bluuebunny/crossref_metadata_2025 huggingface dataset:
An improved histone deacetylase (HDAC) assay and its application for high‐throughput screening of inhibitors against Cryptosporidium parvum enzymes
Authors: Rider Stanley Dean , Zhu Guan
Published: June 2021

High throughput screens (HTS) for HDAC inhibitors are commercially available. The assays involve a two‐part reaction with deacetylation of a substrate followed by development of a chromophore/fluorophore using trypsin‐like proteases. A major obstacle for colorimetric and fluorometric HTS is that compound libraries are replete with visible light absorbing chemicals that can produce false discoveries. The addition of a second fluorogenic substrate that is responsive to trypsin‐like proteases (Bodipy FL casein; Molecular Probes) can reduce false discoveries resulting from general absorbance artifacts or from protease inhibitors. This modification was tested in combination with a commercially available screen (Fluor de Lys; BioMol) and a unique Cryptosporidium parvum HDAC that has been identified as a potential druggable target for antiparasitic chemotherapy. The signal window was unacceptably small for HTS when used with the C. parvum HDAC. To overcome this difficulty, individual buffer components were tested to optimize the assay for use with the parasite enzyme. Adding manganese chloride to the reaction buffer widened the signal window to acceptable levels. The improved assay is suitable for HTS with the C. parvum HDAC.

This research was kindly supported by the NIH‐NIAID through grant R21AI068461.
The acute insulin sensitizing effects of troglitazone are mediated by AMPK activation and inhibition of pyruvate dehyrogenase activity
Authors: Fediuc Sergiu , Pimenta Aurelio da Silva , Gaidhu Mandeep Pinky , So Mandy , Anthony Nicole Marie , Ceddia Rolando Bacis
Published: June 2021

This study investigated the acute effects of troglitazone (Tro) on glucose and fatty acid (FA) partitioning in skeletal muscle. Exposure of L6 myotubes to Tro for 1h resulted in ∼1.3‐ and ∼1.6‐fold increases in palmitate oxidation and CPT‐1 activity, respectively. Tro inhibited basal (∼25%) and insulin‐stimulated (∼35%) palmitate uptake but significantly increased basal (∼2.2‐fold) and insulin‐stimulated (∼2.7‐fold) glucose uptake. Inhibition of AMPK prevented the effects of Tro on palmitate oxidation and glucose uptake. Although Tro exerted an insulin sensitizing effect, it reduced basal and insulin‐stimulated rates of glycogen synthesis, incorporation of glucose into lipids, and glucose oxidation to values corresponding to ∼30%, ∼60%, and 30% of the controls, respectively. These effects were accompanied by an increase in basal and insulin‐stimulated phosphorylation of AktThr308, AktSer473, and GSK3α/β. Tro also powerfully suppressed pyruvate decarboxylation, which was followed by a significant increase in basal (∼3.5‐fold) and insulin‐stimulated (∼5.5‐fold) rates of lactate production. In summary, we provide novel evidence that Tro exerts acute insulin sensitizing effects by increasing FA oxidation, reducing FA uptake, suppressing pyruvate dehydrogenase activity, and shifting glucose metabolism towards lactate production in muscle cells. Funding was provided by NSERC, CIHR, and CDA.
Molecular Cloning and Characterizations of Manduca sexta Spätzle, a possible ligand of Toll
Authors: An Chunju , Jiang Haobo , Kanost Michael R.
Published: June 2021

