bluuebunny/crossref_metadata_2025 huggingface dataset:
PLACENTAL HEME OXYGENASE ACTIVITY REDUCTION IS ASSOCIATED WITH HYPERTENSION IN PREGNANT RATS
Authors: Llinás María Teresa , Volti Giovanni Li , Reverte Virginia , Salazar Francisco , Sáez Fara , Salazar Francisco Javier
Published: June 2021

Reduction in the placental perfusion pressure has long been thought to underlie preeclampsia (PE). It has also been demonstrated that inhibition of heme oxygenase (HO) activity in isolated placenta increases placental perfusion pressure. This finding suggests that the HO‐CO system is involved in regulating blood flow to this organ. However, it is unknown whether the reduction of placental HO activity produces hypertension during gestation, which is a common feature in PE. Therefore, the aim of this study was to determine whether administration of an HO inhibitor during gestation induces an elevation in blood pressure (BP) and whether the hypertension is associated to a reduced placental HO activity. The HO inhibitor, stannous mesoporphyrin (SnMP; 50 micromol/kg i.p) or vehicle (sodium carbonate) were administered to virgin (n=6) and pregnant rats (n=7) at day 14 of gestation. BP was measured 5 days later in conscious rats. SnMP increased (p<0.01) BP in pregnant rats (114 ± 1 vs 100 ± 2 mmHg) but had no effect in virgin rats (121 ± 2 vs 124 ± 3 mmHg). This increase was associated to a reduction in placental HO activity (10.4 ± 0.5 vs 6.9 ± 0.2 ng/bilirubin/μg protein/h, p<0.05) and an important fetal reabsorption (4.0 ± 0.7 vs 1.0 ± 0.3, p<0.05), indicating possible placental ischemia. These results suggest that the HO‐CO system may play an important role in the development of PE.
Differential Regulation of NADPH Oxidase Activity in Lipid Rafts in Renal Proximal Tubule Cells from Rats and Humans
Authors: Yu Peiying , Han Weixing , Villar Van Anthony M
Published: June 2021

We tested the hypothesis that NADPH oxidase activity and Nox subunits are differentially regulated in lipid rafts (LRs) in renal proximal tubule cells (RPTCs) from humans and rats. Nox4 protein was expressed in LRs in rat RPTCs (47±3.1%) but not in human RPTCs (n=3); more Nox2 (40.3±3.7%) and Rac1 (58±3.1%) resided in LRs in human RPTCs than in rat RPTCs (18.2±4.4 and 29.5±4.4%, respectively). Cholesterol depletion with methyl‐β‐cyclodextrin (βCD/2%) decreased oxidase activity (42±7.1%), accompanied by decreased Nox2, Nox4 and Rac1 expression in LRs in rat RPTCs. In contrast, in human RPTCs, βCD treatment markedly increased oxidase activity (154±10.5 vs vehicle=103.1±3.4%, P<0.001 ANOVA, n=4), accompanied by a redistribution of Nox subunits into non‐LR fractions. The effect of βCD in both human RPTCs and rat RPTCs was reversed by cholesterol repletion. The stimulatory effect of βCD treatment on NADPH oxidase activity in human RPTCs was also reversed by inhibitors of NADPH oxidase, apocynin (10μM,100±3.2%) and diphenylene iodinium (5μM, 9.5±3.3%) (P<0.01, ANOVA, n=4–6/group). These studies demonstrate for the first time that NADPH oxidase subunits are differently targeted to LRs in human and rat RPTCs; and disruption of LRs produces opposite effects in humans (stimulation) and rats (inhibition). The results suggest that the integrity of LRs plays an important role in maintaining NADPH oxidase in the inactive state in human kidney cells.
Adrenergic receptor mediated tonic vasoconstriction is not increased in skeletal muscle of older beagles
Authors: DeLorey Darren , Buckwalter John B , Mittelstadt Scott , Kluess Heidi A , Clifford Philip S
Published: June 2021

Aging is associated with an increase in muscle sympathetic nerve activity. Whether this increased efferent sympathetic nerve activity leads to greater tonic vasoconstriction and reduced blood flow to resting and contracting skeletal muscles in old compared to young has not been clearly established. Therefore, the purpose of the present study was to investigate the effect of aging on α1– and α2–adrenergic receptor mediated restraint of skeletal muscle blood flow. Young (n = 6; 22 ± 3 months) and old (n = 7; 119 ± 22 months) beagles were instrumented chronically with transit‐time flow probes on the external iliac arteries and an indwelling catheter in a branch of the femoral artery. On separate days, flow‐adjusted doses of selective α1‐ (prazosin; 0.25 ug/ml/min) and α2–adrenergic‐receptor (rauwolscine; 1.25 ug/ml/min) antagonists were infused at rest, and during treadmill running at 2.5 miles/h and 4 miles/h with 2.5% grade. Hindlimb blood flow (HBF) and mean arterial pressure (MAP) were monitored continuously and vascular conductance (VC) was calculated as HBF/MAP. Infusion of prazosin resulted in similar increases in VC (p > 0.05) in young and old beagles at rest (Young 100 ± 43 % ; Old:, 158 ± 84 %) and at 2.5 miles/h (Young 61 ± 28 % ; Old:, 80 ± 25 %) and 4 miles/h with 2.5% grade (Young: 36 ± 28 %; Old: 57 ± 13 %). Rauwolscine infusion also produced similar increases in VC (p > 0.05) in young and old beagles at rest (Young: 64 ± 49 %; Old: 119 ± 60 %) and at 2.5 miles/h (Young: 64 ± 20 %; Old: 86 ± 32 %) and 4 miles/h with 2.5% grade (Young: 49 ± 20 %; Old: 61 ± 11 %). These results demonstrate that α1– and α2‐adrenergic receptor mediated restraint of skeletal of muscle blood flow does not increase in older compared to young beagles at rest or during dynamic exercise.
Enhanced Diastereoselective Synthesis of L‐threo‐3,4‐dihydroxyphenylserine by Low‐specific L‐threonine aldolase mutants
Authors: Baik Sang‐Ho , Gwon Hui‐Jeong , Lim Sang‐Yong , Kim Dong‐Ho , Harayama Shigeaki
Published: June 2021

