bluuebunny/crossref_metadata_2025 huggingface dataset:
The A563T variation of TRPV5 among African Americans significantly enhances TRPV5‐mediated calcium influx
Authors: Na Tao , Zhang Wei , Jiang Yi , Peng Ji‐Bin
Published: June 2021

The TRPV5 gene, which encodes the renal epithelial Ca2+ channel, exhibits unusual high frequencies of non‐synonymous single nucleotide polymorphisms (SNP) among African Americans. To evaluate the functional impact of the non‐synonymous SNP variations in TRPV5, A8V, C157R, A563T and L712F variations were introduced to TRPV5 individually, and their abilities to mediated Ca2+ influx and Na+ current were tested using X. laevis oocytes. Among these variants, A563T, which localizes in the last transmembrane domain of TRPV5, exhibited a significant increase in Ca2+ influx with a concomitant decrease in Na+ current as determined by radiotracer uptake and two‐microelectrode voltage clamp, respectively. When threonine 563 was replaced by a serine, Na+ current of TRPV5563S was not much altered while the Ca2+ influx was reduced as compared to TRPV5563T. In contrast, when threonine 563 was replaced by a valine, Ca2+ influx of TRPV5563V was not much altered while the Na+ current was increased as compared to TRPV5563T. It appears that the hydroxyl group of Thr563 may hinder the transport of Na+ through the channel while the bulky side chain of Thr563 may facilitate that of Ca2+. In conclusion, the A563T variant of TRPV5 significantly increases Ca2+ influx by altering its cation selectivity. The A563T variation in TRPV5 may contribute to the lower prevalence of hypercalciuria, kidney stone, and osteoporosis among African Americans.

Supported by NIH grant DK072154
Activation of cAMP‐dependent protein kinase (PKA) down‐regulates organic anion transporter 6 function
Authors: Schnabolk Gloriane Willene , Youngblood Geri L. , Sweet Douglas H.
Published: June 2021

Organic anion transporter 6 [Oat6 (Slc22a20)], a member of the Organic Anion Transporter family uniquely expressed in the testis, mediates the transport of sulfated steroid conjugates. Sequence analysis revealed three putative PKA phosphorylation sites within Oat6. In the current study we analyzed the regulation of PKA on Oat6‐mediated transport using a Chinese Hamster Ovary cell line stably expressing murine Oat6 (CHO‐mOat6). The PKA activator 8‐bromoadenosine (8‐Br‐cAMP) inhibited Oat6‐mediated uptake in a dose and time dependent manner. Preliminary results indicated that pre‐incubation (5 min) of CHO‐mOat6 cells with the PKA inhibitor H‐89 fails to block 8‐Br‐cAMP induced inhibition of Oat6. Using site directed mutagenesis, each PKA site was individually altered and transport activity of the mutants analyzed. Two of the three mutants showed unaltered substrate uptake, whereas the third showed a significant decrease. To confirm expression and plasma membrane targeting of the mutant Oat6 proteins a FLAG tag epitope was fused to Oat6. Introduction of the FLAG tag had no effect on Oat6 transport activity. Immunocytochemistry indicated there was no degradation or mis‐targeting of Oat6 as a result of mutation. These findings suggest that Oat6‐mediated transport of steroid conjugates in the testes may be regulated by PKA through an indirect mechanism.

Support: NIDDK RO1‐DK067216, T32 ES012878
The human organic cation transporter 2 (hOCT2) transports cadmium (Cd2+) and mediates Cd2+ induced cell death
Authors: Thevenod Frank , Ciarimboli Giuliano , Wolff Natascha A. , Schlatter Eberhard , Koepsell Hermann
Published: June 2021

Kidney proximal tubule (PT) is a major target of chronic Cd2+ nephrotoxicity and may take up free or bound Cd2+forms. Free Cd2+ uptake pathways are not completely understood. The electrogenic polyspecific OCTs are expressed at the apical and basolateral plasma membrane of PT cells where they translocate small organic cations with one positive charge. Here we investigated Cd2+ as a substrate for OCT using human OCT1‐3 stably transfected in HEK293. 109Cd2+ uptake was only observed in hOCT2 cells, where it was linear at 10 or 100 μM free Cd2+ for < 60 min.However, uptake was saturable with increasing Cd2+ with an apparent Km of 53.7 ± 7.2 μmol/l and Vmax of 481.9 ± 31.34 μmol/l. OCT inhibitors/substrates cimetidine, quinine and choline (IC50 50 nM, 0.4 μM and 0.8 mM) blocked 109Cd2+ uptake. Cu2+ and Zn2+, but not Fe2+ and Al3+, competed 109Cd2+ uptake (IC50 0.5 and 8.0 μM). Uptake of 1 μM ASP+, a model substrate for hOCT, was competed by Cd2+ and Zn2+ (IC50 205 and 60 μM) in hOCT2 cells. 100 μM Cd2+ uptake into hOCT2 cells for 2.5 h significantly increased apoptosis by annexin V labelling. Using MTT assay, cell viability was significantly reduced by 10 or 100 μM Cd2+ for 2.5 h or 16 h in hOCT2 cells. Cd2+ induced cell death was abolished by cimetidine. The data demonstrate for the first time that hOCT2 transports the divalent metal Cd2+, where it could contribute to subsequent toxicity.

