bluuebunny/crossref_metadata_2025 huggingface dataset:
Human cytomegalovirus and mucoepidermoid carcinoma of salivary glands: cell‐specific localization of active viral and oncogenic signaling proteins
Authors: Melnick Michael , Sedghizadeh Parish P , Allen Carl M , Jaskoll Tina
Published: June 2021

Human cytomegalovirus (hCMV) infection is common. Given that hCMV is frequently resident in salivary gland (SG) ductal epithelium, we hypothesized that hCMV would be important to the pathogenesis of SG mucoepidermoid carcinoma (MEC). Previously we found that purified CMV induces malignant transformation in SG cells in an in vitro mouse model, and utilizes a pathogenic pathway previously reported for human MEC. Here we present the histologic and molecular characterization of 39 human SG MECs selected randomly from a repository of cases spanning 2004–2011. IHC assays were performed for active hCMV proteins (IE1 and pp65) and the activated COX/AREG/EGFR/ERK signaling pathway. All four prospective causal criteria for viruses and cancer are fully satisfied: (1) protein markers for active hCMV are present in 97% of MECs; (2) markers of active hCMV are absent in non‐neoplastic SG tissues; (3) hCMV‐specific proteins (IE1, pp65) are in specific cell types and expression is positively correlated with severity; (4) hCMV correlates and colocalizes with an upregulation and activation of an established oncogenic signaling pathway (COX/AREG/EGFR/ERK). To our knowledge, this is the first demonstration of hCMV's role in human oncogenesis that fully responds to all of Koch's Postulates as revised for viruses and cancer.
Mechanisms for luminal cell filling induced by active Rac 1 and K‐Ras
Authors: Kiyokawa Etsuko
Published: June 2021

It has been reported that Rho‐family small GTPases play important roles in cell polarity and cell migration in two‐dimensional environment. However, it has not been shown when and where the GTPases are activated in 3‐dimensional epithelial structures. Based on the principal of Foerster (or Fluorescence) resonance energy transfer (FRET), we visualized the spatio‐temporal changes of RhoA, Rac1, and Cdc42 activity during cysto‐ and tubulo‐genesis of MDCK cells. We found Rac1 activity is homogenous at the entire plasma membrane in early stages of cystogenesis, whereas in later stages Rac1 activity is higher at the lateral than the apical plasma membrane. If Rac1 is activated at the apical membrane in later stages, however, the monolayer cells proliferate into the luminal space. In these cells, tight junctions are disrupted, resulting in mislocalization of polarization markers and disorientation of cell division. These observations indicate that Rac1 suppression at the apical membrane is essential for the maintenance of cyst structure. We also found that the active variant of KRas induced similar luminal cell filling, however, the cell division was not accompanied with this Ras‐induced phenotype. These results suggest that Rac and Ras utilize different mechanism to induce similar phenotypes.
Effect of Postural Change on the Prevalence of Ventilator‐ Associated Pneumonia
Authors: Sato Ryuhei , Miyagawa Tetsuo , Aruga Tohru , Maruyama Ryoko
Published: June 2021

Ventilator‐associated pneumonia (VAP) is the most frequent hospital‐acquired infection among ventilated patients. A few studies have shown that postural change for common secretion management was effective in preventing VAP. The purpose of this study was to evaluate factors associated with the development of VAP, focusing on the relationship between VAP and postural change. We performed a retrospective observational study on 111 patients who had been admitted to the emergency room of a university hospital between January 2008 and September 2010. VAP developed in 32 patients (28.8%); compared to the patients without VAP, the ones who developed VAP had higher rates of blood transfusion, enteral nutrition, and continuous infusion of muscle relaxant. Multiple logistic regression analysis revealed that a longer duration of mechanical ventilation was independently associated with the development of VAP. An increase in postural change within 24 h of endotracheal intubation tended to increase the risk for VAP. Our results suggest that early‐stage postural change for mechanically ventilated patients may affect the incidence of VAP; therefore, a postural change must be carefully performed in a high‐risk group. Further studies should be conducted to examine whether postural change may be a risk factor for VAP in patients requiring mechanical ventilation.
Propofol, a short‐acting general anesthetic, produces depression followed by rebound excitation overshoot of potentials evoked in the prepyriform (olfactory) cortex by stimulation of the lateral olfactory tract (LOT)
Authors: Woolley Dorothy Elizabeth , Hasan Zuheir
Published: June 2021

The role of the prepyriform cortex (PPC) in the anesthetic and subsequent excitatory state produced by propofol was studied in freely behaving rats bearing chronically implanted brain electrodes. Electrical stimulation of the PPC produces seizures more readily than stimulation of most other limbic areas, demonstrating the importance of the PPC in generating seizures in the rat. Propofol enhances the action of GABA on the GABAA receptor, producing an anesthetic state, accompanied by depression of potentials evoked in the hippocampus by stimulation of the dorsal perforant path and the PPC. This is followed by a rebound increase in amplitude and frequencies of the same potentials. Propofol abolished or reduced the amplitude of after‐potentials produced in the PPC by stimulation of the LOT, the principal input to the PPC. This was a typical anesthetic effect and was followed by a significant increase in the amplitude and occurrence of after‐potentials in the PPC. The latter was a typical excitatory effect. Chlordane, a GABA‐antagonist with seizure‐producing effects, had just the opposite effects of propofol during the anesthetic phase. Thus, the PPC is strongly affected by both GABA agonists like propofol and GABA antagonists like chlordane. In turn, they markedly influence seizure‐susceptibility. Supported by research funds from the University of California, Davis.
Varenicline Attenuates Relapse‐like Ethanol Drinking Behavior in Mice
Authors: Sajja Ravi Kiran , Rahman Shafiqur
Published: June 2021