Four mutants that displayed more enhanced diastereoselectivities (de) than the wild‐type L‐TA from Streptomyces avermitilis MA4680 were successfully obtained by an error‐prone PCR followed by a high‐throughput screening. One candidate mutant, T3‐3 which showed almost the same reverse aldol condensation activity as E. coli transformed pLTASA‐2 (wild‐type) but lower L‐erythro‐DOPS synthesis activity, was selected for further improvement by second error‐prone PCR. About 20,000 clones were screened as the same way, and a mutant named T3‐3m5 was finally obtained. Wild‐type L‐TA showed a very poor de of 14%, while the de of the isolated mutants was approximately 20.5%, 26.2%, 21%, 40%, respectively. Most improved diastreoselectivity was achieved from mutant T3‐3m7 showing a 2.7‐fold more increase than that of the wild‐type L‐TA after a second screening. The complete sequences of the obtained mutants were determined. Sequence analysis showed that each of these mutants had a single or several amino acid substitutions: V86I; R241C; Y306C for T2‐2, Y34C for T2‐4, R241C; A287V for T3‐2 and Y39C, Y306C for T3‐3.
Hydrogen Sulfide as an Oxygen Sensor in Trout Chemoreceptors
Authors: Olson Kenneth R. , Healy Michael J. , Qin Zhaohong , Vulesevic Branka , Duff Douglas W. , Whitfield Nathan L. , Yang Guangdong , Wang Rui , Perry Steve F.
Published: June 2021

Oxygen (O2) sensing chemoreceptors elicit cardiorespiratory reflexes in all vertebrates but the oxygen sensing‐signal transduction mechanism(s) is unknown. Here we examined the possibility that H2S is an O2 sensor in rainbow trout where the first pair of gill arches is a primary site of chemoreception and the homolog of the mammalian carotid body. In unanesthetized trout, ambient hypoxia produced a PO2‐dependent increase in plasma [H2S] indicative of inverse coupling of PO2 and H2S. mRNA for the H2S synthesizing enzymes CBS and CSE was present in gill tissue and gills produced H2S enzymatically. Intrabuccal injection of H2S in unanesthetized trout produced a dose‐dependent bradycardia and increased ventilatory frequency and amplitude similar to hypoxia. Removal of the first but not second gill arches inhibited H2S‐mediated bradycardia, consistent with the hypothesized loss of aquatic chemoreceptor function. In isolated zebrafish neuroepithelial cells, the putative chemoreceptive cells of fish, hypoxia and H2S produced a similar ~10 mV depolarization. This suggests that H2S is involved in the O2 sensing/signal transduction pathway in chemoreceptive cells as it is in vascular smooth muscle. This mechanism, whereby [H2S] is governed by the balance between constitutive production and oxidation, tightly couples tissue [H2S] to PO2 and may provide an exquisitely sensitive O2 sensor in a variety of tissues. Support: NSF IOS 0641436 & NSERC
Disuse atrophy delays and reduces amino acid induced activation of key translational signaling proteins in humans
Authors: Glover Elisa Ingrid , Phillips Stuart M , Tang Jason E , Oates Bryan R , Tarnopolsky Mark A , Smith Kenneth , Selby Anna , Rennie Michael J
Published: June 2021

We aimed to determine the effect of muscle disuse on activation by mixed amino acid (AA) feeding of three anabolic signaling proteins: mTOR, eIF2Bε and p70s6k. 10 men and 2 women wore an immobilizing knee brace for 14d, reducing muscle by CSA (5±1%, P<0.001). Subjects then received infusions of mixed AA at either 44 mg kg−1h−1 or 261 mg kg−1h−1. Quadriceps biopsies were taken from both legs when fasted and fed (1, 2, 4 h). After 1 h AA infusion phosphorylation of mTOR (Ser2448), eIF2Bε (Ser 539) and p70s6k (Thr389) in the non‐immobilized leg increased by 35–56% (P<0.05), with a return to baseline by 2 h (p70s6k) or 4 h (mTOR, eIF2Bε). In the immobilized leg mTOR phosphorylation did not significantly increase at all after AA infusion whereas phosphorylation of eIF2Bε increased by only 22% at 2 h (P<0.05); phosphorylation of p70s6k increased by only 32 % at 1 h (P<0.05), but remained elevated until 4 h. p70s6k phosphorylation was 22 % higher (P=0.05) in the immobilized leg in the fasted state. These findings, together with an observed depressed AA‐induced muscle protein synthetic response, suggest a dampened response of the underlying translational control mechanisms with disuse atrophy in humans. We propose that a reduction in the feeding‐induced stimulation of muscle protein synthesis is of major importance in causing muscle loss with disuse in humans. Support: NSERC, CIHR, UK BBSRC, and EC EXEGENESIS
Neurogenesis from Neuroglia: Role of Epidermal Growth Factor in Neuroglial Transdifferentiation into Neuronal Precursor Cells
Authors: Gouw Arvin , Cancedda Laura , Tseng Lisa , Mahuron Kelly , Manandhar Shila , Wadhwani Hemant , Poo Mu‐ming , Timiras Paola
Published: June 2021