Funded by DFG TH 345/8‐1 and TH 345/10‐1
Urinary excretion of both D‐ and L‐lactate using a calf‐infusion model
Authors: Zello Gordon Alexander , Janzen Alisha , Abeysekara Saman , Hamilton Don , Naylor Jonathan M. , Lohmann Katharina L.
Published: June 2021

D‐lactic acidosis is recognized as a complication of gastrointestinal diseases in humans and ruminants. Absorbed D‐lactate can be excreted via the kidney; however, competition between D‐ and L‐lactate in the renal mechanisms may effect its elimination. The objective of this study was to measure D‐ and L‐lactate clearance in a calf infusion model. Five calves (age 14–31 days, weight 59 ± 8 kg) were infused with DL‐ and L‐lactic acid (300 mmol/L, 2 mL/kg/h for 8 hours) in a randomized cross‐over design. Calves were fitted with intravenous catheters for infusion and blood collection, and with suprapubic bladder catheters for urine collection. Samples for blood gas analysis, serum and urine D‐ and L‐lactate concentration, urine volume and serum and urine creatinine were collected every 2 hours for 24 hours. D‐ and L‐lactate clearances were calculated for each 2 hour interval and compared to creatinine clearance. With infusion of DL‐lactate, average serum D‐lactate concentration at steady state was 2.9 ± 1.6 mmol/L (range 1.8–5.7 mmol/L), and average urine D‐lactate concentration was 4.0 ± 1.7 mmol/L. Average D‐lactate clearance during steady state was 7.4 ± 3.4 mL/min. Percent excretion of infused D‐lactate increased during the 8 hour infusion, with minimum and maximum excretion averaging 0.6 ± 0.1% and 10.4 ± 4.4%. L‐lactate clearance could not be calculated, likely due to rapid metabolism of infused L‐lactate. Therefore, L‐lactate elimination is more efficient than D‐lactate since it can both be metabolized and excreted from the body. (Supported by NSERC Canada)
Role of the T‐type calcium channel in the spontaneous phasic contraction of pregnant rat uterine smooth muscle
Authors: Lee Si‐eun , Ahn Duck‐sun , Kang Bok‐soon , Lee Young‐ho
Published: June 2021

We investigated the role of the T‐type Ca2+ channel in spontaneous phasic contraction (SPC) of the rat myometrium. SPC and [Ca2+]i were measured simultaneously in longitudinal strips of female rats in late pregnancy. Expression of T‐type Ca2+ channel subunits was examined by using RT‐PCR technique. The SPC and [Ca2+]i were completely inhibited by removal of external Ca2+and treatment of nifedipine. T‐type Ca2+ channel blocker, 1μM mibefradil and 5μM NNC 55‐0396 evoked concentration‐dependent inhibition of SPC and [Ca2+]i and these blockers decreased the amplitude and frequency of SPC as well as [Ca2+]i. On the other hand, 100μM nickel, a blocker of T‐type Ca2+ channel, decreased the frequency but not the amplitude of SPC. Pretreatment of mibefradil and NNC 55‐0396 significantly attenuated the KCl‐induced increase in contraction and [Ca2+]i, respectively, but nickel had no effect on KCl‐induced increase in contraction and [Ca2+]i. All of three T‐type Ca2+ channel blockers decreased the slope of rising phase of SPC and [Ca2+]i. In RT‐PCR analysis, T‐type Ca2+ channel subunits, Cav3.1 and Cav3.2, were expressed in pregnant rat myometrium. The present study demonstrates that T‐type Ca2+ channel involves in the generation of SPC of pregnant rat myometrium. Furthermore, Ca2+influx through T‐type Ca2+ channel may play a key role for the slow depolarization.
Probing endogenous coenzyme Q10 (CoQ10) redox status in intact pulmonary arterial endothelial cells using the CoQ10 homolog, coenzyme Q1 (CoQ1)
Authors: Krenz Gary , Audi Said H. , Bongard Robert D. , Lindemer Brian J. , Merker Marilyn P.
Published: June 2021

The exogenous quinone, CoQ1, is reduced predominately via mitochondrial complex I (CI) in intact, cultured bovine pulmonary arterial endothelial cells (PAECs), wherein the hydroquinone (CoQ1H2) product can be oxidized at complex III or appear in the extracellular medium. We hypothesized that insofar as CoQ1 is a homolog of the endogenous CI electron acceptor, CoQ10, CoQ1 reduction kinetics might reflect competition with oxidized CoQ10 for reduction at CI and provide information regarding CoQ10 redox status. Cell‐mediated CoQ1 reduction rates vs CoQ1 concentration (1–50 μM) were obtained in the presence of cyanide to eliminate CoQ10 as a CoQ1 competitor. A CoQ10 competition model was fit to cell‐mediated CoQ1H2 oxidation and CoQ1 reduction vs time and CoQ1 reduction rates vs CoQ1 concentration curves in the absence of cyanide. The model fit was consistent with CoQ10 competing with CoQ1 for reducing equivalents at CI. The 95% nonlinear confidence region for CoQ10 participating in competition for reducing equivalents at CI was 7–31 mM. The nonlinear confidence region provides an estimate of CoQ10 redox poise, % CoQ10H2 / (CoQ10H2 + CoQ10) of 57–64%, which is consistent with values reported for total tissue CoQ10 in various organs. The study reveals the potential utility of CoQ1 as a probe of CoQ10 redox status, which may be altered in oxidative stress and lung disease, in PAECs. Funding: HL‐65537,‐24349 and Dept of Veterans Affairs.
Contribution of ASIC to Increased Pulmonary Vascular Smooth Muscle Resting [Ca 2+]i and Store‐Operated Ca2+ Entry Following Chronic Hypoxia
Authors: Jernigan Nikki L. , Walker Benjimen R. , Resta Thomas C.
Published: June 2021

Increases in basal pulmonary vascular smooth muscle (VSM) intracellular Ca2+([Ca2+]i) and store‐operated Ca2+ entry (SOCE) have been implicated in mediating the vasoconstrictor and arterial remodeling components of chronic hypoxia (CH)‐induced pulmonary hypertension. The goal of the present study was to characterize a role for the unique class of ion channels, acid sensing ion channels (ASICs), in regulating basal VSM [Ca2+]i and SOCE in the hypertensive pulmonary circulation. We hypothesized that enhanced Ca2+ entry through ASIC contributes to elevated basal VSM [Ca2+]i and increased SOCE following CH. Inhibition of ASIC with amiloride or benzamil attenuated SOCE and associated vasoconstriction in fura‐2‐loaded, endothelium‐disrupted, pressurized small pulmonary arteries from control rats. Furthermore, siRNA knockdown of ASIC1, but not ASIC2 or ‐3, diminished SOCE in primary cultures of rat pulmonary VSM cells. Resting [Ca2+]i and SOCE were enhanced in isolated small pulmonary arteries from CH rats (4 wk at 0.5 atm) vs. controls. Benzamil reduced basal [Ca2+]i in arteries from both groups, but caused a greater reduction in resting [Ca2+]i in arteries from CH rats compared to controls. In addition, benzamil attenuated the increase in SOCE in CH arteries and normalized responses between groups. Consistent with these findings, CH upregulated pulmonary vascular ASIC1, but not ASIC2 or ‐3 expression as determined by Western blotting. These studies support a novel role of ASIC to mediate elevated basal VSM [Ca2+]i and increased SOCE following CH.
Electrophysiological characterization of cells isolated from endarterectomized tissue from patients with chronic thromboembolic pulmonary hypertension (CTEPH).
Authors: Firth Amy L , Yao Weijuan , Ogawa Aiko , Platoshyn Oleksandr , Remillard Carmelle V , Sacks Richard S , Auger William R , Madani Michael , Thistlethwaite Patricia A , Jamieson Stuart W , Yuan Jason X‐J
Published: June 2021

