070950)
ATP Potentiates Insitol‐1,4,5‐triphosphate‐induced Calcium Release in Permeabilized Parotid and Pancreatic Acini
Authors: Park Hyungseo , Betzenhauser Matthew J. , Won JongHak , Wagner Larry E. , Knowlton Lyndee M. , Yule David I.
Published: June 2021

A pivotal event underlying exocytosis from exocrine cells is Ca2+ mobilization from intracellular stores initiated by Insitol‐1,4,5‐triphosphate (InsP3) binding to InsP3 receptors (InsP3R). InsP3R gating is regulated by a diverse array of regulatory inputs. We aimed to investigate the effects of ATP on InsP3‐induced Ca2+ release in permeabilized parotid and pancreatic acini. ATP dependency is known to differ between InsP3R subtypes. Type II InsP3R is predominantly present in parotid gland, and both Type II and III InsP3R are expressed in pancreas. Following the SERCA driven filling of Ca2+ stores, 0.1 μM to 10 μM InsP3 was infused with or without ATP in β‐escin permeabilized acini. InsP3 increased the Ca2+ release rate in a concentration‐dependent manner in both parotid and pancreatic acini. ATP (5 mM) significantly enhanced Ca2+ release at sub‐maximal [InsP3]. Next, the ATP dependency of the InsP3‐induced Ca2+ release was tested. ATP dose‐dependently potentiated the 0.3 μM InsP3‐induced Ca2+ release rate in both parotid and pancreatic acini. However, the ATP concentration dependency was shifted to the left in parotid acini. These results provide evidence that ATP can enhance InsP3‐induced Ca2+ release in both parotid and pancreatic acini and that the complement of InsP3R in parotid acini is more sensitive to this form of regulation.
Ectopic Hepcidin Production in Anemia of Cancer
Authors: Yu Alice , Pastuszka Martha Katherine , Ganz Tomas , Rivera Seth
Published: June 2021

Anemia is a frequent manifestation of almost all cancers. In some cases, the etiology of the anemia is clear (bleeding, bone marrow infiltration, or chemotherapy). When none of these factors are present, the anemia is referred to as anemia of cancer (AC). Hepcidin is a hormone synthesized in the liver that inhibits iron absorption from the diet and iron release from stores. In 2 patients with hepcidin secreting hepatic adenomas, AC resolved when the tumors were removed (Weinstein et al, Blood, 2002). However, it is unclear whether tumors not of hepatic origin can make hepcidin or whether this mechanism contributes to AC. We created two models of metastatic lung cancer by injecting two syngeneic lung cancer cell lines (TC‐1 and LCC) into the peritoneal cavities of C57Bl6 mice. Our data shows that hepcidin production is increased in both the liver and the tumor from TC‐1 injected mice. Inflammatory markers, SAA‐1 and FGN‐ã, are also elevated, indicating that the rise in hepatic hepcidin is likely due to inflammation. LLC‐injected mice developed less severe anemia and the increase in hepcidin mRNA was not significant. LLC tumors expressed measurable amounts of hepcidin but significantly less than TC‐1 tumors or control livers. Inflammatory induction of hepcidin likely plays an important role in AC. Some tumors express hepcidin, which could contributing to AC.
The Role of Potassium Channels in Neuronal Differentiation of Stem Cells from Umbilical Cord Matrix
Authors: Lemming Matthew T. , Hoynowski Steven M. , Xu Yan , Mitchell Kathy E.
Published: June 2021