In the adult, neurogenesis occurs primarily in the hippocampus, olfactory bulb, and subventricular zone. Unlike neurons, neuroglia proliferate throughout life in response to brain aging and damage. We investigated the role of Epidermal Growth Factor (EGF) in transdifferentiating neuroglia into precursor cells and eventually neurons. C6 rat glioma cells, a mixture of astrocyte and oligodendrocytes, was treated with 50ng/mL EGF for 30 days. EGF increases proliferation, but reduces overtime the activity of Glutamine Synthetase (GS) and 2′3′‐cyclic nucleotide 3′ phosphohydrolase (CNP), the enzyme markers for astrocytes and oligodendrocytes, respectively. Simultaneously, immunofluoresence imaging showed that after 14 days of EGF treatment, neuroglial cells expressed the neuronal marker, Nestin. By 21 days, electrophysiological recordings show a resting membrane potential resembling that of neurons rather than that of neuroglia and some isolated action spikes. Thus, EGF treatment of neuroglial cells induces dedifferentiation of these cells and their transdifferentiation to exhibit certain neuronal characteristics. Supported by NIH Grant AG 19145‐05.
Acute intermittent hypoxia (AIH) induces serotonin‐dependent long‐term facilitation (LTF) of glossopharyngeal nerve (IX) activity in rats
Authors: Cao Ying , Ling Liming
Published: June 2021

Although IX is mainly a sensory nerve, it innervates stylopharyngeus and some other upper airway (UA) muscles. Our recent study showed that the UA was less collapsible during peak inspiration vs. expiration, and this phasic difference in UA collapsibility was (~1/3) reduced by bilateral IX section, suggesting that IX may play a role in neuromuscular control of UA patency. Also, the AIH‐induced LTF of UA dilator muscles, as some speculated, may help stabilize UA patency in obstructive sleep apnea patients. We hypothesized that AIH induces serotonin‐dependent LTF in IX activity. Adult male Sprague‐Dawley rats were anesthetized, vagotomized, paralyzed and ventilated. The left IX activity was measured before, during and after 5 episodes of 3‐min isocapnic hypoxia (12% O2) with 3‐min hyperoxic intervals (50% O2) to determine baseline, hypoxic response and LTF, respectively. Either saline (vehicle) or ketanserin (a specific 5‐HT2 receptor antagonist, 2 mg/kg, i.p.) was injected ~30 min before AIH. At 45 min post‐hypoxia, the peak amplitude of integrated IX activity had increased by 32 ± 4% (above baseline) in vehicle rats (n=8) but only 2 ± 2% in ketanserin rats (n=6). Ketanserin injection did not significantly decrease baseline activity or hypoxic IX response (90 ± 5% vs. 78 ± 4% above baseline). These data demonstrated that AIH induces serotonin‐dependent LTF of IX activity in anesthetized rats. (Supported by NIH HL64912)
Strain‐ and Wheel Type‐Dependent Variations in Running Wheel Activity in Mice
Authors: Hare Brendan D. , Herrera Gerald M.
Published: June 2021

Numerous studies have shown positive effects of running wheels on behavioral measures, such as conditioned responding and memory. In the present study, we examined running wheel activity in mice housed under various conditions. Two inbred mouse strains were examined (C57Bl6 and BalbC; 5–7 weeks old). Mice were singly housed under two different conditions. One group was housed in ventilated racks and had access to low‐profile wireless running wheels. The second group was housed in conventional cages and had access to “pet‐store” running wheels. On the first day of wheel access, running levels were similar in BalbC and C57Bl6 mice housed in ventilated racks with low‐profile wheels (4.26±1.11 km vs 4.88±1.31 km, respectively). However, BalbC mice in conventional cages with “pet‐store” wheels ran significantly less than C57Bl6 mice on the first day they had access to wheels (1.67±0.66 km vs. 5.16±1.30 km, p<0.05). Running activity increased over several days in all mice. After 7 days, running levels were similar in BalbC and C57Bl6 mice in both ventilated racks with low‐profile wheels (8.53±1.22 km and 6.17±1.22 km, respectively) and in conventional cages with “pet‐store” wheels (5.42±1.26 km and 7.89±1.15 km, respectively). Mice in different housing conditions run similar levels when given access to wheels having different designs, however strain differences exist.
Reguation of oxidative stress by resveratrol in repetitively loaded muscles.
Authors: Ryan Michael James , Always Stephen E.
Published: June 2021

Resveratrol has been suggested to have several beneficial health effects in lower animals and mammals including anti‐cancer, anti‐viral, anti‐inflammatory, and anti‐aging. The purpose of this study was to examine the effects of dietary supplementaiont with resveratrol on oxidative stress induced in mice by repetitive isometric contractions. We hypothesized that resveratrol supplementation would lower indices of oxidative stress and increase the muscles’ adaptive response to loading. Young adult (3 mo) mice were randomly assigned to either a control (unsupplemented) diet or a diet supplemented with 0.05% resveratrol. The plantar flexors of one limb were activated by 20, five‐second isometric contractions (10v, 100 Hz, 200 ìs pulses) for 3 consecutive days. The contralateral limb served as the intra‐animal control. We measured H2O2 production, the GSH/GSSG ratio, aconitase, xanthine oxidase (XO), GPx, Catalase, Mn SOD and CuZn SOD activity, along with mRNA for GPx, Catalase, Mn SOD and CuZnSOD immediately after the last exercise session in control and loaded muscles. The data suggest a link between resveratrol supplementation and the attenuation of increased XO activity associated with repetitive loading. Furthermore, the decrease in XO activity attenuates the increase in oxidative stress related to repetitive loading in mice.
Pronounced fall in renal sympathetic activity after blockade of endogenous angiotensin‐(1‐7) in the paraventricular nucleus during hyperosmolar conditions
Authors: da Silva Ana Quenia Gomes , Santos Robson A.S. , Fontes Marco Antonio Peliky
Published: June 2021