Despite having well defined clinical directives, the pathogenic mechanisms involved in CTEPH are currently unknown. Ion channels play critical roles in regulating the contractility, proliferation and migration of vascular smooth muscle cells. This study characterizes the electrophysiological profile of cells isolated from endarterectomized and biopsy tissue from patients undergoing pulmonary thromboendarterectomy (PTE). Using patch clamp techniques, currents were recorded from isolated cells using conditions specifically designed to isolate K+, Ca2+ and nonselective cation currents (NSCC).

Four kinetically different voltage‐dependent K+ currents, which were inhibited by 4‐AP and TEA, were recorded in the PTE cells. Very large NSCC were present in virtually all cells and had a linear current‐voltage relationship reversing close to 0mV. The currents were sensitive to extracellular H+ and La3+, but relatively insensitive to 2‐APB and niflumic acid. No voltage‐dependent Ca2+ currents were detected.

The electrophysiological profile may suggest a novel pathway in the enhancement of intracellular Ca2+ in cells isolated from PTE tissue. NSCC may be enhanced; the resultant rise in cytosolic Na+ may activate the Na+–Ca2+ exchange in the reverse mode to further increase Ca2+ and contribute to vasoconstriction and remodeling of the pulmonary arteries in CTEPH patients.
Endothelin‐1 (ET‐1) induces hypoxia‐inducible factor 1 (HIF‐1) in Pulmonary Arterial Smooth Muscle Cells (PASMCs).
Authors: Pisarcik Sarah , Sylvester J T , Semenza Gregg L , Shimoda Larissa A
Published: June 2021

Numerous cellular responses to hypoxia are mediated by the transcription factor, HIF‐1. Recent data suggest that, under certain conditions, HIF‐1 may require feed forward for full expression (Peng, et al. J Physiol, 2006). Recently, ET‐1 was found to increase HIF‐1 levels in tumor cells (Spinella, et al. Cancer Res 2007). Since hypoxia increases ET‐1 levels in the lung, we hypothesized that during moderate prolonged hypoxia ET‐1 might feed forward and amplify activation of HIF‐1 in PASMCs. Primary cultures of rat PASMCs were treated with ET‐1 (10−8M; 48 hr) or exposed to hypoxia (4% O2; 60 hrs). Protein levels of the oxygen‐sensitive α subunit of HIF‐1 (HIF‐1α) were markedly increased in nuclear extracts from both hypoxic cells and cells treated with ET‐1 under non‐hypoxic conditions. Time course studies revealed that accumulation of HIF‐1α in response to ET‐1 required greater than 4 hr of exposure. Real‐time PCR revealed that ET‐1 (10−10‐10−8 M; 48 hr) increased Hif1a mRNA expression. ET‐1 also decreased mRNA and protein expression of prolyl hydroxylase 2 (PHD2), the protein responsible for targeting HIF‐1α for degradation. The induction of HIF‐1α by moderate prolonged hypoxia (4% O2; 60 hr) was blocked by BQ‐123, an ET‐1 receptor subtype A antagonist, whereas BQ‐123 had no effect on the induction of HIF‐1α by severe acute hypoxia (1% O2; 4 hr). These results suggest that ET‐1 induces HIF‐1α by upregulation of HIF‐1α synthesis and downregulation of PHD2‐mediated degradation. Furthermore, the sustained induction of HIF‐1α in PASMCs during moderate prolonged hypoxia may require amplification by ET‐1. Funded by: HL67191
Ixolaris, a tissue factor inhibitor, reduces vascular pruning and delays death in rats with established pulmonary arterial hypertension
Authors: White R James , Swarthout Robert F , Meoli David F
Published: June 2021

We have observed increased tissue factor (TF) expression in pulmonary arterial hypertension (PAH) patients and in a rat model of severe PAH. We hypothesized that TF, the glycoprotein that initiates coagulation and facilitates angiogenesis, contributes to PAH pathogenesis and that blocking TF activity with the tick peptide Ixolaris (IXO) would delay disease progression.

Methods/Results: 34 rats underwent left pneumonectomy followed 7 days later by SQ injection of 50 mg/kg monocrotaline (MCT). On day 11 after MCT (d11, RV pressures generally 60 mmHg), we randomized rats to receive SQ injection of IXO (20 mcg/kg daily) or normal saline (NS) until death. The NS group had 50% mortality by d28; IXO treated animals died an average of 10 days later (p<0.03). In a similar experiment, treatment began at d11 and continued 10 days before rats were acutely vasodilated with sodium nitroprusside and sacrificed for heart weights to measure RV hypertrophy and CT scans to visualize vascular pruning. IXO animals had less RV hypertrophy (p<0.01). IXO prevented macrovascular pruning visualized by CT (Figure, represents 3 pairs of rats). Histology showed reduced neointimal formation in pre‐capillary arterioles.
Expression of functional Angiotensin II (Ang II) receptors types, AT1R and AT2R, in RVLM neuronal cultures from adult rat brain
Authors: Cuadra Adolfo Ernesto , Gillies Robert , Sumners Colin , Raizada Mohan K.
Published: June 2021