Nicotinic acetylcholine receptors (nAChRs) have been implicated in ethanol drinking behaviors. We have shown that nAChR ligands reduce binge‐like ethanol drinking and preference in mice. Here, we examined the effects of varenicline, a high affinity α4β2* nAChR partial agonist, on ethanol deprivation effect (EDE), a model for relapse‐like ethanol drinking in C57BL/6J mice. After habituation to 15% (v/v) ethanol drinking using a 24 h two‐bottle choice procedure, mice were exposed to repeated cycles of ethanol deprivation (5 day) and re‐exposure (2 day). Following 3rd deprivation cycle, animals received repeated intraperitoneal injections of saline (control) or varenicline (0.5 or 3.0 mg/kg), starting at 12 h prior to ethanol re‐exposure. Control mice exhibited a significant increase in ethanol intake (% baseline) during 3rd re‐exposure (150% at 4 h and 155% at 24 h access). Varenicline (0.5 or 3.0 mg/kg) significantly decreased ethanol intake (% control) at post 4 h (50% or 66% respectively) and 24 h (33% or 50% respectively). In addition, varenicline (3 mg/kg) suppressed locomotor activity in mice compared to control. Effects of varenicline on ethanol clearance and chronic ethanol‐induced ΔFosB expression are being studied. Overall, the study indicates that varenicline attenuates EDE in mice by targeting α4β2* nAChRs and suggests an important role of nAChRs in relapse‐like ethanol drinking behaviors. (Supported in part by SDSURF‐JRF grant)
Buspirone is a potent antagonist at D3 and D4 Dopamine Receptors and attenuates the reinforcing effects of cocaine in a primate model
Authors: Roof Rebecca A , Bergman Jack , Furman Chersyse A. , Conroy Jennie L , Mello Nancy K , Skolnick Phil , Sibley David R
Published: June 2021

Multiple lines of evidence indicate that dopamine plays a pivotal role in the reinforcing effects of cocaine. This evidence has led to the exploration of dopamine blockers as pharmacotherapies for cocaine addiction. While neither D1 nor D2 receptor antagonists have proven effective in the clinic, medications that selectively target D3 and D4 receptors have not been explored for this indication. Buspirone, a 5HT1A partial agonist approved for the treatment of anxiety, has been reported to bind with variable affinity to dopamine receptor subtypes. We now show that buspirone, and two of its metabolites, binds with high affinity to D3 and D4 receptors and with lower affinity to D2 and D1‐like receptors. Live cell assays also revealed that buspirone, and its metabolites, function as antagonists at both D3 and D4 receptors. In behavioral studies, doses of buspirone that had limited effects on food‐maintained responding produced a marked downward shift in the dose‐effect function for cocaine‐maintained behavior, reflecting substantial decreases in self‐administration of cocaine. These results support the further evaluation of buspirone as a candidate medication for the management of cocaine addiction.
Kallikrein site targeted ligands are potent PAR2 antagonists
Authors: Flynn Andrea , Hoffman Justin , Tillu Dipti V. , Patek Renata , Vagner Josef , Price Theodore J. , Boitano Scott
Published: June 2021

Protease‐activated receptor‐2 (PAR2) is G protein‐coupled receptor activated by trypsin‐like proteases including kallikreins and those from the fungus Alternaria alternata. Proteases cleave PAR2 at distinct sites to reveal a subset of tethered ligands, activating the receptor and signaling pathways. Peptides/peptidomimetics that mimic these sequences can activate PAR2 without the off‐target effects of proteases. While kallikrein 14 can activate PAR2, no cognate peptides have been described. We have previously shown synthetic tethering of PAR2 trypsin cleavage site ligands with a palmitate anchor (Palm) and three polyethylene glycol (Peg3) linkers increases peptidomimetic potency. We designed two ligands (SSKRGS‐NH2, 2‐aminothiazol‐SKRGS‐NH2 (2‐at‐SKRGS)) and two synthetic tethered ligands (STLs) (SSKRGSOPeg3‐Palm, 2‐at‐SSKRGSO‐Peg3‐Palm) based on the kallikrein 14 cleavage site. While none stimulated PAR2 Ca2+ or MAPK signaling, we found 2‐at‐SKRGS and both STLs inhibited Alternaria protease‐induced Ca2+ signaling. None of the ligands inhibited Alternaria protease activity, suggesting action at PAR2 to inhibit signaling. Serine substitution increased Ca2+ signaling inhibition, which was further increased by STL addition. These ligands are the first developed from the PAR2 kallikrein site and identified using the STL approach. NIH grants NS073664, HL007249.
Long‐term tissue integration of porous biopolymers as a material platform for metabolic biosensor
Authors: Schroeder Thies , Wisniewski Natalie , Boico Alina , Le Nga , Cho Eugenia , Helton Kristen , Gant Rebecca , Klitzman Bruce
Published: June 2021

Implantable devices induce inflammation and collagen formation, which perturb the metabolic microenvironment and reduce mass transport. This study tested whether novel porous biomaterials improve tissue integration and normalize the glucose gradients. Five materials 700μm thick were implanted into rat dorsal subcutis: cotton cloth, solid silicone elastomer, solid polyHEMA, polyHEMA with 40μm pores, polyHEMA with 80μm pores. Specimens with surrounding tissue were explanted and frozen in liquid nitrogen 1, 4, and 8 weeks after implant. Serial sections were quantified for microvascular density (CD31 antibody), collagen (Masson's trichrome), immune cells (nuclear counterstain) and spatial glucose concentration (bioluminescence). Preliminary results indicate neovascularization of 80μm pore size polyHEMA after 1 week, and complete microvascular infiltration of the 80μm matrix after 4 weeks of implantation. Glucose concentrations in the porous polyHEMA after 4 weeks were comparable to those in the surrounding tissue. At that time, a moderate collagen deposition was observed in the 40 and 80 μm porous polyHEMA. While dense vascularization was also observed with cotton, it had the highest immune involvement. These preliminary findings suggest tissue integrates into porous polyHEMA matrix with a high microvessel density after 4 weeks and a more homogeneous glucose distribution.
Ventral and Dorsal Hippocampus of rats similarly modulate autonomic responses evoked by restraint stress
Authors: Scopinho América Augusto , Lisboa Sabrina F , Corrêa Fernando M.A. , Guimarães Francisco S , Resstel Leonardo B.M. , Joca Sâmia R.L.
Published: June 2021

Introduction

The hippocampus is a brain structure divided into dorsal (DH) and ventral (VH) portions. Stimulation of these areas evokes autonomic changes. During restrain stress (RS) hippocampus neurons are activated, besides causing hormonal changes and autonomic responses such as increased heart rate (HR) mean arterial pressure (MAP) and reduction of tail temperature. Thus, the aim of our study was to investigate the involvement of VH and DH on the autonomic responses, caused by the RS.