Angiotensin‐(1‐7) participates in the maintenance of sympathetic outflow exerted by hypothalamic paraventricular nucleus (PVN) neurons. Plasma levels of Ang‐(1‐7) are increased in plasma hyperosmolar conditions, which may alter PVN neuronal activity. We investigated if endogenous Ang‐(1‐7) in the PVN contributes to the maintenance of sympathetic output during plasma hyperosmolality. Male Wistar rats (~300g) were anesthetized (Urethane; 1.2g/Kg) and prepared for MAP, HR and RSNA recording. After 30 minutes of hypertonic saline (HTS; 2.5M) intravenous infusion, A‐779 (1nmol; n=6), Losartan (1nmol; n=6) or vehicle (NaCl 0.9%, 100nl; n=5) were microinjected into the PVN. In all groups, HTS infusion evoked a large increase in MAP (in mmHg, A779: 16±4; Los: 26±0; vehicle: 20±6; P<0.01) and a decrease in RSNA (A779: −18±3%; Los: −12±7%; vehicle: −22±5%; P<0.01) compared with isotonic saline group (ITS; 0.15M; n=4; −7±3mmHg; +13±11%). Microinjections of A‐779 after HTS infusion caused a greater fall in MAP (in mmHg, A779: −40±9 vs Los: −16±7; vehicle: −7±3; P<0.01) and RSNA (A779: −26±6% vs Los: −12±5%; vehicle: −2±3%; P<0.01) compared to both Losartan and vehicle groups; HR was not affected. These results suggest that during plasma hyperosmolality, Ang‐(1‐7) exerts a pronounced excitatory effect on PVN neurons, particularly over the sympathetic vasomotor output. Financial Support: CNPq, Pronex and Fapemig.
Expression of Apoptosis‐Inducing Factor During Hypoxia in the Cerebral Cortex of Guinea Pig Fetus at Term Gestation.
Authors: Ashraf Qazi M , Delivoria‐Papadopoulos Maria
Published: June 2021

The mitochondrial protein Apoptosis Inducing Factor (AIF) has been shown to translocate to the nucleus and induce apoptosis. AIF is an important caspase‐independent death regulator in multiple neuronal injury pathways. The present study tests the hypothesis that hypoxia results in increased expression of AIF in the neuronal nuclei of the cerebral cortex of guinea pig fetus at term gestation. To test this hypothesis, we studied guinea pig fetuses at 60 days gestation in normoxia (Nx, n=7) and hypoxic (Hx, n=8) groups. Hypoxia in the fetuses was induced by exposing the pregnant guinea pigs to a FiO2 of 0.07 for 60 min. Cerebral tissue hypoxia was documented biochemically by determining the tissue levels of ATP and phosphocreatine (PCr). Neuronal nuclei were isolated and separated and probed with specific AIF antibody. Protein bands were detected and analyzed by imaging densitometry. ATP levels (μmoles/g brain) were 4.1 ± 0.8 in the normoxic group and 1.9 ± 0.3 in the hypoxic group. PCr levels were 3.7 ± 0.5 in the normoxic group and 1.7 ± 0.3 in the hypoxic group. Expression of AIF was 62.7 ± 3.4 (Nx) and 173.2 ± 7.6 (Hx). The data show that expression of AIF was higher in hypoxic group as compared to normoxic group. These results demonstrate an alternate pathway of programmed cell death following hypoxia in the cerebral cortex of guinea pig fetus at term. (Funded by St. Christopher's Foundation for Children)
Decreased anti‐apoptotic ARC and XIAP as well as elevated pro‐apoptotic Smac and procaspase‐8 protein in soleus muscle of hypertensive rats
Authors: Quadrilatero Joe , Rush James W.E.
Published: June 2021

Elevated skeletal muscle apoptosis plays a significant role in age‐ and disease‐related muscle wasting and dysfunction. Recently, we have demonstrated increased apoptosis in skeletal muscle of hypertensive rats. In this report we extend our previous findings by evaluating several pro‐ and anti‐apoptotic proteins involved in caspase‐mediated apoptotic signaling in skeletal muscle of normotensive Wistar‐Kyoto (WKY) and spontaneously hypertensive rats (SHR). Protein levels of apoptosis repressor with caspase recruitment domain (ARC) and X‐linked inhibitor of apoptosis protein (XIAP) were significantly lower by 59% (p<0.001) and 44% (p<0.05), respectively, in soleus muscle of SHR compared to WKY rats. In contrast, protein levels of the IAP inhibitor, second mitochondria‐derived activator of caspases (Smac), and pro‐apoptotic procaspase‐8 were elevated by 33% (p<0.005) and 32% (p<0.005), respectively, in hypertensive rats. Interestingly, western blot analysis revealed that ARC protein migrated at 30kDa in WKY and 32kDa in SHR soleus muscle; possibly suggesting a posttranslational modification in hypertensive rats. This data confirms our previous findings and suggests that alterations in proteins involved in caspase‐mediated signaling may be responsible for elevated apoptosis in skeletal muscle during hypertension. Research supported by the Heart & Stroke Foundation of Ontario and NSERC Canada.
Quantitative Measurement of Apoptotic and Mitotic Nuclear Events Using Automated Classification of Bright Field and Nuclear Fluorescent Imagery with the ImageStream MultiSpecral Imaging Flow Cytometer
Authors: Morrissey Philip , Henery Shannon , George Thaddeus , Venkatachalam Vidya , Wardhani Aster , Ortyn William , Basiji David
Published: June 2021

Both apoptotic and mitotic cells exhibit nuclear condensation which can be measured in a quantitative manner using a simple DNA dye and the ImageStream multispectral imaging flow cytometry. To distinguish apoptotic from mitotic cells additional morphological measurements such as textured brightfield imagery and increased texture and intensity of dark field scatter imagery may also be required. Distinguishing the sometimes minimal difference in level of nuclear condensation between apoptotic and mitotic cells in an automated manner can be problematic when applying only individual intensity and texture image analysis parameters. Here we present an automatic classifier which generates complex weighted combinations of intensity and texture features applied to multiple images of each cell to optimize distinction and quantitation of apoptotic and mitotic events in the same sample. Imagery from Ramos, Jurkat, and Colo205 cell lines were acquired on the ImageStream multispectral imaging flow cytometer after incubation with mechanistically distinct apoptosis inducing drugs (Camptothecin and Staurosporine) and staining with simple nuclear dyes. The accuracy of an automatic classifier which can measure the apoptotic index and mitotic index of samples based on brightfield, nuclear, and darkfield scatter imagery without the need for additional fluorescent labels using the IDEAS analytical software package is demonstrated.
Regulation of platelet‐activating factor (PAF) receptor by p85α subunit of phosphatidylinositol 3‐kinase (PI3K) in enterocytes
Authors: Lu Jing , Caplan Michael S , Jilling Tamas
Published: June 2021