Drosophila Spätzle is a component of the signaling pathway required for dorsal‐ventral patterning and immune responses. It is produced as an inactive precursor that is activated by a specific proteolytic cleavage. The active carboxyl‐terminal fragment binds to Toll receptor and stimulates signal transduction. To test whether a Spätzle ortholog also exists in the tobacco hornworm, we have cloned a full‐length cDNA of Manduca sexta Spätzle by RT‐PCR using degenerate primers and rapid‐amplification of cDNA ends. The 2589‐bp cDNA contains an open reading frame of 918 bp, which encodes a 33.1 kDa polypeptide, 17% and 44% identical to Drosophila and Bombyx mori Spätzle, respectively. The expression of Spätzle gene is up‐regulated in larval fat body and hemocytes after immune challenge. We have expressed the pro‐Spätzle in Drosophila S2 cells and purified it by copper‐chelating and nickel affinity chromatography. The purified recombinant protein is a disulfide‐linked homodimer. M. sexta hemolymph protease 8 (HP8), a homolog of Drosophila Easter, can cut recombinant pro‐Spätzle in vitro. We are testing whether injection of pro‐Spätzle and Spätzle into the larvae results in elevated mRNA levels for several antimicrobial genes. We propose that M. sexta Spätzle functions in a manner similar to Drosophila Spätzle by acting as a ligand to activate a Toll pathway. Supported by NIH Grants GM41247 and GM58643.
Antigen‐specific TGF‐beta‐induced regulatory T cells modulate mouse splenic dendritic cell function
Authors: Lu Tangying (Lily) , Kim Yong Chan , Andersson John , Shevach Ethan M.
Published: June 2021

It is widely accepted that CD4+CD25+Foxp3+ regulatory T cells (Treg) can suppress the activation of CD4+CD25− T cells. Although Treg might exert their suppressive effects directly on responder T cells, recent studies have raised the possibility that they may act on dendritic cells (DC). To examine the modulatory effect of Treg on DC, antigen‐specific Foxp3+ Treg were generated in vitro by stimulation of Foxp3− T cells in the presence of TGF‐β. Peptide‐pulsed DC were then cultured alone, with Treg alone, with naïve T cells (Tn) of the same specificity, or with Treg and Tn. DC cultured in the absence of T cells spontaneously matured in culture as demonstrated by upregulation of CD86 expression. CD86 upregulation was enhanced 3‐fold by culture with Tn and slightly inhibited by co‐culture with Treg alone. The Tn‐induced upregulation of CD86 was completely inhibited by co‐culture with Treg. DC co‐cultured with Tn and Treg also failed to induce the proliferation of Tn both in vitro and in vivo. The inhibitory effects of Treg on DC maturation could be reversed by the addition of SB216763, a glycogen synthase kinase‐3 (GSK3) inhibitor, but not by anti‐IL‐10, ‐TGF‐β, or ‐CTLA4. These studies demonstrate that the immunosuppressive effects of Treg may be mediated via suppression of DC maturation/function and may involve modulation of the GSK‐3/β‐catenin pathway. Supported by NIH/NIAID.
Negative feedback regulation of T cells: Interleukin 2 and FOXP3 reciprocity
Authors: Popmihajlov Zoran , Smith Kendall A
Published: June 2021

TCR activation of human T cells induces FOXP3 expression, but the mechanism remains unknown. We investigated the role of IL2 in the TCR/CD3‐induction of FOXP3 expression by normal human T cells in vitro. Freshly isolated PBM Cells were stimulated in vitro with anti‐CD3(OKT3) with or without anti‐CD25 and/or anti‐IL2 antibodies; human rIL2 was supplied exogenously. Four‐color flow cytometry was used to determine the expression of FOXP3. Anti‐CD3‐activation increased FOXP3 expression in both CD4+ and CD8+ T cells to a peak at 24h and by 96h was back to baseline levels. Both anti‐CD25 and anti‐IL2 caused a dose‐dependent inhibition of anti‐CD3‐stimulated FOXP3 expression, to a maximal suppression of 80% in CD4+ and >90% in CD8+ T cells, while the addition of rIL2 restored and prolonged FOXP3 expression. When anti‐CD3‐activated PBMCs were monitored daily for 4 days, FOXP3 and IL2 remained mutually exclusive, indicating that FOXP3 expression restricts but does not suppress IL2 expression upon TCR/CD3‐restimulation. TCR‐activated FOXP3 expression is primarily regulated by IL2/IL2R signaling and occurs only transiently in a subset of both CD4+ and CD8+ T cells. These results are consistent with an IL2‐dependent negative feedback loop that regulates the T cell immune response via FOXP3, so that FOXP3 cannot be considered solely a phenotypic marker for human T‐Reg cells.