Potassium channels play a critical role in neurogenesis and establishing electrical excitability of neurons in model organisms. Much less is known about the involvement of potassium channels in human development. We have investigated the potential role of potassium channels in neurogenesis of stem cells isolated from human umbilical cord matrix. We hypothesize that potassium channels expressed in stem cells play an important role in both development and repair in the brain. Here we demonstrate expression of a number of potassium channels during the course of neuronal differentiation of stem cells derived from human umbilical cord matrix based on immunodetection and whole cell patch clamp analysis of potassium currents. We explored the effects of blocking potassium channels that were identified on neuronal differentiation. Our findings show that blocking different potassium channels had varying effects with blocking the large conductance calcium activated potassium channel being most profound. Neuronal differentiation was inhibited based on the lack of morphological changes into neuronal phenotypes and the altered expression of a number of genes associated with neuronal differentiation. These results suggest that potassium channels play a critical role in differentiation of stem cells into neuronal phenotypes. Funded by NIH Grant Number P20 RR016475, NIH (R01NS036124) and the State of Kansas.
Nuclear localization signal peptides enhance transfection efficiency of chitosan/DNA complexes delivery
Authors: Rojanarata Theerasak , Opanasopit Praneet , Apirakaramwong Auayporn , Ngawhiranpat Tanasait , Ruktanonchai Uracha
Published: June 2021

The purpose of this study was to investigate the effect of nuclear localization signal “KPKKKRKV” (NLS) on the enhancement of transfection efficiency of chitosan/DNA complexes delivery. The gene transfer efficiency of chitosan/DNA complexes with and without NLS was evaluated in a human cervical carcinoma cell line (Hela cells). The chitosan/DNA complex containing NLS increased transfection efficiencies in a NLS‐dose dependent manner on Hela cells, compared to control (chitosan/DNA complex or NLS). Cytotoxicity of the NLS/chitosan/DNA complexes increased as the amount of the peptide increased, however, over 80 % average cell viability was observed for complexes at the effective concentration of the peptide for transfections. Therefore, the NLS is expected to be a potent transfection enhancing agent without a covalent modification of pDNA or chitosan. Our findings suggest that the high gene expression with the negligible cytotoxicity can be achieved by adding the NLS peptide to chitosan/DNA complexes at optimal ratio. This research was supported by the Thailand Research Fund and Silpakorn University Research and Development Institute, Thailand.
Numerical Modeling of Coil Compaction in the Treatment of Cerebral Aneurysms using Porous Media Theory
Authors: Khanafer Khalil , Berguer Ramon , Schlich Marty , Bull Joseph
Published: June 2021

A numerical study was conducted to quantify the reduction in blood velocity and pressure resulting from the placement of endovascular coils within a cerebral aneurysm using physiological velocity waveforms. The flow characteristics within the aneurysm sac were modeled using the volume‐averaged porous media equations. We studied the effects of narrow and wide aneurismal necks on the velocity fields and pressure within its sac in the absence of the coils. Wide‐neck aneurysms experience higher velocity and pressure than narrow‐neck aneurysms within the sac at peak systole. Our study shows that velocity fields are significantly affected by the presence of endovascular coil within the aneurysm sac. Moreover, we estimated that a volume density of 20% platinum coil in the aneurysmal sac was sufficient to cause sufficient blood flow arrest in the aneurysm to allow for thrombus formation. The present model, using porous media theory, permits the study of fluid motion across small spaces of variable and complex geometry. Additional refinements will be required to describe the permeability of a coil inserted in a patient based on its porosity, geometry and shape.
Central nitric oxide participates in the lipopolysaccharide‐induced tolerance in periodontal tissues of rats
Authors: Tedeschi Valéria Pontelli Navarro , Branco Luiz Guilherme Siqueira
Published: June 2021