PAF and intestinal epithelial apoptosis play important roles in the development of necrotizing enterocolitis. We revealed that activation of the PAF receptor (PAFR), a member of the G‐protein coupled receptor (GPCR) family, induces apoptosis through a PI3K/Akt‐dependent pathway in enterocytes. Since direct binding of p85a subunit of PI3K to certain GPCRs was shown to disrupt GPCR recycling and signaling, we sought to study the interaction between PAFR and the p85a subunit of PI3K in intestinal epithelial apoptosis. We revealed that intestinal epithelial cells (IEC‐6) over‐expressing p85a subunit had significantly lower basal levels of caspase 3 activity compared to controls and failed to respond to PAF. Over‐expression of p85a in IEC‐6 cells resulted in sustained activation of Akt and prevented PAF‐induced Akt dephosphorylation. In live cells, over‐expressed p85a in IEC/p85a cells induced the formation of large perinuclear vesicles that were decorated with p85a. When it was transfected into p85a‐expressing cells, PAFR was recruited to these vesicles and there was a co‐localization between p85a and PAFR. This formation of a complex was confirmed by immunoprecipitation. In summary, p85a and PAFR interaction could be a mechanism to regulate PAFR endocytosis and PAFR‐activation‐induced apoptosis.

This may provide insights to design therapeutic interventions for NEC. Supported by NIH HD37581 and DK062960.
Chemosensory function of amphibian skin epithelium: Amiloride inhibition of basolateral membrane depolarization in response to increased bath osmolality correlates with afferent neural activity and behavior.
Authors: Hillyard Stanley D. , Willumsen Niels J.
Published: June 2021

Cutaneous water absorption by dehydrated toads is associated with initiation of water absorption response behavior (WR) in which the toads press the skin to a rehydration surface. WR initiation is suppressed in toads given a hyperosmotic (250 mM) NaCl solution. The activity of spinal nerve 6 increased when the skin was exposed to this solution and the basolateral membrane potential (Vb) of principal cells in the isolated skin depolarized. These results support our hypothesis that the chemosensory properties of the skin are analogous to taste cells of the mammalian tongue where depolarization of Vb also stimulates neural activity. The frequency of WR initiation increased from 21% to 50% of trials in toads given 250 mM NaCl with 10 μM amiloride, the time toads allowed ventral skin contact was greater in the presence of amiloride, and the depolarization of Vb was greatly reduced, relative to NaCl alone. The frequency of WR initiation was also greater with 250 mM Na gluconate as the rehydration solution (40% vs 21% with NaCl) and was increased to 80% of trials when 10 μM amiloride was added to the Na gluconate solution. Inhibition of the transcellular pathway with amiloride and the paracellular pathway with gluconate have similar behavioral responses, and variability in the behavioral responses corresponds with variability in Vb and paracellular conductance.
Rho Kinase inhibitors impair the response of chicken ductus arteriosus to oxygen and other vasoconstrictors
Authors: Villamor Eduardo , van der Sterren Saskia , Agren Pia , Zoer Bea , Blanco Carlos E , Cogolludo Angel L , Perez‐Vizcaino Francisco
Published: June 2021

Accumulated evidence demonstrates that RhoA/Rho kinase‐mediated increases in calcium sensitivity of the contractile apparatus contributes to ductus arteriosus (DA) contraction. We hypothesized that the contribution of Rho kinase to DA tone is developmentally regulated. In the 19‐day DA (total incubation 21 days), the contractions induced by oxygen, KCl, the inhibitor of KV channels 4‐aminopyridine (4‐AP), the α1‐adrenoceptor agonist phenylephrine, and endothelin‐1 were markedly impaired by the presence of the Rho kinase inhibitors Y‐27632 (10μM) and hydroxyfasudil (10μM). The 15‐d DA did not respond to oxygen or phenylephrine and the response to KCl and 4‐AP was also impaired by Y‐27632 and hydroxyfasudil. During contractions induced by KCl (125 mM), Y‐27632 and hydroxyfasudil (1nM‐10 μM) produced a concentration‐dependent relaxation with a respective ‐log EC50 of 5.7±0.2 and 5.58±0.2 in the 19‐d DA; and 5.0±0.2 and 4.98±0.2 in the 15‐d DA (P<0.05 vs. 19‐d). In conclusion, our data indicate that activation of Rho kinase is a key signalling pathway in oxygen‐, as well as membrane depolarization‐ and receptor stimulation‐induced contraction of chicken embryo DA and that a developmental increase in this pathway occurs during prenatal life.

Supported by a grant from “Fundación de Investigación Médica Mutua Madrileña”
Expression of heat shock proteins in aging bivalve mollusks
Authors: Ivanina Anna , Sokolova Inna , Sukhotin Alexey
Published: June 2021

The mechanisms of aging are not well understood in animals with infinite growth such as fish, reptiles, amphibians and numerous invertebrates, including mollusks. Aging in aerobes is accompanied by a progressive cellular oxidative damage counterbalanced by antioxidant protection and repair systems. An important aspect of aging process involves functional changes in the molecular chaperones responsible for folding/refolding of newly synthesized and damaged proteins and for sequestering and degradation of proteins that are damaged beyond repair. The aim of our study was to detect the effects of age on heat shock proteins (HSPs) in two mollusks – oysters Crassostrea virginica and clams Mercenaria mercenaria. Oysters of five age classes (7 mo, 10 mo, 1.5, 2, 2.5 and 4 years old), and clams of three age classes (1, 2 and 4 years old) were studied. Expression of HSPs in gill tissue was detected using standard immunoblotting methods with monoclonal antibodies against HSP70, HSP90 and HSP60. Mitochondrial heat shock protein (HSP60) decreased with age in mollusks suggesting an age‐related decline in mitochondrial chaperone protection. In contrast, changes in cytosolic chaperones were species‐specific. HSP90 and HSP70 increased with age in oysters and clams, respectively. HSP70 expression in oysters did not change, and HSP90 declined in clams with aging. Supported by UNC Charlotte and NSF CAREER award (IBN‐0347238).
Skeletal muscle lipids in Weddell seals (Leptonychotes weddellii): Differences in age class and possible response to resource limitations
Authors: Trumble Stephen J. , Hawke Thomas J. , Pearson Linnea E. , Kanatous Shane B.
Published: June 2021