Supported by NIAID and the Belfer Foundation.
Vascularized bone marrow transplantation (VBMT) across a fully allogeneic barrier induces immune chimerism with absence of graft versus host disease (GVHD)
Authors: Strande Louise F , Lefebvre Daniel , Tai Chau , Matthews Martha , Marra Steven , Hewitt Charles
Published: June 2021

Our laboratory developed a model in rats to study cardiac allotransplantation with simultaneous VBMT. We hypothesized VBMT would induce chimerism with the absence of GVHD.

Lewis female rats received VBMT (n=9) or VBMT with heterotopic heart transplant (n=5) from ACI male donors. Recipients received 2mg/kg FK506 immunosuppression for 14 days. Sacrifice was electively between 21 to 280 days or at time of heart allograft rejection. To demonstrate chimerism, PCR was performed to identify the donor Y‐specific sequence.

None of the rats exhibited clinical signs of GVHD. Four rats expelled the VBMT between days 42 to 201, representing probable technical failure. Remaining rats had no signs of VBMT failure. Histology revealed necrotic bone for the failed VBMTs and areas of viable bone marrow in the successful VBMTs. Two of 5 hearts continued beating up to 280 days. Histology of the cardiac allografts showed viable myocardium. Chimerism was demonstrated in all rats, including the failed VBMTs.

We conclude VBMT is capable of inducing chimerism across a fully allogeneic barrier with the absence of GVHD. We hypothesize that VBMT serves as an extra‐thymic site of immune education via it's intricate microstromal network and modulates the host immune response which may then serve as the foundation for solid organ allograft transplantation tolerance induction. Supported by American Heart Association Grant #0555795T.
ABCA1 is upregulated in athero‐protected regions of arteries in response to brief hypercholesterolemia treatment in vivo
Authors: Civelek Mete , Grant Gregory R , Irolla Chrysta R , Stoeckert Christian J , Karanian John W , Chiesa Oscar A , Pritchard Wiiliam F , Davies Peter F
Published: June 2021

Endothelial cells (EC) in atherosclerosis‐prone regions of arteries display distinct gene expression profiles compared to athero‐protected regions. We investigated the differential alteration of EC phenotype at these sites during brief hypercholesterolemia. Eleven adult male swine were fed a normal chow diet (n=6) or an isocaloric high fat diet (n=5) for a period of 2 weeks. EC RNA was isolated from 3 protected (carotid, descending thoracic, renal arteries) and 3 susceptible (aortic arch, renal branch, abdominal aorta) arterial regions, linearly amplified and hybridized to porcine microarrays. Fifty eight lipid metabolism related genes were differentially expressed in ECs from all regions in response to hypercholesterolemia. However, ATP Binding Cassette A1 (ABCA1), an endothelial transmembrane protein that regulates phospholipids and cholesterol homeostasis inside the cell in response to changing levels of cholesterol in the blood, was specifically upregulated in athero‐protected regions. This response was also validated at the protein level by immunohistochemistry. Region‐specific upregulation of ABCA1 transcript and protein in ECs demonstrates a site‐specific differential EC phenotypic response to hypercholesterolemia. Supported by grants from NHLBI.
Upregulation of PIP3‐Dependent Rac Exchanger 1 (P‐Rex1) Promotes Prostate Cancer Metastasis
Authors: Qin Jianbing , Xie Yan , Wang Bo , Wolff Dennis W. , Scofield Margaret A. , Dowd Frank J. , Hoshino Mikio , Tu Yaping
Published: June 2021

Activation of Rac by guanine‐nucleotide exchange factors (GEFs) plays an important role in cell migration, a critical component of tumor metastasis. We investigated the role of P‐Rex1, a novel specific GEF for Rac, in human prostate cancer metastasis. P‐Rex1 was almost undetectable in a normal prostate epithelial cell line (PrEC) and two prostate cancer cell lines with little metastatic ability (LNCaP and CWR22Rv1), but was clearly detectable in more metastatic prostate cancer cell lines (DU‐145, PC3‐LN4 and PC‐3). Studies of human prostate tumor specimens revealed a four‐fold increase in P‐Rex1 protein expression in localized cancer cells and a five‐fold increase in lymph node prostate metastasis when compared to normal prostate tissues. Rates of migration and invasion in transwell chamber assays for various prostate cancer cell lines were directly correlated with their P‐Rex1 expression levels. Moreover, manipulating P‐Rex1 levels in these cells caused parallel changes in migration and invasion. In vivo studies found that the tumor growth rate of a stable CWR22Rv1 cell line expressing P‐Rex1 was unchanged while metastasis was promoted. Altogether, our results identify P‐Rex1 as an important regulator of prostate cancer metastatic progression and suggest that it may be targeted for prevention and treatment of prostate cancer metastasis.