Methods

Microinjections of CoCl2 (cobalt chloride; 1mM/ 500nL) or vehicle in the VH or DH of Male Wistar were made. Ten minutes after the microinjection of drugs, the animals were submitted to the RS for an hour. During RS cardiovascular parameters and skin temperature were registered.

Results

The injections of CoCl2 into the VH (n=6) significantly reduced the increase of both MAP (F1,480=449.5; P < 0.001) and HR (F1,480 = 429.8, P<0.0001) and temperature decreases (F1,144 = 273.2, P<0.0001) evoked by RS, when compared to the control animals (n=4). In the same way, the bilateral microinjections of CoCl2 in DH (n=7) reduced the increase of both MAP (F1,165= 211, P<0.05) and HR (F1,165= 161, P<0.05) and temperature decreases (F1,165= 164, P<0.05) when compared to the control animals (n=6).

Conclusion

These results suggest that show that VH and DH similarly modulate autonomic responses to RS.

Financial Support: CAPES and FAPESP.
Investigating the contribution of the adrenal medulla and sympathetic nerves in the regulation of dexamethasone‐induced hypertension
Authors: Soto-Pina Alexandra Estela , Rani Sheela , Franklin Cynthia , Hinojosa-Laborde Carmen , Strong Randy
Published: June 2021

Chronic treatment with dexamethasone (DEX), a synthetic glucocorticoid produces hypertension in humans. Our studies show that DEX treatment in Fisher 344 rats increases mean arterial pressure (MAP) and reduces heart rate (HR) (+16 ± 2 mmHg, and −39±15 beats/min). We tested the hypothesis that these effects are caused by sympathoadrenal activation. Our aim was to determine whether combined adrenal medullectomy (ADMX) and chemical sympathectomy with 6‐hydroxydopamine (6‐OHDA) reduces the cardiovascular effect of DEX. Fischer 344 male rats were implanted with radiotelemetry transmitters and MAP was recorded continuously during the study. After MAP baseline recording, animals were subjected to ADMX surgery, followed by treatment with 6‐OHDA (20 mg/kg, i.p.) and DEX (0.1 mg/day, in drinking water for 7 days). The results indicate that combined ADMX and 6‐OHDA decreased baseline MAP by −14 ± 3 mmHg, and increased HR by 85 ± 8 beats/min. Subsequent DEX treatment increased MAP from post ADMX and 6‐OHDA level by 22 ± 2 mmHg. HR increased by 49 ± 7 beats/min. These results indicate that the adrenal medulla and peripheral sympathetic nerves are involved in the maintenance of baseline blood pressure, but they may not be required for the elevation of blood pressure induced by DEX. Funding support: Department of Veterans Affairs and American Heart Association.
TRPV4 channels tune astrocytic endfoot Ca2+ to optimize neurovascular coupling
Authors: Dunn Kathryn M , Baylie Rachael L , Nelson Mark T
Published: June 2021

A rise in intracellular calcium ([Ca2+]i) in astrocytes is required to communicate the energy demands of neurons to the cerebral microcirculation to precisely match local cerebral blood flow to neuronal metabolism. This “neurovascular coupling” (NVC) is essential for neuronal viability and homeostasis. A moderate rise in [Ca2+]i in the perivascular “endfoot” terminals of astrocytes in response to neuronal activation produces vasodilation of the adjacent parenchymal arteriole, whereas high endfoot [Ca2+]i can produce vasoconstriction. Thus, precise regulation of astrocytic [Ca2+]i is necessary for proper function of the cerebral microcirculation. Transient receptor potential vanilloid 4 (TRPV4) channels are Ca2+‐permeant plasma membrane cation channels that participate in intracellular Ca2+ signaling in a variety of cell types. We previously reported that both the synthetic TRPV4 agonist, GSK1016790A, and the endogenous agonist, 11,12‐EET, increase the frequency and amplitude of perivascular astrocytic endfoot Ca2+ oscillations in brain slices. Here we provide evidence from multiphoton confocal microscopy in brain slices and cerebral blood flow (CBF) measurement by laser Doppler flowmetry in vivo, that Ca2+ entry through TRPV4 in astrocytic endfeet contributes to NVC. Incubation of brain slices with the selective TRPV4 antagonist, HC‐067047 (1 μM), attenuated the parenchymal arteriolar vasodilation to neuronal stimulation by 42% (n=5). This effect was accompanied by a reduction in pre‐stimulation endfoot [Ca2+]i and in the evoked increase in endfoot [Ca2+]i in response to stimulation. Superfusion of HC‐067047 over the somatosensory cortex in mice had no effect on resting CBF, but reduced the CBF response to contralateral whisker stimulation by 9% (n=7). These results indicate that TRPV4 channels tune perivascular astrocytic enfoot [Ca2+]i to optimize NVC. This study was supported by National Heart, Lung, and Blood Institute grants HL‐44455, T32 HL‐07944, and HL‐095489.
Cerebrovascular oxidative stress and endothelial dysfunction in response to aldosterone is Nox2‐mediated
Authors: Chrissobolis Sophocles , Faraci Frank M. , Sobey Christopher G.
Published: June 2021