In this study, we investigated the intramuscular triglyceride (IMTG) profiles and oxidative metabolism substrates of a deep diving mammal, the Weddell seal. We sampled pups (4–6 wks), juveniles (12–15 mo) and non‐lactating adults (< 15 mo) and tested for age‐class, gender and yearly differences. No gender differences were observed in IMTG or oxidative metabolism substrates. While pup (6g/100g) muscles had consistently three times the IMTG stores than juveniles (2g/100g), no differences were observed between yearly IMTG stores. Also, no yearly differences were found in oxidative metabolism substrates (lipoprotein lipase, citrate synthase, lactate dehydrogenase) among age classes. However, the IMTG stores of 2006 sampled adults (2g/100g) decreased 550% from 2005 to levels (11g/100g). This finding appears to correlate with the receding ice coverage in the McMurdo Sound area and its impact on the population density of adults (increase of 62% in 2006). Assuming breeding aged or lacatating females had similar IMTG values implies these adult animals may be faced with a physiological bottleneck similar to that of juveniles. These data suggest that population density of Weddell seals which may be directly impacted by climate change will have dramatic effects on their reproductive success.
Molecular and immunological characterization of Na+/H+ antiporter (NHE3) in the gills of a marine teleost (Myoxocephalus octodecemspinosus)
Authors: Claiborne James B. , Edwards Susan , Kratochvilova Hana , Diamanduros Andrew , Lanier Curtis , Hyndman Kelly , Evans David , Cutler Christopher , Foster Makesha
Published: June 2021

The marine long horn sculpin compensates for metabolic acidosis by excretion of net acid equivalents across the branchial epithelium. Transfer of H+ is dependent on external Na+ and can be inhibited by amiloride. We have evidence that at least four orthologs of mammalian Na+/H+ exchangers are expressed in the gills (NHE1–3, 8). In this study, we have cloned a full open reading frame for gill NHE3. mRNA (Northerns; 4.3–4.8 kb) and protein expression (Westerns; ~104 kd) band sizes were similar to mammalian NHE3. In situ hybridization with anti‐sense probe recognized cells at the base of the lamellae and in the interlamellar space. This location was confirmed with florescent immunohistochemistry using species specific antibodies. Strong apical NHE3 signal was detected in gill cells within the interlamellar region. Na+/K+‐ATPase was colocalized to these cells as well. Sculpin specific antibodies against H+‐ATPase detected cytoplasmic expression of this protein in a separate subpopulation of gill cells. Paralleling in vivo net H+ efflux, NHE3 mRNA signal increased two hours after an infused acid load while apical NHE3 protein expression was detected in both control and acid loaded animals. Thus it appears that an NHE3 ortholog is coexpressed in Na+/K+‐ATPase rich gill cells and likely contributes to H+ excretion for acid‐base regulation. Supported by NSF‐IBN‐0616187 to JBC.
Making Cents of Osirix: Workstation Class Medical Imaging in the Gross Lab
Authors: Fogarty Edward Francis
Published: June 2021

This seminar is a descriptive and illustrative session detailing an innovative approach to teaching anatomy in the medical school curriculum. Clinical medical imaging anatomy data is an important resource for the education of medical students that has been underutilized in the basic science and clinical years of medical education. In the past, this has been due to the cumbersome and expensive nature of film based archives not being readily available to students and teaching faculty. With the advent of Picture Archiving Communication Systems, the cost of bringing these complex imaging databases into use is declining since digital archiving is exponentially less costly than film‐based archiving. Expensive workstations from the clinical imaging industry have limited the use of imaging data for teaching purposes. Recent developments in open‐source DICOM‐based imaging software on the Mac OS have made consumer grade computer systems available for medical image processing. Three‐dimensional renderings of anatomy from this workstation‐class software and virtual dissection techniques will be performed. The involvement of teaching professionals from within the diagnostic radiology community as well as the gross anatomy department is critical to the success of such a program, but the capital cost of the cross‐disciplinary approach is now so small with this particular implementation that it can be easily justified in any budget.
More is not always better: using the dog model to identify what dose of selenium provides the best protection against prostate cancer
Authors: Waters David J , Shen Shuren , Kengeri Seema , Chiang Emily C , Combs Gerald F , Morris J Steven , Bostwick David G
Published: June 2021

In 2001, the NCI launched SELECT to evaluate whether daily supplementation with selenium (Se) or vitamin E prevents prostate cancer. But very little was known about the anticancer mechanism of Se or what dose of Se might offer the most potent cancer‐protective effects. We hypothesized that Se regulates the accumulation of genotoxic damage within the prostate and that the relationship is non‐linear, i.e. more Se is not better. We conducted a randomized feeding trial in which 69 elderly beagles (equivalent to 65 year‐old men) received adequate or supranutritional Se intake for 7 months. We used the aging dog prostate to mimic the aging human prostate, enabling us to study the effects of Se on prostatic cells in an appropriate context. Se supplementation significantly decreased the accumulation of DNA damage in the prostate (alkaline Comet assay). When we examined the relationship between toenail Se level and prostatic DNA damage, we discovered an intriguing U‐shaped dose response curve: more was not better. Further, we showed that the Se level that minimizes DNA damage in the aging dog prostate remarkably parallels the Se level that minimizes prostate cancer risk in 2 large human studies. From the U‐shaped dose response, it follows that not all men will necessarily benefit from increasing their Se intake; baseline nutrient status should be required for all individuals in prevention trials to avoid oversupplementation.
Dysregulation of lipid metabolism in cardiac muscle of mice with cachexia
Authors: Tian Min , Asp Michelle L. , Wendel Angela A. , Belury Martha A.
Published: June 2021