Our previous studies have demonstrated that AT1R signaling pathway regulates RVLM neuronal activity. Their increased expression and coupling to PI3K has been linked to increased Ang II‐mediated neuronal activity in the SHR‐RVLM. These findings, obtained from mixed cultures of neonatal rat hypothalamus, may not reflect those of the RVLM in adults. Thus, our objectives in this study were to establish neuronal cultures from the RVLM of adult rats, and use them to study the effects of Ang II. GFP‐AAV viral vector was injected into the RVLM of SD rats two weeks prior to isolation of neurons for anatomical confirmation. Cells were dissociated, plated onto culture dishes and allowed to establish for 7–14 days. Presence of neuron specific markers, neuN and synaptophysin in GFP expressing cells confirmed the presence of RVLM neurons. Certain fluorescent neurons expressed AT1R. Ang II effect was tested on Ca2+ currents (IBa) recorded from GFP expressing neurons. Ang II caused a 30% increase in IBa that was attenuated by 2 μM losartan. However, pretreatment with AT2R antagonist, PD123319, resulted in 2.5 fold augmentation of Ang II‐mediated increase in IBa. Together, these observations demonstrate that both AT1R and AT2R are present on the same neuron and that inhibition of the AT2R accentuates the AT1R‐mediated stimulation of IBa. They suggest a functional coupling of AT1R and AT2R in the RVLM. (Supported by NIH HL 33610 and HLT6312)
DOCA‐salt hypertension persists in mice with ouabain‐resistant α1 and α2 Na, K‐ATPase (NKA)
Authors: Loreaux Elizabeth L , Oshiro Naomi , Lingrel Jerry B , Lorenz John N
Published: June 2021

It has been proposed that endogenous cardiac glycosides may play an important role in the development of various forms of human and experimental hypertension, including DOCA‐salt hypertension and ACTH‐induced hypertension. We have previously shown that ACTH‐induced hypertension is blunted in mutant mice that express a ouabain‐resistant isoform of the α2NKA. In the present study, therefore, we sought to explore whether the development of DOCA salt hypertension is altered in mutant mice variably expressing ouabain‐resistant and ‐sensitive α1 and α2 NKA isoforms. Mice were uninephrectomized and instrumented with telemetry implants for the continuous measurement of blood pressure. After recovery, DOCA pellets were implanted and animals were given saline to drink. Mean arterial pressure increased comparably in α1‐resistant/α2‐sensitive mice (wild type, 110±1 to 149±5 mmHg) vs α1‐resistant/α2‐resistant mice (108±4 to 146±3 mmHg). In separate groups of DOCA‐salt‐treated mice instrumented with indwelling arterial catheters, treatment with digoxin antibody (digibind) had no effect on blood pressure in either α1‐resistant/α2‐sensitive or α1‐resistant/α2‐resistant mice. We also evaluated DOCA‐salt hypertension by telemetry in α1‐sensitive/α2‐resistant mice, and found that blood pressure increased similarly to the other two genotypes (106±3 to 153±9). These data suggest that the elevated pressure in DOCA‐salt treated mice is not dependent on the interaction of endogenous cardiotonic steroids with α1 or α2 NKA.
The exercise pressor reflex in hypertensive humans
Authors: Sausen Mark Thomas , Delaney Erin , Stillabower Michael , Farquhar William
Published: June 2021

During dynamic handgrip exercise (DHE), there is an increase in blood pressure (BP) mediated by “central command” (a neural drive originating in the brain) and the “exercise pressor reflex (EPR)” (a neural drive originating in skeletal muscle). The EPR has been shown to be overactive in hypertensive rats.

Purpose:

To examine the EPR in hypertensive (HTN) humans during DHE and post‐exercise ischemia (PEI).

Hypothesis:

The exercise‐ and ischemia‐induced increase in BP will be greater in HTN compared to normotensive (NTN) adults.

Methods:

12 HTN (64±1 yrs) and 15 NTN (65±1 yrs) adults were recruited. Beat‐to‐beat mean arterial pressure (MAP), systolic BP (SBP), and diastolic BP (DBP) were measured non‐invasively at the finger (Finometer). DHE at 60% of maximal voluntary contraction was performed for 3 minutes followed by 2 minutes of PEI (upper‐arm occlusion cuff inflated to 200 mmHg to trap metabolites and isolate the EPR). A repeated‐measures ANOVA was used to examine differences between conditions and groups.

Results

(mean±SEM): DHE resulted in increased MAP that was not different between groups (HTN: Δ=16±3 vs. NTN: Δ=12±3 mmHg). During PEI the change in MAP, SBP, and DBP from baseline was greater in HTN than NTN (MAP: Δ=12±3 vs. Δ=6±1 mmHg, SBP: Δ=17±4 vs. Δ=9±2 mmHg, DBP: Δ=7±2 vs. Δ=3±1 mmHg; p<0.05).

Conclusion:

These findings suggest HTN humans have an overactive EPR. Supported by NIH grant RO3 AG‐23836.
Co‐culture model of the human bronchial epithelium : cytokine‐dependent reduction of ciliary and barrier function
Authors: Kirkpatrick Charles James , Pohl Christine , Hermanns M. Iris
Published: June 2021

The upper respiratory tract is one of the first lines of defence against inhaled pollutants or toxic material. Two of the essential elements of this protection are coordinated ciliary movement and barrier function of the bronchial epithelial cells. Normal human bronchial epithelial cells (NHBE) from primary culture can be co‐cultured with human lung fibroblasts (Wi‐38) as a bilayer on Transwell filter plates. Using the air‐liquid interface (ALI) technique we show that NHBE form a pseudostratified epithelium and express both tight (TJ) and adherens junction (AJ) proteins after 14 to 17 days in vitro. Mucus production and cilia formation developed within 21 days. Additionally, these cilia showed a beat frequency about 16 to 20 Hz. The differentiated state of the bronchial cells was maintained up to three months, correlating with average transepithelial resistance‐values between 600‐800 ohm.cm2 and prolonged ciliary beating. Exposure to TNF‐alpha and IFN‐gamma induced significant reduction in barrier functions (reduced expression of ZO‐1, occludin, E‐cadherin, beta‐catenin : immunofluorescence, Western blot, RT‐PCR; increased permeability for FITC dextrans) and ciliary beat frequency. These data demonstrate a relevant co‐culture model to investigate pathomechanisms of proximal respiratory tract injury. (Supported by the German Defence Ministry)
The expression of PKA and VASP within migrating VSMCs in the rat model of type 2 diabetic macroangiopathy
Authors: Zhang De‐Ling , Ouyang JingPing , Zhang YeMin , Wang Nian , Su Hui , Liu YongMing
Published: June 2021