Aldosterone is thought to be an important contributor to hypertension. There is an association between high aldosterone levels and stroke risk that is blood pressure‐independent. High aldosterone levels may increase stroke risk through effects on the cerebral vasculature involving oxidative stress (via Nox2‐oxidase) and endothelial dysfunction. We tested whether aldosterone produces cerebrovascular oxidative stress and endothelial dysfunction, and Nox2‐oxidase involvement. Aldosterone treatment (0.28 mg/kg/day, 2 wks) had no effect on blood pressure. Aldosterone impaired dilation of isolated, pressurised basilar arteries to acetylcholine (ACh; endothelium‐dependent agonist) compared to vehicle treatment in control mice, indicating aldosterone causes endothelial dysfunction. In contrast, aldosterone had no effect on ACh responses in Nox2‐deficient mice, indicating this effect is Nox2‐dependent. Basal and Nox2‐stimulated superoxide levels were greater in cerebral arteries of aldosterone vs vehicle‐treated mice, effects that were abolished in Nox2‐deficient mice. These data provide the first evidence suggesting aldosterone causes oxidative stress and endothelial dysfunction in brain via Nox2‐oxidase.
Interactive Changes between Macrophages and Adipocytes
Authors: Xie Linglin , Ortega Teresa M. , Mora Silvia , Chapes Stephen K.
Published: June 2021

We tested the hypothesis that communication between macrophages and adipocytes affects insulin response and macrophages function interactively. Firstly, we looked at the impacts of adipocytes on macrophages activation and differentiation. We observed that preadipocytes and adipocytes increased PEC‐C2D macrophage IL‐6 transcript levels, while preadipocytes inhibited IL‐1b transcript levels in co‐culture. We therefore explored the impact of white adipose tissue (WAT) and brown white adipose tissue (BAT) on C2D cells in vivo. After intraperitoneal injection, C2D cells migrated into WAT and BAT. A higher percentage of C2D macrophage cells isolated from WAT expressed Ly‐6C, CD11b, Mac‐2 and F4/80 compared to C2D cultured in vitro, with upregulated transcription levels of inflammatory related genes, but those for BAT isolated C2D is decreased. Secondly, we studied the impact of macrophages on insulin response and differentiation in 3T3L1 adipocytes. Macrophages and recombinant TNF‐a, IL‐1b, and IL‐6 decreased GLUT4 transcript levels in 3T3L1 adipocytes. IL‐6 and TNF‐a, but not IL‐1b, inhibited Akt phosphorylation within 30 min of insulin stimulation. Lastly, adipocyte adipogenesis was inhibited by macrophages or by recombinant TNF‐a, IL‐6, and IL‐1b. These data suggest that macrophages and adipocytes interactively affect insulin signaling, adipocyte differentiation, and macrophage function.
In vitro evaluation of anti‐diabetic potential of Piper sarmentosum Roxb. extract
Authors: Krisanapun Chutwadee , Wongkrajang Yuvadee , Temsiririrkkul Rungravi , Phornchirasilp Srichan , Peungvicha Penchom
Published: June 2021

Diabetes is a chronic disease among many people worldwide. The oral anti‐diabetic medications still have side effects, so anti‐diabetic plants are still in research. Piper sarmentosum Roxb., abundance in Asia and traditional usage as remedy for diabetes, is very interesting. The purpose of this study was to evaluate the water extract from aerial part of P. sarmentosum on 2 mechanisms. The yield of the extract was about 16% w/w of dry aerial part. The inhibition of glucose absorption was conducted by the everted sac method using murine intestine. The results revealed that the extract concentration of 5 and 10 mg/ml could significantly lower glucose absorption (p<0.05) like sodium fluoride, used as positive control, did. The enhancement of glucose utilization was performed using rat diaphragm which represented the muscle cell. Pieces of diaphragm were incubated in Kreb solution with or without the extract for 90 min. The rest of glucose in the media was measured and used for the calculation for muscle glucose consumption. The extract concentration of 2.5–5 mg/ml could enhance glucose consumption about 2–2.5 times in comparison with the control value (without the extract). These results suggest that the hypoglycemic effect of P. sarmentosum may result from the inhibition of glucose absorption and the enhancement of glucose consumption. This work was support by National Research Council, Thailand.
Genotoxic Stress Results in Selective P‐body Formation in S. cerevisiae
Authors: Segal Scott P. , Rathmann Blaine , Koch Anthony , Sutter Chad , Michelson Cara , Wilson Ted
Published: June 2021

Survival mechanisms to cope with oxidative stress are conserved across all eukaryotes. However, our understanding of these mechanisms at the level of DNA and mRNA is poorly understood. Exposure to oxidizing agents can result in damage to lipids and nucleic acids, and can illicit a stress response. The genotoxic agent sodium dichromate oxidizes guanines resulting in formation of OxoG bases, or causes double strand breaks. In contrast, peroxides, allow for strong oxidation of lipids and nucleic acids.

Control of translation may play a role as part of the DNA oxidative stress response. Upon exposing yeast to sodium dichromate, we saw a selective increase in P‐bodies, with a treatment as low as 15 uM, whereas stress granules fail to form. P‐bodies and stress granules are cytoplasmic structures in which non‐translating mRNA are found. Whereas P‐bodies contain RNA degradation factors, stress granules contain translation initiation factors. This suggests that stress granules contain stored RNA poised for translation. Moreover, the response occurs within 10 minutes of exposure, suggesting a model in which either damaged mRNA or mRNA transcribed from damaged genes is selectively degraded rather than stored. Currently, we are determining whether a co‐treatment with anti‐oxidants can abrogate this effect. This study provides novel insights into how yeast and perhaps other higher eukaryotic cells respond to DNA oxidation.
Blood brain barrier KCa3.1 channels: evidence for a role in brain Na uptake and edema during ischemic stroke
Authors: Chen Yi-Je (Jay) , Yuen Natalie , Wallace Breanna K , Wulff Heike , O'Donnell Martha E
Published: June 2021