Approximately 30% of all cancer deaths are caused by cachexia, a condition of severe muscle loss and abnormal lipid metabolism. The objective of this study was to elucidate events that lead to impaired lipid metabolism and muscle wasting in cardiac muscle of mice exhibiting cancer‐induced muscle atrophy. In male CD2F1 mice injected with C26 tumor cells, body weight and mass of quadriceps, gastrocnemius and heart were significantly reduced after 18 days compared to mice injected with vehicle. In the highly oxidative cardiac muscle of mice bearing tumors, phosphorylated‐AMP dependent kinase (AMPK) was elevated. However, a rate limiting enzyme in β‐oxidation, carnitine palmitoyl transferase‐1β (CPT‐1β) mRNA expression was reduced by 40% while peroxisome proliferator‐activated receptor α (PPARα) mRNA was reduced by 35%. In contrast, mRNA levels of lipoprotein lipase (LPL) and adipose triglyceride lipase (ATGL) were induced, suggesting increased fatty acid uptake and lipolysis. Unexpectedly, uncoupling protein 2 (UCP2) mRNA was induced 5.3 fold. Activated AMPK with increased fatty acid uptake without induction of markers of β‐oxidation suggest that downstream events of AMPK are dysregulated in cachexia‐induced atrophy of cardiac muscle. Further work will determine whether induction of UCP2 alleviates reactive oxygen species (ROS) and results in thermogenesis. Supported by Cognis Corporation and OARDC.
Development of a simulation model to determine the most appropriate micronutrient composition of a fortified blended food
Authors: Fleige Lisa , Murphy Suzanne P , Sahyoun Nadine
Published: June 2021

Current micronutrient fortification levels in P.L. 480 fortified blended foods (FBF) may not be appropriate for all food aid beneficiaries, particularly infants/young children and pregnant/lactating women. We developed a modeling exercise to simulate fortification levels targeted to different genders/ages, but not exceeding the upper limits (UL) of the Dietary Reference Intakes for any one group. Two models were developed: a complementary food for ages 6–36 months and a supplementary food for all other groups. For each micronutrient, the age and gender group with the highest daily Recommended Nutrient Intake (RNI) relative to energy intake was identified, and the effect of providing different percentages of that RNI was simulated. The modeling exercise also examined consumption of the FBF at 25% (the usual level), 50% and 100% of daily energy intake to assess whether any ULs would be exceeded. Results indicated that if the FBF were fortified at 75% of the RNI, and consumed at 50% of daily energy intake, most micronutrients would not exceed the UL for any group, although at 100% of daily energy intake, several micronutrients were undesirably high. We conclude that fortifying an FBF to 75% of the RNI would be appropriate for most micronutrients, but the potential for excess intake of some micronutrients requires further evaluation. Supported by the Bill & Melinda Gates Foundation.
Sun Exposure, Vitamin D Intake and Vitamin D Status in Davis, CA.
Authors: Hall Laura Marie , Stephensen Charles B. , Aronov Pavel A. , Hammock Bruce D. , Kimlin Michael G. , Slusser James R.
Published: June 2021

The contribution of sun exposure to vitamin D status is difficult to assess. The objective of our study was to measure the contribution of sun exposure, dietary intake and level of skin pigmentation to vitamin D status as measured by serum 25OH vitamin D (25OHD). Our study was conducted with 4 cohorts of healthy young adults each followed for 7â8 wks in the Fall, Winter, Spring and Summer (2006â07) in Davis, CA (38.55 N). Subjects had a wide range of sun‐exposure behavior and skin pigmentation. A total of 72 subjects (â¼18 per season) enrolled and completed the study. Skin pigmentation was measured using a reflectance spectrophotometer. Dietary vitamin D intake was assessed by food records. Sun exposure was assessed using UVB dosimeter badges and questionnaires. Serum 25OHD was measured by LC‐MS. Multiple linear regression analysis was used to predict serum 25OHD using continuous variables for sun exposure (UVB dosimeter), skin pigmentation and diet, and dummy variables for cohort. Sun exposure, skin pigmentation and diet were all significant predictors of serum 25OHD (p < 0.05). These results show that the contribution of these variables to vitamin D status can be assessed using simple methods in free‐living adults. These methods may help nutritionists make more accurate individualized recommendations for vitamin D intake based on skin pigmentation and sun behavior.
C5a plays a role in PMN infiltration into the DSS inflamed colonic mucosa
Authors: Stadnyk Andrew , Rostom Reem , Farooq Shukkur , Stillie RoseMarie
Published: June 2021

A role for complement in colitis has been implicated yet it is unclear whether it promotes or possibly inhibits inflammation. As C5a is a potent PMN chemoattractant and PMN infiltrate the mucosa during dextran sulphate sodium (DSS) colitis, we hypothesized that C5aR−/− mice would experience less colitis due to reduced PMN infiltration. Balb/c and C5aR−/− mice were fed 5% DSS in their drinking water for 5 days then were euthanised on day 6 and their colons examined. Balb/c mice experienced limited clinical illness. Their mid‐colons were inflamed with PMN infiltrates in the mucosa, crypt loss, and small focal ulcers. In contrast, C5aR−/− mice experienced a more severe colitis, with greater ulcers, but interestingly PMN remained primarily in the submucosa. Staining for F4/80 indicated that a substantially greater number of macrophages infiltrated the C5aR−/− colon versus the Balb/c mice. In order to rule‐out the possibility that PMN were damaging the colon from the submucosa, C5aR−/− mice receiving DSS were made neutropenic using injections of Gr‐1 antibody. Despite abolishing PMN infiltration, these mice still demonstrated high numbers of mucosal F4/80+ cells and colon damage. Thus while C5a does play a role in PMN recruitment into the mucosa, our hypothesis was incorrect and C5aR−/− mice in fact experience heightened colitis, with macrophages possibly mediating the inflammation. Supported by the CCFC.
Intracellular signals mediated by crosslinking Monocyte PECAM promote transendothelial migration in the presence of a transmigration blockade
Authors: Florey Oliver , Muller William
Published: June 2021