Vasodilator‐stimulated phosphoprotein (VASP), an important substrate of PKA, plays a critical role in the regulation of actin‐based cell motility in vitro. But the role of VASP in cell migration in vivo is not entirely clear. In present study, the relationship between VSMC migration and VASP expression in vivo were investigated. Eight‐week‐old male SD rats were fed with normal diet (control group, n=10) and fed with high fat diet and intraperitoneally injected with STZ (25mg/kg) to induce a model of type 2 diabetes (DM group, n=12). After feeding for 8 weeks, DM group showed significantly increased levels of the plasma glucose, HbAlc and blood cholesterin. Insulin sensitivity was reduced, identified with a glucose test using HOMA‐IR index. Morphological changes of aorta macroangiopathy in DM group were observed by HE staining and transmission electron microscope. The thickness of media was increased and the VSMCs in the media proliferated, the elastic lamella within the intima was destroyed and VSMCs in the media migrated to the intima. Immunohistochemistry showed that expression of PKA and phosphorylated VASP were distributed in the cytoplasm of VSMCs. Compared with diffused distribution within media in control group, the number of VSMCs expressed PKA was increased and mainly distributed in the region near the intima in DM group. Similarly, the distribution of VSMCs expressed VASP was consistent with that of PKA. Our study indicates that the rat model of type 2 diabetic macroangiopathy was successfully established, in which the expression of PKA and phosphorylated VASP were correlated with the migration of VSMCs. (The research is supported by national natural science foundation of China granted NO.30470680)
Lack of beta‐3 adrenergic receptor function in ventricles of ob/ob mice
Authors: Romano Fred D. , Malicay Margaret , Goeppinger Heidi , Sondgeroth Korie , Wahler Gordon M. , Smith Jacquelyn M.
Published: June 2021

Type 2 diabetes mellitus (T2DM) is associated with ventricular dysfunction. Previous studies have shown that activation of the β3‐adrenergic receptor (β3AR) mediates a decrease in ventricular function in rodents. Since the β3AR is upregulated in hearts from rats with T1DM, we hypothesized that β3AR‐mediated inhibitory mechanisms contribute to the myocardial depression of T2DM. The contractile response (myocyte shortening) to the β3AR agonist, CL316243, was determined in ventricular myocytes obtained from 12 to 16 week old ob/ob mice, a model for T2DM, and lean (+/?) littermates. Basal shortening was induced by electrical stimulation (0.5Hz). CL316243 (100nM) increased myocyte shortening in lean mice by 17.4%, but had no effect on shortening of myocytes from ob/ob mice. SR59230A (100nM), a β3AR antagonist, reduced the effect of CL316243 on shortening in lean myocytes. The β1/β2AR antagonist, nadolol (10μM) had no effect on CL316243 shortening in lean myocytes, consistent with β3AR‐mediated shortening of myocytes. Analysis of β3AR mRNA transcripts indicated that mRNA levels were reduced in the ob/ob mouse ventricles as compared to lean. These data suggest that the absence of the β3AR positive inotropic response in the ob/ob mouse heart could contribute to the myocardial depression seen in T2DM. Supported by the MWU Master of Biomedical Sciences Program and Office of Research and Sponsored Programs.
Pressure overload exacerbates insulin resistance in obesity and type 2 diabetes
Authors: Marsh Susan A , Dell’Italia Louis J , Chatham John C
Published: June 2021

Insulin resistance and type 2 diabetes are associated with a high fat diet, hypertension and obesity. Hypertensive diabetic patients have an increased risk for progressing to heart failure, a condition also associated with insulin resistance in non‐diabetics. Young male rats were divided into control (CON; 12% kcal fat/19% protein/69% carbohydrate), high fat diet (HF; 60/19/21%) and type 2 diabetic (DIA; high fat diet + low dose streptozotocin) groups and after 8 weeks underwent sham (SH) or pressure overload (PO) surgeries. Fasting glucose levels were elevated in DIA (10.8 ± 1.3 mmol/l) compared to CON (3.5 ± 0.1 mmol/l) and HF (3.9 ± 0.2 mmol/l); there were no differences between SH and PO. Mortality was higher in DIA‐PO (30%) and CON‐PO (25%) compared to HF‐PO (8%) and there was 100% survival in all SH groups. Four weeks after surgeries, insulin sensitivity was 20% lower in DIA‐PO compared to DIA‐SH and this difference widened to 42% after 8 weeks; at both time points, HF‐SH were more insulin resistant than CON‐SH. At 4 weeks insulin sensitivity in CON‐PO and HF‐PO was not different from their respective SH; however, at 8 weeks insulin sensitivity in HF‐PO was 35% lower than HF‐SH. These data suggest that insulin resistance and mortality are exacerbated by pressure overload in type 2 diabetic animals, which may contribute to increased heart failure and mortality in diabetic patients.
Manganese modulates genes involved in regulation of cell cycle
Authors: Taka Equar , Reams Renee R , Heiman Ann S , Soliman Karam , Darling‐Reed Selina F
Published: June 2021