Our previous studies provided evidence that ischemia induces cerebral edema in part by stimulating activity of a luminal blood brain barrier (BBB) Na‐K‐Cl cotransporter (NKCC), leading to increased secretion of Na, Cl and water from blood into brain through functional coupling of NKCC with abluminal Na/K pump and Cl channels. K channels support NaCl transport across other epithelia by maintaining membrane potential, allowing continued Cl efflux via Cl channels. However, little is known about BBB K channels. In the present study we have identified the presence of the calcium‐activated KCa3.1 channel on cultured bovine cerebral microvascular endothelial cells (CMEC) by patch‐clamp, Western blot and immunocytochemical staining methods. Using radioisotopic flux methods and the KCa3.1‐specific blocker TRAM‐34, we also found a dose‐dependent TRAM‐34 inhibition of K+ flux in the CMEC. Further, we found that the CMEC TRAM‐34‐sensitive K+ flux was stimulated by oxygen‐glucose deprivation. Finally, in magnetic resonance (MR) Na spectroscopy and MR imaging in vivo studies we found that middle cerebral artery occlusion‐induced rat brain Na uptake and edema were significantly attenuated by TRAM‐34 (40 mg/kg IP). Our findings suggest that KCa3.1 blockade constitutes an attractive approach for reducing cerebral edema formation in the early stages of ischemic stroke or during brain surgery.

Supported by AHA & NIH
Pulmonary abnormalities due to Niemann‐Pick Type C2 (NPC2) gene‐targeted deficiency in mice
Authors: Bates Sandra R. , Tao Jian-Qin , Ning Yue , Yu Kevin J. , Gao Ling , Styer Christina , Huang Shaohui , Feinstein Sheldon I. , Roszell Blair R.
Published: June 2021

NPC2 is a luminal protein shown to facilitate cholesterol transport. Patients deficient in this protein demonstrate cholesterol accumulation in lysosomes, neuropathy and lung alveolar proteinosis. This work examined the characteristics of lung disease in the NPC2 gene‐targeted mice. Compared to wild type mice, the NPC2‐deficient mice demonstrated: a) by histology, areas of both severe and mild pathology characterized by thickened septae, “nests” of foam‐filled macrophages, and alveolar macrophages containing excessive surfactant‐like material; b) by electron microscopy, alveolar spaces with alveolar proteinaceous material and type II cells with smaller lamellar bodies (mean area 50% of normal); and c) by biochemical analysis, a 2‐fold elevation in phospholipid (PL) and cholesterol in lung tissue; a 3‐fold enrichment in PL and cholesterol in isolated alveolar macrophages; a 2‐fold increase in protein, a 4‐fold increase in PL and a striking increase in cholesterol (16‐fold) in the broncho‐alveolar lavage fluid, observations characteristic of alveolar proteinosis; with a similar enrichment in lipid profile found in isolated lamellar bodies. Thus, near absence of NPC2 has a detrimental effect on the surfactant system demonstrated by abnormal lung morphology and surfactant composition indicating that the activity of NPC2 is important for the maintenance of normal lung homeostasis. [HL 19737 and NRSA T32‐07748]
Role of CFTR and sphingolipids in hypoxic pulmonary vasoconstriction
Authors: Yu Hanpo , Wang Liming , Kapus Andras , Kuebler Wolfgang M.
Published: June 2021

Background

Hypoxic pulmonary vasoconstriction (HPV) optimizes ventilation‐perfusion matching in the lung. As cystic fibrosis patients suffer from ventilation‐perfusion mismatches, we probed for a potential role of cystic fibrosis transmembrane conductance regulator (CFTR) in HPV.

Methods

HPV was quantified in isolated lungs as increase in perfusion pressure in response to hypoxia. In cultured human pulmonary artery smooth muscle cells (PASMC), Ca2+ concentration ([Ca2+]i) was imaged, and caveolae were isolated and probed for translocated Ca2+ entry channels.

Results

HPV was attenuated by CFTR inhibitors or in lungs of CFTR−/− mice. In PASMC, CFTR inhibition blocked the hypoxia‐induced [Ca2+ ]i increase and the caveolar translocation of TRPC6 channels. CFTR's role in HPV was not attributable to Cl− transport, as modulation of extracellular Cl− did not alter HPV. Instead, the role of CFTR was related to sphingolipid signaling in HPV. In isolated lungs, inhibition of either neutral sphingomyelinase (SMase) or sphingosine kinase attenuated HPV. Exogenous SMase mimicked hypoxia, in that it caused pulmonary vasoconstriction, increased PASMC [Ca2+]i, and translocated TRPC6 to caveolae, yet responses to SMase were blocked by CFTR inhibition.

Conclusion

Our findings identify a critical role for CFTR in HPV that relates to sphingolipid signaling by a mechanism currently under investigation.
The effects of oxytocin on gastric motility are mediated by different neuronal pathways in an iodoacetamide model of functional dyspepsia
Authors: Holmes Gregory M , Travagli R. Alberto
Published: June 2021

Emotional stress triggers functional dyspepsia (FD) and gastric dysmotility. Oxytocin (OXY) has a recognized antistressor role and OXY microinjection in the dorsal vagal complex induces gastric relaxation via a vagally‐mediated pathway. Our in vitro data (see abs. by Babic et al.) show that FD induces a plastic rearrangement in the gastric‐related vagal circuits so that OXY perfusion modulates the otherways unresponsive GABAergic inputs to gastric‐projecting preganglionic vagal (DMV) neurons.

The aim of the present study was to investigate the in vivo mechanisms of action of OXY in a FD model.

Following induction of FD, we monitored the gastric motility response induced by DMV microinjection of OXY (12.5–300pmoles). In control rats, the OXY‐mediated decrease in gastric tone peaked at a dose of 300pmoles and was antagonized by systemic administration of the NOS inhibitor, L‐NAME, which per se did not alter gastric tone. In FD rats, the dose‐response to OXY‐mediated decrease in gastric tone was shifted leftward, L‐NAME increased gastric tone and did not attenuate the OXY response.