Endotoxin tolerance is defined as an attenuation of the febrile response to repeated administrations of LPS. In this study, we tested the hypothesis that LPS tolerance occurs after repeated local injection of LPS into the periodontium of rats and that nitric oxide plays a role in this tolerance. Rats were given a gingival injection of saline or LPS (100 μg/kg) on 3 days. Body temperature was measured with a datalogger. Another group was treated with L‐NMMA, 500μg/rat i.c.v. 30 min before LPS in the 3rd day. Fos immunohistochemistry was performed 3 h after the injections in non‐tolerant rats (day 1) and in tolerant rats (day 3), after i.c.v. saline or L‐NMMA. On day 1 we observed a polyphasic febrile response after LPS injection, on day 2 this response was sensitized and on day 3 it was completely abolished, characterizing the development of LPS tolerance. This tolerance could be prevented with the pretreatment with L‐NMMA (p<0.05). Additionally, immunohistochemistry detected a reduction in LPS‐induced Fos‐like expression in the caudal subnuclei of the spinal trigeminal nucleus and in the preoptic area of hypothalamus in tolerant rats compared with non‐tolerant rats, and an enhanced of Fos positive cells in both areas after the reversion of tolerance with L‐NMMA (p<0.05). These results indicate that the LPS tolerance may occur locally in the oral cavity and central NO plays a role in this mechanism. Finantial support: FAPESP.
Short term consumption of diets high in fat and/or sugar in young animals increase cardiovascular risk factors prior to the onset of obesity
Authors: Marsh Susan A , Rosiers Christine Des , Bouchard Bertrand , Asselin Caroline , Dell’Italia Louis J , Chatham John C
Published: June 2021

The incidence of obesity, type 2 diabetes and cardiovascular disease in children is increasing and is exacerbated by a “western” diet. We examined the effect of short term consumption of high fat and/or sugar diets on cardiovascular risk factors in young male rats that received control (CON; 12% kcal fat/19% protein/69% carbohydrate), western (WES; 40/15/45%) or high fat (HF; 60/19/21%) diets for 2 weeks. Insulin sensitivity was lower in WES (34%) and HF (68%) than CON while blood glucose remained similar. Serum cholesterol and triglyceride levels were elevated in HF (43%) and WES (40%) respectively as was plasma xanthine oxidase activity (HF: P<0.05; WES: P=0.059) compared to CON; however there were no differences in blood levels of the lipid peroxidation product 4‐hydroxynonenal bound to proteins. Despite a lower left ventricular (LV) weight (P<0.05) and a trend towards higher collagen content in WES (P=0.053), there were no differences in LV morphology or function. Arterial stiffness tended to be higher in both WES (−5 ± 3%; P=0.081) and HF (−5 ± 2%; P=0.097) compared to CON (−12 ± 1%) with no differences in mean arterial pressure. LV mitochondrial Krebs cycle enzyme activities (citrate synthase, aconitase) were depressed in both groups. These data highlight early changes in some cardiovascular risk factors after short‐term consumption of diets high in fat and/or sugar in young animals prior to the onset of obesity.
Modulatory effect of carotid chemoreceptors tonic activity on the eupneic drive to breathe and on the central response to hypoxia and hyperoxia
Authors: Blain Gregory , Smith Curtis A. , Henderson Kathleen S. , Dempsey Jerome A.
Published: June 2021

We assessed the contribution of carotid chemoreceptors tonic activity (CC) on control of eupnea and on the ventilatory response to brain hypoxia and hyperoxia in the unanesthetized awake dog. We used extracorporeal blood perfusion of the reversibly isolated carotid sinus to maintain normal (normoxic, normocapnic perfusate) or to inhibit (hyperoxic, hypocapnic perfusate) CC, while the systemic circulation, and therefore the central nervous system (CNS), was normoxic, hypoxic (PaO2 = 28, 35 and 44 Torr) or hyperoxic (PaO2 > 600 Torr). We found that 1) CC inhibition during normoxia was accompanied by immediate hypoventilation (from 3.8 to 2.5 L/min) and hypercapnia (from 41 to 48 Torr) that was sustained for more than 5 min. 2) Severe CNS hypoxia (PaO2 = 28 Torr), per se, caused immediate and sustained hyperventilation (> + 1.7 L/min) and hypocapnia (> − 2.5 Torr), all due to breathing frequency increase, whether the isolated carotid body was maintained normal or inhibited. 3) CNS hyperoxia (PaO2 > 600 Torr), per se, caused hyperventilation (+ 0.8 L/min) and hypocapnia (− 4 Torr) solely via increase in tidal volume, when the isolated carotid body was maintained normal. We conclude that the CC contributes substantially to the maintenance of the tonic eupneic drive to breathe in the intact awake dog and that central severe hypoxia or hyperoxia can significantly increase ventilatory drive independent of the CC. NHLBI/NIH.