Manganese (Mn) is a neurotoxic metal. Excessive accumulation of Mn in the globus pallidus and striatum leads to decreased dopamine and cell death in the Parkinsonian‐like syndrome called manganism. Efforts to understand Mn neurotoxicity have focused on oxidative stress, apoptosis, and cell cycle; however, the molecular targets of Mn neurotoxicity remain unknown. Thus, we hypothesized that manganese causes neurotoxicity by modulating genes involved in regulating cell cycle. The hypothesis was tested using rat pheochromocytoma (PC12) cells which are a widely accepted model for neuronal studies. Using microarray gene expression profiling and GeneSifter data analysis, we identified 223 genes whose expression level was altered by >1.5 or < −1.5–fold. Of these genes, 146 were up‐regulated while 77 were down‐regulated. Gene modulation was noted in several categories including: cellular processes (99 genes), metabolism (71 genes), development (45 genes), signal transduction (22 genes), and apoptosis (16 genes). Using Real‐time PCR, mRNA expression was confirmed for cyclin G1, c‐Jun, Jun‐B, Jun‐D and cdk4 known to be involved in cell cycle progression. Flow cytometry showed that Mn caused S‐phase cell cycle arrest. In conclusion, our findings uncovered the effects of Mn on genes involved in cell cycle, metabolism, development, signal transduction and apoptosis in PC12 cells. Supported by ATSDR and MBRS.
Exercise‐induced plasticity in the hypoglossal nucleus of young and old rats
Authors: Moeser Adam E , Connor Nadine P , Behan Mary
Published: June 2021

Breathing and swallowing problems affect a large percent of the elderly and may be related to age‐associated dysfunction of the tongue. The goal of this study was to determine whether changes in the rat tongue motor control system could be modified or reversed by resistance training. Hypoglossal motoneurons that innervate the tongue receive a robust, excitatory serotonergic (5HT) input. Previous studies have shown age‐associated changes in 5HT and the 5HT2A receptor in the hypoglossal nucleus. We hypothesized that: (1) 5HT input to the tongue decreases with age; (2) tongue exercise augments 5HT input to the hypoglossal nucleus at all ages, but to a greater extent in young than in old rats. Young (9–10 months), middle‐aged (24–25 months), and old (32–33 months) male F344/BN rats were exposed to progressive resistance tongue exercise for 8 weeks. Immunoreactivity for 5HT was measured in digital images of sections through the hypoglossal nucleus using ImageJ software. Preliminary data suggest there is a significant decrease in 5HT immunoreactivity in the hypoglossal nucleus with age, and exercise results in a significant increase in 5HT at least in young rats. NIH AG18760 (MB) and DC008149 (NC).
Effects of long term losartan treatment on D2 receptor modulation of breathing in the dystrophic hamster in air and following intermittent hypoxic bouts
Authors: Schlenker Evelyn Heymann , Richards Abby
Published: June 2021

Angiotensin II (A) acting through A1 receptors and hypoxia release dopamine in several brain regions and the carotid body. Blockade of dopaminergic receptors (D2) modulates ventilation in the hamster. We tested the hypothesis that two months of losartan (L, an A1 receptor blocker, 50 mg/kg) treatment would affect D2‐modulated control of breathing during exposure to air and five minutes each of three bouts of 10% O2 interspersed with air (IH) differently compared to tap water‐treated dystrophic (D) hamsters. Ventilation was evaluated in conscious control (C), CL, D and DL hamsters injected with vehicle or domperidone, a D2 receptor antagonist (Dom) that does not cross the blood‐brain barrier. During baseline air exposure, frequency (f) was higher in the C versus the D groups. Dom normalized f and increased tidal volume in the DL group relative to CL. Dom doubled minute ventilation (MV) in DL relative to the D group, but did not affect C or CL groups. To evaluate the effects of IH, the ratio of MV during the third bout of IH to baseline (H/B) was determined. In DL, Dom relative to vehicle had no effect on MV H/B but in D hamsters Dom increased MV H/B (1.2 ± 0.12 vs. 1.8 ± 0.14, P=0.02). Losartan had no effect on MV H/B in the control groups. These results suggest that in dystrophic hamsters breathing air or hypoxia losartan alters D2‐modulated control of breathing by acting on the carotid body.
Macrophage Depletion Suppresses Sympathetic Hyperinnervation following Myocardial Infarction
Authors: Wernli Gwenaelle , Hasan Wohaib , Smith Peter G.
Published: June 2021

Myocardial infarction is followed by sympathetic axon sprouting in the peri‐infarct region and this has been associated with arrhythmias leading to sudden cardiac death. Inflammatory cells expressing nerve growth factor, a potent sympathetic neurotrophic protein, are frequent residents of the infarct, leading to the hypothesis that these cells may contribute to sympathetic sprouting. Our goal was to determine if macrophages are obligatory for sympathetic hyperinnervation. Ovariectomized female rats received left coronary artery ligation (CAL) or sham operation, followed by injections of saline vehicle or clodronate liposomes to kill macrophages. Sham‐operated myocardium contained sympathetic axons (0.9 ± 0.06% of section area), few myofibroblasts and no macrophages or T‐cells. In the saline‐injected CAL group, sympathetic innervation was increased two‐fold and the peri‐infarct contained myofibroblasts (134,374 ± 9,710 μm2/mm2 section), macrophages (1,195 ± 97/mm2) and T‐cells (412 ± 27/mm2). Treatment for 7 days with clodronate liposomes reduced sympathetic innervation to control levels. Macrophages were reduced by 69% and myofibroblasts area was reduced by 23%, but the number of T‐cells was comparable to untreated CAL rats. These results indicate that macrophages are required for the development of sympathetic hyperinnervation following myocardial infarction. Supported by HL079652
Brachial arterial sympatholytic response in competitive rock climbers
Authors: Baldi James Christopher , Cassuto Nick A , De Young Lindsey G , Kinsey Eric W , Greenlee Kellen C
Published: June 2021

Training studies of vascular reactivity often involve brachial arterial function in leg‐trained athletes. This study examined brachial arterial function in competitive rock climbers. We hypothesized that hand‐grip exercise would cause a greater reduction in sympathetically mediated vasoconstriction in climbers. Competitive male rock climbers and healthy control subjects reported to the lab on two separate days. Brachial arterial flow (Doppler ultrasound), blood pressure, heart rate and vascular conductance were measured at rest, during a cold pressor test (CPT) or during 30% maximal hand grip exercise with and without a CPT. Test order was randomized. Preliminary results show similar reductions in vascular conductance in the groups during the CPT (P < 0.05). Exercise elicited a greater increase in arm blood flow and vascular conductance in climbers (P < 0.05). When CPT was added to exercise, reductions in arm flow and conductance (P < 0.05) were not different, but absolute flow remained higher in climbers despite similar workloads. These findings suggest that training increases absolute blood flow during exercise, but does not alter relative sympathetic vasoconstriction in the trained limb.
The use of OsiriX® software and 3d Reconstructions from diagnostic images as an aid to cadaver dissection and surgical planning.
Authors: Swift Andrew
Published: June 2021