These data show that in control rats, the gastric relaxation induced by OXY is mediated by activation of a NANC vagal pathway. Following FD induction, the gastric relaxation mediated by OXY is no longer mediated by activation of a NANC pathway, but rather by inhibition of a vagal cholinergic input.

Funding: NIDDK 55530
Selective effects of inhaled methacholine on 3rd generation bronchial blood flow and airway dimensions in awake sheep
Authors: White Saxon William , McLeod Damian , Parsons Gibbe , Gunther Robert , Cottee David , Quail Anthony
Published: June 2021

Evolution and natural selection in mammals has ensured mechanisms for preventing absorption of inhaled molecules from lung airways. The postulate that inhaled cholinergic compounds selectively access airway wall structures was examined in six sheep instrumented at left thoracotomy under general anaesthesia (i.v. propofol 5 mg.kg−1, 3% isoflurane‐oxygen) each with pulsed‐ Doppler blood flow transducers mounted on bronchial artery, and sonomicrometer probes (Bishop et al, Pulm Pharmacol Ther 20, 178–189, 2007) on high air‐flow resistance 3rd generation lingular lobe bronchus. Continuous measurements of bronchial blood flow (Qbr) and conductance (Cbr), plus bronchial hemi‐circumference (CIRCbr) and wall thickness (THbr) were made in recovered, standing, awake sheep. Dose‐response studies were performed using i.v. methacholine (MCh: 0.125–2.0 μg.kg−1), or nebulised MCh (1–32 mg.kg−1) inhaled by mask. MCh i.v. at the highest dose caused 233% rise in Qbr (P< 0.05), and 286% in Cbr (P< 0.05). CIRCbr fell to 90% of control (P< 0.05); THbr did not change. Inhaled MCh at highest dose caused rise in ventilation, and proportional rise in aortic pressure and Qbr; CIRCbr fell to 91% (P< 0.05); Cbr and THbr did not change. Thus cholinoceptor agonists penetrate the airway wall ex‐ or endogenously to maximally contract the bronchial wall, but by unknown selective mechanisms do not penetrate the systemic vascular bed.
Blockade of P2X purinergic receptors of the lateral parabrachial nucleus reduces hypoxia‐induced tachypnea
Authors: Menezes Miguel Furtado , Menani José Vanderlei , De Paula Patrícia Maria
Published: June 2021

In the present study, we investigated the involvement of the purinergic receptors of the lateral parabrachial nucleus (LPBN) on cardiorespiratory responses induced by hypoxia (HYP) in awake rats. Male Holtzman rats (290–310 g, n=9) with cannulas implanted in the LPBN were used. Mean arterial pressure (MAP), heart rate (HR) and ventilation (VE) were recorded before and after bilateral injections of saline (SAL) or PPADS (a selective P2X purinergic receptor antagonist) into the LPBN under normoxia (NORM, 21% O2) or HYP (7% O2). HYP reduced MAP (95±2 mmHg, vs. NORM: 100±1 mmHg, p<0.05) and increased HR (438±10 bpm, vs. NORM: 396±6 bpm, p<0.05) and VE (1026±84 ml/min/kg, vs. NORM: 620±62 ml/min/kg, p<0.05). Bilateral injections of PPADS (4 nmol/200 nl) into the LPBN in HYP reduced the increase of respiratory frequency (fR, 10±6 cpm, vs. SAL: 28±5, p<0.05) and slightly increased tidal volume (VT, 2.3±0.5 ml/kg, vs. SAL: 1.7±0.6 ml/kg, p>0.05), without changing VE (896±96 ml/kg/min, vs. SAL: 795±74 ml/kg/min). Bilateral injections of PPADS into the LPBN did not change MAP and HR in HYP and VE, MAP and HR in NORM. The results suggest that the reduction of HYP‐induced tachypnea by the blockade of purinergic receptors into the LPBN is counterbalanced by the increase in VT and, therefore, HYP‐induced hyperventilation is not affected, suggesting that activation of purinergic receptors in the LPBN facilitate HYP‐induced tachypnea.

Supported by FAPESP, CAPES and CNPq
Angiotensin converting enzyme 2 attenuates angiotensin II‐mediated phosphorylation of MAP kinase and Akt in neurons
Authors: Sriramula Srinivas , Pedersen Kim Brint , Lazartigues Eric
Published: June 2021

Our lab has reported that overexpression of angiotensin converting enzyme 2 (ACE2) in the brain blunts the development of angiotensin‐II (Ang‐II)‐induced hypertension and sympathetic activation. Yet, the signaling mechanisms are still elusive. Hypothesis: Ang‐II‐mediated phosphorylation of MAP kinase and Akt in neurons is attenuated by ACE2 overexpression. A Neuro‐2a cell line overexpressing ACE2 was generated by stable transfection with an expression plasmid for human ACE2 (N2AhACE2). Control and N2AhACE2 cells were treated with 100 nM Ang‐II or vehicle, in presence and absence of inhibitors. Phosphorylation of p38 MAP kinase (Thr180/Thr182) and Akt (Ser473) by Ang‐II in control cells (2‐ and 1.5‐fold increase, respectively, p<0.05) (Western blot), was prevented by ACE2 overexpression. Pretreatment with DX600 (ACE2 inhibitor) for 30 min had no effect in control cells, but restored Ang‐II‐induced phosphorylation in N2AhACE2. In addition, Ang‐II‐induced expression of AT1R (1.8‐fold increase vs. vehicle, p<0.05) was blunted by Losartan (AT1R antagonist), or ACE2 overexpression. Furthermore, pretreatment with A‐779 (Ang‐(1–7) receptor antagonist) restored phosphorylation in N2AhACE2. These data suggest that ACE2 counteracts Ang‐II‐induced phosphorylation of p38 MAP kinase and Akt, by both, decreasing AT1R signaling and increasing Ang‐(1–7) receptor signaling in neurons. (Support: HL093178)
Quantifying the ellipticalness of the lateral femoral condyle in Gorilla, Pan and Homo
Authors: Sylvester Adam David
Published: June 2021

Articular surfaces of limb bones are thought to be informative for understanding animal locomotion because their size and shape are related to habitual postures and movements. Here I present a method for quantifying an aspect of the shape of femoral condyles, applying this technique to hominid femora.