(Supported by Nebraska State LB595 to Y. Tu.)
Calcium extrusion by plasma membrane calcium pump is impaired in absence of intact caveolae
Authors: El‐Yazbi Ahmed , Cho Woo Jung , Schulz Richard , Daniel Edwin
Published: June 2021

Plasma membrane calcium ATPase (PMCA) is an important calcium extrusion mechanism in smooth muscle cells with PMCA4 as the predominant isoform. PMCA is localized in lipid rafts and caveolae. In this study we examined the effects of blocking PMCA4 function in small intestinal tissue from wild type (Cav1+/+) and caveolin‐1 knockout (Cav1−/−) mice and in bovine airway smooth muscle tissue before and after caveolae disruption. Small intestinal tissues from Cav1+/+ mice treated with the PMCA4 inhibitor caloxin 1c2 (5 μM) showed a higher contractile tone to carbachol (10 μM) when compared to tissues treated with 5 μM of a control scrambled peptide. This effect of caloxin 1c2 was not seen in tissues from Cav1−/− mice. Immunohistochemistry and Western blotting showed that PMCA was co‐localized with caveolin‐1 in Cav1+/+ tissues. In bovine tracheal smooth muscle tissue, caveolae disruption by cholesterol depletion led to the diminution of caveolin‐1 and PMCA4b immunoreactivities, previously co‐localized in the smooth muscle plasma membrane, and to the loss of the increase in carbachol‐induced contraction by caloxin 1c2. Our results suggest that the calcium removal function of PMCA4 in smooth muscle cells is dependent on its presence in intact caveolae. We suggest that this is due to the close spatial arrangement that allows calcium extrusion from a privileged cytosolic space between caveolae and sarcoplasmic reticulum.
The endocannabinoid anandamide is a substrate of cytochrome P450 2D6
Authors: Snider Natasha Tasheva , Sridar Chitra , Hollenberg Paul
Published: June 2021

The endocannabinoid anandamide, an arachidonic acid derivative, activates the cannabinoid receptors CB1 and CB2. These receptors and the enzymes involved in the synthesis and inactivation of their endogenous ligands represent novel targets for the treatment of many nervous system and peripheral disorders. We recently demonstrated that anandamide is a substrate for the liver and kidney cytochrome P450 (CYP) enzymes 3A4 and 4F2. Several CYP isoforms are present in the brain and have important roles in the oxidation of endogenous substrates. The objective of the current studies was to determine if anandamide is a substrate for the human polymorphic CYP 2D6, one of the major CYP isoforms in human brain. We found that recombinant CYP 2D6 converted anandamide to 20‐hydroxyeicosatetraenoic acid ethanolamide (20‐HETE‐EA) and 5,6‐, 8,9‐, 11,12‐, and 14,15‐epoxyeicosatrienoic acid ethanolamides (EET‐EAs) with apparent Km values of 1–2 μM. CYP 2D6 further metabolized the epoxides of anandamide to form several di‐oxygenated derivatives. To our knowledge, anandamide and its epoxides are the first eicosanoid molecules to be identified as CYP2D6 substrates, raising the possibility that this enzyme could be involved in the metabolism of other endogenous mediators with similar structural properties. Supported in part by the National Institutes of Health (Grants CA‐16954 and T32 GM007767).
(+)‐Methamphetamine (METH) pharmacokinetics in timed‐pregnant rats during early‐ and late‐stage pregnancy
Authors: White Sarah Jane , Laurenzana Elizabeth M. , Hendrickson Howard P. , Owens S. Michael
Published: June 2021