OsiriX® software allows the trained user to render a 3 dimensional virtual representation based on images captured during diagnostic CT and MRI scans. OsiriX® software can assign levels of transparency and color to select tonal values and stack each slice relative to its position during the initial scan to create “3D Reconstructions” that can be manipulated and exported as image files. These 3D Reconstructions improve the understanding of anatomic relationships as they exist in the living body. They may be used to supplement anatomy instruction, provide an adjunct to traditional dissection, and improve surgical planning.

Traditional dissection often requires the removal, displacement or distortion of superficial anatomy in order to access deeper structures. The non‐destructive and reversible nature of virtual dissections allows a trained user to remove or isolate structures selectively, permitting the visualization of a particular region or tissue. Measurements of distance and volume are also possible. Diagnostic images of both living patients and cadavers destined for dissection can be reconstructed and used to correlate diagnostic images to the actual anatomy of a patient or cadaver.

Cost and practical concerns of setting up a 3D workstation, usability of the OsiriX® software, as well as patient confidentiality issues will be discussed. Several case studies will be presented.
High dietary intake of sodium selenite does not affect gene mutation frequency in rat colon and liver
Authors: Zeng Huawei , Uthus Eric O , Ross Sharon A , Davis Cindy D
Published: June 2021

Gene mutations have been implicated in the etiology of cancer. In the present study, we utilized Big Blue transgenic rats to evaluate the in vivo mutation frequency of the λ cII gene in rats fed either a Se‐deficient (0 μg Se/g diet) or selenium‐supplemented diet (2 μg Se/g diet) (n=6 rats/diet) and injected with dimethylhydrazine (DMH) (25 mg/kg body weight, i.p.). There were no differences in body weight (252∼264 g) between the Se‐deficient and Se‐supplemented rats, but liver glutathione peroxidase and thioredoxin reductase activities were significantly lower (p<0.0001) in Se‐deficient rats (17.20 and 1.24 U/mg) compared to rats supplemented with Se (607.68 and 4.59 U/mg, respectively). Gene mutation frequency was significantly lower in liver (140∼146 mutants per 106 λ cII gene sites) than in colon (579∼604 mutants per 106 λ cII gene sites), and there were no differences in gene mutation frequency in DNA from colon mucosa or liver from rats fed the Se‐deficient diet compared to those fed the Se‐supplemented diet. While previous studies have shown that selenium is protective against DMH‐induced preneoplastic colon cancer lesions, the current results suggest that it does not inhibit DMH‐induced mutations in the λ cII gene.
Analyses of procyanidins in foods using Diol phase HPLC
Authors: Gu Liwei , Hager Aaron C. , Robbins Rebecca J. , Prior Ronald L.
Published: June 2021

Separation of procyanidins using silica‐based HPLC suffered from poor resolution for higher oligomers and low sensitivity due to the fluorescence quenching effects of methylene chloride in the mobile phase. Optimization of a published Diol‐phase HPLC method resulted in near baseline separation for pentamers to decamers at 29 min to 50 min and the elution of polymers with molecular weights higher than decamers at 66 min. By using excitation and emission wavelengths of 230 nm and 321 nm, the sensitivity was increased by 5 fold. Flavonoids other than proanthocyanidins did not generate signals at these wavelengths. Dimers and trimers in grape seed procyanidins eluted as a cluster of 5 peaks of isomers on normal phase HPLC, whereas they merged into a large peak with shoulders using the Diol‐phase HPLC. Hexamers, heptamers, and octamers can be resolved on Diol‐phase HPLC but not on normal‐phase HPLC. Separation of procyanidins on Diol‐phase HPLC was according the degree of polymerization and was not sensitive to isomeric differences for procyanidins of the same degree of polymerization. Proanthocyanidins in foods that contained predominantly procyanidins, such as sorghum, pear, and apple, eluted as well separated oligomers and polymers using Diol‐based HPLC.
Absorption of Zinc (Zn) from High Zn & Control Wheat
Authors: Hambidge K Michael , Rosado Jorge L , Miller Leland V , Hotz Christine , Westcott Jamie E , Garcia Olga P , Gonzalez Karla , Ortiz‐Monasterio Ivan , Pfeiffer Wolfgang , Krebs Nancy F
Published: June 2021

Background: One goal of HarvestPlus is to increase the Zn content of crops by selective plant breeding.

The objective of this study was to assess the efficacy of moderately high‐Zn wheat by measuring Zn absorption from a quantity of wheat (300 g) likely to be consumed daily by adult women who depend on wheat as a major food staple.

Study Design: Randomized double blind cross‐over design within extraction groups (95% and 80%). 28 healthy women were randomized to consume tortillas prepared from either high or control Zn wheat on day 1 or 2 as their primary source of energy and Zn. Total absorbed Zn (TAZ, mg) from high Zn wheat tortillas was compared with control wheat tortillas and with the predicted TAZ from a model of TAZ as a function of total ingested Zn (TIZ, mg) and phytate (TIP, mg) at 2 extraction levels. TIZ was measured by extrinsic labeling with stable Zn isotopes and a dual isotope tracer ratio technique. Salient data, means (SD), are given in the table.