Three‐dimensional surface models were created for 50 modern human, 30 chimpanzee and 29 gorilla femora. Software was developed that fit separate cylinders to each of the femoral condyles. These cylinders were constrained to have a single longitudinal axis, but could have different radii. The cylinder fit to the lateral condyle was allowed to assume an elliptical cross‐section. The shape of the elliptical cylinder (ratio of the axes of the ellipse) and the angle between the major axis of the ellipse and the femoral shaft were measured. Species were compared using ANOVA and post hoc multiple comparisons tests.

Human lateral femora condyles are more elliptical than those of chimpanzees (p<0.01) and gorillas (p<0.01), however the latter two do not differ. Human femora exhibit a narrow range of the angle between the elliptical cylinder and the femoral shaft (80–110 degrees). Most chimpanzee femora fall in two categories for this angle (25–60 and 90–140 degrees), while gorilla show no preferred angle. This suggests that, unlike gorilla, human and chimpanzee femoral condyles have functionally distinct regions.

Grant Funding Source: N/A
Expression and involvement of the Mas‐related gene receptor MrgD in intestinal inflammation in the mouse
Authors: Avula Leela R. , Buckinx Roeland , Alpaerts Katrien , Adriaensen Dirk , Anderson David , Van Nassauw Luc , Timmermans Jean-Pierre
Published: June 2021

Some members of the Mas‐related gene receptor (Mrg) family have been suggested to play a role in mediating IgE‐independent mast cell activation, as well as in neuroimmune communication. In a previous study, we observed an increased expression of some Mrgs, such as MrgD, in the murine ileum during intestinal inflammation. To further unravel the distribution and involvement of MrgD in intestinal inflammation, we compared the ileum of non‐inflamed and Schistosoma mansoni‐infected wild‐type and MrgD−/− mice. In the wild‐type mice, in the non‐inflamed ileum, immunohistochemistry revealed no MrgD immunoreactivity (IR), whereas in the inflamed ileum, MrgD IR was observed in 5% of the myenteric neurons. Neurochemical coding revealed that the MrgD‐expressing neurons were of the intrinsic primary afferent type. Moreover, MrgD IR was detected in mucosal mast cells (MMCs) in the inflamed ileum. In the MrgD−/− mice, no MrgD IR was detected, while an increased infiltration of MMCs was observed in the inflamed ileum. The expression of MrgD in sensory neurons and MMCs in the inflamed ileum of wild‐types, and the increased MMC infiltration observed after MrgD deletion, suggest that MrgD is involved in nociceptive and mast cell responses during intestinal schistosomiasis. Future work should aim at elucidating the mechanisms underlying MrgD‐mediated effects during intestinal inflammation. Supported by FWO grant G.0179.08
Biochemical Genetics of the Mitochondrial Replicase: Clustering of Pathogenic Mutations into Five Functional Modules in Human DNA Polymerase γ
Authors: Kaguni Laurie S , Farnum Gregory A , Euro Liliya , Palin Eino , Wartiovaara Anu
Published: June 2021

Mutations in Pol γ represent a major cause of human mitochondrial diseases, especially those affecting the nervous system in adults and in children. More than 160 POLG1 disease mutations have been identified, which are nearly uniformly distributed along the length of the POLG1 sequence. We have assessed structure‐function relationships for these mutations by evaluating biochemical data on site‐directed mutagenesis of the human, Drosophila and yeast Pol γs, and their close homologues from the family A DNA polymerase group. We have done so in the context of a molecular model of Pol γ in complex with primer‐template DNA that we developed based upon the crystal structure of the apoenzyme form. We present evidence that 130 pathogenic mutations cluster within five distinct functional modules in the catalytic core of Pol γ. Among these is a novel structural module found only in the Pol γ subfamily of the family A DNA polymerases, which we propose affects the interplay between pol and exo function to achieve the high fidelity of DNA synthesis that is characteristic of Pol γ. Our results suggest that cluster prediction can be used to evaluate both the likely biochemical defects and the relative pathogenicity of new Pol γ variants.
Entrance to a lipoxygenase substrate cavity is defined
Authors: Gaffney Betty J. , Bradshaw Miles , Freed Jack , Borbat Peter
Published: June 2021

A persistent puzzle about lipoxygenase enzymes is how specificity for position and stereochemistry of oxygen addition to polyunsaturated fatty acids is achieved. In lipoxygenase structures, a curved substrate channel approaches the centrally located active site, with ends of the curve near different surface locations. A long‐standing suggestion is that varied specificity results from reversed placement of the polar and methyl ends of the substrate within highly similar protein structures. We approach this problem by placing a spin label on the polar end of a lipoxygenase substrate analog and use paramagnetic distance geometry to locate the substrate spin relative to selected protein positions. Soybean seed lipoxygenase‐1 has been substituted with spin labels at five sites. A five‐point representation of the structure is obtained by measuring ten inter‐site distances by pulsed dipolar EPR spectroscopy (PDS) in doubly spin labeled enzyme constructs. Refinement of spin label coordinates results from modeling and fits to the native protein structure. Finally, the substrate analog polar end is found with five more distance measurements between substrate and protein spins. The spin of the substrate analog is just outside the conjunction of helices‐2 and ‐11. Detailed spin labeling of helix‐2 reveals flexibility permitting entrance to the internal cavity. 1NIH GM65268 and 2NIH RR016292
Effects of Lysophosphatidic Acid on CCN1 Expression in Human Prostate Cancer Cell Lines
Authors: Zhang Zhihong , Bailey Chrystal R , Meier Kathryn E
Published: June 2021