The purpose of these studies was to better understand the potential pharmacological impact of METH use through pharmacokinetic studies of METH and (+)‐amphetamine (AMP, a pharmacologically active metabolite) in female rats during early‐ and late‐stage pregnancy. Rats received an i.v. dose of METH (1 mg/kg) on either gestational day 7 (GD7), just prior to organogenesis, or on GD21, 1–2 days before parturition. METH and AMP concentrations in serum and urine were quantitated by LC‐MS/MS. As gestation progressed from GD7 to GD21, METH pharmacokinetic values significantly increased (p<0.05) for area under the METH serum concentration versus time curve (18,075 vs. 21,156 kg‐min/ml) volume of distribution (5 vs. 7 L/kg), and terminal elimination half‐life (86 vs. 116 min). In contrast, systemic and non‐renal clearance significantly decreased. In addition, the AMP metabolite half‐life nearly doubled from GD7 (100 min) to GD21 (191 min). These data suggest that METH and AMP pharmacokinetics are dependent upon gestational time. If similar changes were found in humans, they could impact mothers and their unborn children. (Funded by NIDA grant DA07610 and GlaxoSmithKline Fellowship).
Prostaglandins do not modulate cardiovascular and renal responses to LPS in conscious lambs
Authors: Qi Wei , Fewell James E , Smith Francine G
Published: June 2021

It is well recognized that core temperature responses to LPS are abolished when endogenously produced prostaglandins are removed. The present study was designed to evaluate the role of prostaglandins in the cardiovascular and renal responses to LPS. Core temperature, mean arterial pressure, heart rate, renal blood flow and various parameters of renal function were measured for 60 min before (C) and for 5 h after I.V. injection of 0.03 μg·kg−1of the LPS SAE (i.e., EC100 for core temperature response) (N=10, group I), after pretreatment with indomethacin (N=6, 10 mg·kg−1, group II, to inhibit prostaglandin production) or after vehicle (N=9, group III). The increase in core temperature after LPS was abolished by pretreatment with indomethacin. In contrast the mean arterial pressure, heart rate, renal blood flow, urinary flow and Na+ excretion responses to LPS were not altered by indomethacin pretreatment. Therefore, in young lambs, cardiovascular and renal responses to LPS appear to be independent of endogenously produced prostaglandins.
Effect of exogenous hypercapnia on lung's pro‐ and anti‐inflammatory cytokines during development of pulmonary oxygen toxicity in neonatal rats
Authors: Torbati Dan , Totapally Balagangadhar R. , Raszynski Andre
Published: June 2021

Exogenous Hypercapnia, the intentional addition of CO2 to the ambient gas, is being explored as a possible means of protecting the lung from ongoing damage due to hyperoxia or ventilator‐induced lung injury. We studied the effect of hypercapnia on lung's inflammatory cytokines during the development of pulmonary oxygen toxicity in one‐day old rats exposed to air (normoxia; 21% O2), Hyperoxia (>98% O2), or Hyperoxia/Hypercarbia (HHC; 95% O2 + 5%CO2), for 96 hours (n=15/group). After euthanasia, the lungs were homogenized for determination of IL‐1 beta, IL‐6, TNF‐alpha, and IL‐10 content by ELISA procedures. Production of IL‐1‐beta and TNF‐alpha were significantly reduced during hyperoxia and HHC, as compared to normoxia (P<0.001; ANOVA followed by Tukey‐Kramer). These decreases were more severe in the HHC rats as compared to hyperoxia‐treated rats (p<0.05). IL‐10 production was significantly increased only in hyperoxic rats, as compared to normoxia. No significant changes occurred in IL‐6 production in all groups, compared to each other. These data suggest that exogenous hypercapnia may modulate the onset of pulmonary oxygen toxicity in neonatal rats by modifying inflammatory response to hyperoxia.
Purinergic P2X7 Receptor: A Pivotal Role in Surfactant Regulation?
Authors: MISHRA AMARJIT , CHINTAGARI NARENDRANATH REDDY , LIU LIN
Published: June 2021