Conclusion: A significant increase in TAZ is achieved with selection of moderately high Zn wheat. The increase in absorbed Zn is compatible with that predicted from a model of TAZ as a function of TIZ and TIP. Variation in extraction between 95% and 80% does not alter TAZ. Supported by HarvestPlus & IAEA.
Mammalian thioredoxin reductases: roles in redox homeostasis and analysis of cellular targets
Authors: Turanov Anton A , Kehr Sebastian , Carlson Bradley A , Hatfield Dolph L , Gladyshev Vadim N
Published: June 2021

Thioredoxin reductase (TR) and thioredoxin (Trx) constitute a major cellular redox system. In the present study, we characterized subcellular localization of mouse TR3 forms which resided in mitochondria and cytosol. We generated recombinant selenoprotein forms of TR1 and TR3 and found that these enzymes were inhibited by zinc. A proteomic method for identification of targets of TRs in mammalian cells and tissues was developed. We found that Trx1 was the major target of TR3 and TR1 in rat and mouse liver cytosol and that Trx1 was the major target of TR1 in rat brain cytosol and could be enriched on the TR affinity columns from mouse serum. TR3 knockdown cells had increased sensitivity to hydrogen peroxide, whereas TR1 knockdown cells were more sensitive to diamide. To further examine the roles of TRs in redox homeostasis, we used knockout mice, in which selenocysteine tRNA gene was disrupted specifically in the liver. We found that the levels of cytosolic TR1 and mitochondrial TR3 were significantly lower in the knockout samples compared to wild type, whereas the levels of cytosolic Trx1 and mitochondrial Trx2 were elevated. Trx1 was mostly in the oxidized state in the knockout samples. Overall, these data provide important insights into the roles of mammalian TRs in redox homeostasis.
SVCT1 and SVCT2 Genotypes Modify the Association between Dietary Vitamin C and Serum Ascorbic Acid Concentrations in Men
Authors: Cahill Leah , Fontaine‐Bisson Bandicte , El‐Sohemy Ahmed
Published: June 2021

Clinical trials have established that individuals have different serum ascorbic acid (vitamin C) responses to dietary vitamin C. This may be due, in part, to genetic differences. In humans, vitamin C transport proteins 1 and 2 (SVCT1 and SVCT2) are required for vitamin C absorption and uptake, respectively. Our objective was to determine if polymorphisms in the SVCT1 and SVCT2 genes alter fasting serum ascorbic acid concentrations in response to dietary vitamin C. We recruited 533 women and 235 men between the ages of 20â29 yrs who were all non‐smokers. Dietary, lifestyle and anthropometric data were obtained and blood samples collected to determine fasting serum ascorbic acid concentrations by HPLC and to isolate DNA. Real‐time PCR was used to genotype for two polymorphisms in SVCT1 (rs4257763 G>A and rs6596473 G>C); and two polymorphisms in SVCT2 (rs6139591 G>A and rs2681116 C>T). Overall, the diet‐serum correlation was significant for men (r=0.21, p<0.01), but significant correlations were only observed for the following genotypes: rs4257763GG (r=0.32, p<0.01), rs6596473CC (r=0.44, p<0.01), rs6139591GG (r=0.39, p<0.01) and rs2681116 TT (r=0.66, p<0.001). No significant associations were observed for women in any of the genotype groups. Our data suggest that the correlation between dietary vitamin C and serum ascorbic acid concentrations in men differs between the genotypes of common polymorphisms that affect the absorption and uptake of ascorbic acid. Supported by the Advanced Foods & Materials Network.
Cinnamon affects immune responses by regulating anti‐ and pro‐inflammatory gene expression and energy metabolism in macrophages
Authors: Cao Heping , Urban Joseph F. , Anderson Richard A.
Published: June 2021

Tristetraprolin (TTP/ZFP36) family proteins have anti‐inflammatory effects by destabilizing pro‐inflammatory mRNAs. TTP expression is induced by insulin and cinnamon polyphenol extracts (CPE) in adipocytes, by lipopolysaccharide (LPS) in macrophages, and by green tea polyphenol extract in rat muscle and liver cells. This study compared the effects of CPE, insulin, and LPS on the expression of genes encoding TTP, pro‐inflammatory cytokines, and glucose transporter (GLUT) families in RAW264.7 cells. CPE increased TTP mRNA and protein levels to a lesser extent than LPS. CPE (100 ìg/ml, 4 h) increased TTP, TNF, COX2, and IL6 mRNA levels up to 2, 6, 3, and 3‐ fold, respectively. LPS (0.1 ìg/ml, 4 h) increased TTP, TNF, GM‐CSF, COX2, and IL6 mRNA levels by 39, 48, 1256, 532, and 1868‐ fold, respectively. CPE and LPS increased the expression of GLUT1 (the major GLUT form in macrophages) by three and two‐fold, respectively. Insulin (100 nM, 0.5–4 h) did not exhibit significant effects on the expression of these genes. CPE increased TTP gene expression more rapidly than the pro‐inflammatory cytokines measured, and the net increase in TTP mRNA was larger than those of pro‐inflammatory cytokines. These results suggest that CPE may affect immune responses by regulating anti‐ and pro‐inflammatory gene expression and energy metabolism in macrophages (supported in part by USDA‐ARS Human Nutrition Research Program).
Incorporation of Vitamin D into a Comprehensive Food and Nutrient Database
Authors: Jasthi Bhaskarani , Schakel Sally F , Harnack Lisa
Published: June 2021

Growing evidence suggests that vitamin D may be important for more than bone health, with the vitamin potentially playing a role in preventing colorectal and other cancers, diabetes, arthritis and even multiple sclerosis. Human studies examining these relationships must rely on food and nutrient databases to estimate intake of this vitamin. Unfortunately there are a number of obstacles to adding vitamin D to comprehensive databases. Most notably, limited nutrient composition information and variability in the vitamin D content of foods pose special challenges. The systematic approach used by the Nutrition Coordinating Center (NCC) at the University of Minnesota to include vitamin D values for foods in its nutrient database may be a useful model for others. The approach used involved first compiling vitamin D values available from a variety of sources. This compiled data was then used to assign vitamin D composition values for foods in the NCC database. For foods where several values were available, one best value was generally selected on the basis of several criteria. However, for some foods, such as many species of fish in the database, values were averaged from a variety of sources because the variation in values observed reflect natural (true) variation in vitamin D composition. Manufacturers' label claims for vitamin D were used for fortified foods and vitamin supplements. To minimize missing values imputation procedures were developed and applied, with published data on the effects of cooking technique and manufacturing processes on vitamin D composition taken into account in these procedures.