Lysophosphatidic Acid (LPA) is a lipid growth factor that increases proliferation and migration of prostate cancer cells through the G protein‐coupled receptors (GPCRs) LPA1 and LPA3. CCN1 is an inducible secreted matricellular protein that facilitates adhesion signaling, thereby enhancing cancer cell migration and survival. Knockdown of CCN1 in Du145, PC‐3, and LNCaP cells has been shown to limit proliferation. This study addressed the hypothesis that LPA acts through LPA GPCRs to increase the expression of CCN1 in human prostate cancer cells. The results showed that LPA increases expression of CCN1 protein in Du145 and PC‐3 prostate cancer cell lines, with maximal levels reached by 16 hours (Du145) and 4 hours (PC‐3) respectively. Treatment of Du145 and PC‐3 cells with Ki16425, an LPA antagonist acting at LPA1 and LPA3, blocks LPA‐induced expression of CCN1. Knockdown of LPA1 receptors, using siRNA, results in a decrease in LPA‐induced CCN1 expression. These studies provide the basis for continued studies of the role of CCN1 in prostate cancer cells, and for the development and testing of LPA antagonists as potential therapeutic agents in prostate cancer (Supported by UW Grant Number UL1RR025014 from the National Center for Research Resources).
Conformational switching on AKAP7 modulates the phosphorylation dynamics of anchored PKC
Authors: Redden John M , Greenwald Eric , Saucerman Jeff , Dodge-Kafka Kimberly
Published: June 2021

The spatiotemporal fidelity of signal transduction is maintained in large part by scaffolding proteins. In particular, A‐Kinase Anchoring Proteins (AKAPs) accelerate and amplify the phosphorylation of substrate proteins by anchored kinases. However, the mechanisms underlying these phenomena remain unclear. Our present study endeavors to clarify this mechanism utilizing a combination of mathematical modeling, biochemistry, and FRET microscopy. We propose a model wherein enzymes on a scaffold undergo a “conformational switch” into an active intermediate. This model predicts anchored enzymatic reactions to be accelerated, amplified, and insulated from inhibition in comparison to those occuring in solution. We are exploring a novel interaction between AKAP7α and Protein Kinase C (PKC) as a model system to validate these predictions. Our preliminary data comparing the FRET responses of a genetically encoded PKC activity reporter (CKAR) to an AKAP7α‐CKAR fusion protein reveal that both the speed and strength of the CKAR substrate phosphorylation is enhanced when it is tethered to AKAP7α. Additionally, PKC bound to AKAP7α appears to be less susceptible to several classes of PKC inhibitors, including the ATP competitive inhibitor Gö6976 and the substrate inhibitor PKC 20–28. Collectively, our findings provide strong theoretical and molecular evidence that more narrowly defines the anchoring hypothesis.
Blocking of CDCP1 In Vivo Cleavage Presents Akt‐Dependent Survival of Cancer Cells and Inhibits Their Metastatic Colonization via PARP1‐Mediated Apoptosis
Authors: Casar Berta , Hooper John D , Quigley James P , Deryugina Elena I
Published: June 2021

The CUB domain‐containing protein 1 (CDCP1) is a transmembrane molecule implicated in cancer progression. We have established a novel mechanism for CDCP1‐induced signaling in vivo and demonstrated that specific processing of cell surface CDCP1 by an extracellular serine protease is a prerequisite for CDCP1‐mediated survival of cancer cells during metastasis. The in vivo cleavage of CDCP1 triggers a multistep signaling cascade involving activation of Src, Src phosphorylation of membrane‐retained CDCP1 fragment and CDCP1‐associated PKCδ, and activation of Akt, ultimately increasing tumor cell survival. Preventing the in vivo cleavage of CDCP1 with unique anti‐CDCP1 antibodies, serine protease inhibitors or genetic modulation of the cleavage site in the CDCP1 molecule completely abrogated survival signaling and induced PARP1‐mediated apoptosis, resulting in a substantial inhibition of metastatic colonization. In plasminogen knockout mice, the complete lack of CDCP1 cleavage was accompanied by the reduction of tumor cell survival, which were both rescued by in vivo supplied plasmin, indicating that plasmin is the crucial serine protease executing cleavage of CDCP1 during tissue colonization. Together, our findings indicate that in vivo blocking of CDCP1 cleavage upstream of CDCP1‐induced pro‐survival signaling provides a potential mechanism for therapeutic intervention into metastatic disease.
Myocardial bridges: From anatomy to clinical practice
Authors: de Prates Nadir Eunice Valverde Barbato , Cabral Richard Halti
Published: June 2021

Myocardial bridge (MB) continues to be an important concern from anatomy to clinical practice. Since its first anatomical description, there are increasingly many published scientific papers describing patients with MB who present symptoms of angina, myocardial infarction and sudden death. Milking effect is described as narrowing during systole and widening during diastole in the segment wit MB during catheter angiography. Recent reports have described the utility of electrocardiograph (ECG)‐gated multidetector computer tomography (CTA) for non invasive evaluation of coronary artery disease. Intravascular ultrasound is an alternative imaging method which provides morphological and functional in formation for MB. However this method is invasive and is not commonly used in daily practice. So, in order to contribute with some answers we have developed macroscopic, microscopic and biochemical studies of human and non‐human hearts from fetuses to adult specimens. According to our results we found that MB is not acquired during life once it was found in fetuses (28%), Gender has no influence in MB incidence (55% male vs 44% female). Moreover, differently from what is found in adults, the light and scanning microscopic l analysis in children revealed a well‐defined adipocyte layer, located immediately under the MB and perhaps functioning as a protective cushion for the vessel wall against systolic constriction since it was not described any symptom in children. The distribution and composition of glycosaminoglicans: choindritin sulfate, dermatan sulfate and heparin sulfate were also evaluated in coronary artery under MB. This analysis showed an increase in 47% of these components. All these important aspects of MB described in the last years make until now, the need of hardly study this anatomical variation.