PECAM is known to play an important role in the transmigration of leukocytes across the endothelium, yet the molecular mechanisms behind this process remain to be elucidated, particularly regarding PECAM on the leukocyte. Inhibiting the homophilic interaction between leukocyte PECAM and endothelial PECAM using blocking antibodies has been shown to reduce leukocyte transmigration. Using an in vitro system, we show that we can overcome a block of transmigration mediated by either anti‐PECAM or anti‐CD99 by cross‐linking PECAM on the surfaces of the arrested monocytes. Crosslinking CD99, another protein involved in monocyte transmigration, did not reverse the inhibition. We found no evidence of a soluble factor released from leukocytes that may explain these results, as pre‐conditioned media from PECAM crosslinked leukocytes did not reverse the transmigration block. This leads us to postulate that crosslinking leukocyte PECAM induces intracellular signals that are important for transmigration and bypasses the requirement for homophilic PECAM interaction. Crosslinking PECAM on monocytes induces a rapid increase in tyrosine phosphorylation of several proteins as well as a src and tyrosine kinase‐dependent increase in intracellular calcium and F‐actin content. This is the first evidence that leukocyte PECAM plays an active role in transmigration beyond the requirement for homophilic engagement.
Pentoxifylline attenuates fMLP‐induced oxidative burst in human neutrophils through PKA dependent and independent mechanisms: Effect on Ras, Raf, and MAPK activation
Authors: Deree Jessica , Costantini Todd , Woon Tiffany , Loomis William H , Coimbra Raul
Published: June 2021

Pentoxifylline (PTX) has been proven to be an inhibitor of fMLP‐induced neutrophil (PMN) oxidative burst and is thought to function by increasing cAMP and Protein kinase A (PKA). We hypothesized that PTX diminishes production of the respiratory burst by both PKA dependent and independent mechanisms.

Human PMNs (1 x 106 cells/sample) were isolated and stimulated with fMLP (1μmol/L) and PTX. PMN activation was determined by the cytochrome C reduction method in the presence and absence of p38 MAPK (SB203580 10 mM), ERK 1/2 (PD98059 50 mM), and PKA inhibitors (H89 10 mM). Western blot analysis of Ras, c‐Raf, p38 MAPK, and ERK 1/2 was performed in PMNs exposed to fMLP and PTX.

PTX produced a decrease in the reduction of cytochrome C that was diminished but not abrogated by H89 exposure. The reduction in Ras activation seen with PTX was not effected by the presence of H89. The attenuation in phosphorylation of c‐Raf p38 MAPK, and ERK 1/2 observed with PTX was only partially reversed with H89.

PTX attenuates fMLP induction of oxidative burst through effects on Ras, c‐Raf, p38 MAPK, and ERK 1/2. The attenuation of early events by PTX in the cascade leading to production of the oxidative burst is PKA independent, while the effects of PTX on signaling distal to c‐Raf phosphorylation rely on both PKA‐dependent and independent mechanisms.

Source of Support: Department of Surgery, University of California San Diego
Biomarkers for ALS disease progression
Authors: Bowser Robert , Ryberg Henrik , Darko Sam , An Jiyan , Wilson Meghan , Lacomis David
Published: June 2021

Amyotrophic lateral sclerosis (ALS) is a fatal neurodegenerative disease resulting from a loss of motor neurons in the brain and spinal cord. ALS is a rapidly progressive disease, with the typical time from diagnosis till death of 2–5 years. There are no current diagnostic tests for ALS or surrogate markers of disease progression. Surrogate markers of ALS disease progression would be valuable measurements for drug efficacy in clinical trials and also identify new targets for drug therapy. To identify protein biomarkers that correlate to clinical measurements of disease progression, we prospectively collected cerebrospinal fluid (CSF) and blood plasma every 6 months from 25 ALS patients. We also collected CSF and plasma from control subjects over a 2‐year time frame. ELISA and mass spectrometry were used to identify protein alterations that correlate to disease progression. We identified protein alterations that correlate to clinical parameters of disease progression within ALS patients, and also proteins that identified subpopulations of ALS patients based on site of disease onset.
Retinoid signaling alterations in amyotrophic lateral sclerosis
Authors: Kolarcik Christi Lynn , Bowser Robert
Published: June 2021

Amyotrophic lateral sclerosis (ALS) is characterized by the progressive degeneration and death of motor neurons (MNs) in the brain and spinal cord. Previous reports demonstrated decreased transthyretin (TTR) levels in cerebrospinal fluid of ALS patients. As TTR impacts retinoid signaling, we examined this pathway using immunohistochemistry and immunoblot. Differences in cytoplasmic proteins including cellular retinol binding protein (CRBP) and cellular retinoic acid binding proteins (CRABP)‐I and II were observed. CRBP immunoreactivity was higher in ALS spinal cord MNs while CRABP‐I was decreased in ALS. CRABP–II was concentrated to the nucleus in ALS MNs while control MNs exhibited a diffuse cytoplasmic pattern. We also assessed the expression and distribution of retinoic acid nuclear receptors. RARβ exhibited increased immunoreactivity in MN nuclei of ALS spinal cord, and RXRβ was increased in MNs of ALS spinal cord. Preliminary studies utilizing a motor neuron‐enriched culture system have evaluated the functional significance of these alterations. Our results indicate that retinoic acid signaling is altered in ALS and components of this pathway may represent novel therapeutic targets.

Sources of research support: NIH grant ES013469 (RB) and NIH T32 EB001026 (CK)