P2X7 receptors (P2X7R) are specifically expressed in lung alveolar type I cells (AT1). We hypothesize that the activation of P2X7R on AT1 cells releases ATP and stimulates surfactant secretion from alveolar type II cells (AT2) via a paracrine manner. To test this hypothesis, we isolated primary rat alveolar epithelial cells by releasing them from lung tissues using enzymatic digestion. Immunophenotyping and cytometric analysis with cell specific markers showed 32% and 64% AT1 and AT2, respectively. Surfactant lipid secretion was measured on overnight cultured AT1 and AT2 cells in presence of 3′‐O‐(4‐benzoyl) benzoyl adenosine 5′‐triphosphate (BzATP), a known potent P2X7R agonist. BzATP significantly increased surfactant lipid secretion in the mixed cells in a dose dependent manner when compared to AT2 cells cultured alone and was significantly inhibited by P2X7R antagonist, Brilliant Blue G (BBG). However, when ATP was scavenged with apyrase and ADA, BzATP evoked surfactant secretion was significantly decreased. BzATP‐mediated secretion was also reduced in the presence of suramin and staurosporine, indicating the possible involvement of P2Y2R and downstream PKC‐mediated signaling pathway. Our results suggest that AT1 communicate in a paracrine fashion with AT2 cells in modulating surfactant secretion via P2X7R activation and ATP release.[NIH; HL 052146]
Low tidal volume ventilation increases distal lung fluid absorption in preterm guinea pigs via SGK1 dependent ENaC regulation
Authors: Koshy Shyny , Li Tianbo , Folkesson Hans G
Published: June 2021

The clinical outcome in ARDS patients improved with the introduction of low tidal volume (TV) ventilation. Thus, our objective was to determine why low TV ventilation induced lung fluid absorption in preterm guinea pigs at risk of developing respiratory distress. We ventilated preterm guinea pigs at 66–68 days gestation with low TV (6 ml/kg) and compared with traditional TV (12 ml/kg). Low TV ventilation resulted in a 23 ± 10% (±SD) distal lung fluid absorption, which when compared to −1 ± 3% in conventional TV ventilated fetuses appeared as an induction of fluid absorption. The increased distal lung fluid absorption in low TV ventilated fetuses was associated with an increased αENaC mRNA. We also measured serum and glucocorticoid‐regulated kinase‐1 (SGK1) and Na, K ATPase and correlated changes in SGK1 expression to induction of lung fluid absorption. αENaC and Na,K ATPase expression were both induced when SGK‐1 was elevated. Thus, our data suggest that low TV ventilation increases fetal lung fluid absorption by SGK1‐dependent ENaC regulation. (Funded by March of Dimes Birth Defects Foundation Research Grant No. 6‐FY03‐64 (HGF); Travel award from Peter K. Lauf Student Travel Scholarship (SK)
NKCC1 Is Phosphorylated in Rat Inner Medullary Collecting Duct (IMCD)
Authors: Kassai Eliza M , Yu Ming‐Jiun , Knepper Mark A , Chou Chung‐Lin
Published: June 2021

Previously, we demonstrated a bumetanide‐inhibitable increase in IMCD cell height in response to vasopressin (AVP) in isolated perfused tubules in which the bath osmolality was increased to 490 mOsm (lumen 290 mOsm). This suggests that AVP induced cell swelling was not due to apical water entry, but rather basolateral solute uptake via NKCC1, followed by water entry. To address the role of NKCC1 we used an N‐terminal directed antibody recognizing total NKCC1 (α‐NT) and a phospho‐directed antibody targeted to Thr‐212 and Thr‐217 in the N‐terminal tail of human NKCC1 (R5). Using the α‐NT antibody for immunofluorescence, we confirmed that NKCC1 is localized to the basolateral membrane of the rat IMCD cell. To test if NKCC1 is phosphorylated, we used the R5 phospho‐antibody in immunoblotting. The antibody recognized 170 and 150 kDa bands in IMCD suspensions under control conditions (290 mOsm). Hypertonicity (490 mOsm) caused the band density of the lower band to increase by 2‐fold (P<0.05, n=3). There was no obvious change in mobility of proteins recognized by a COOH‐tail antibody. A dephosphorylation experiment using λ‐phosphatase confirmed that the R5 antibody recognized only phosphorylated proteins. AVP alone did not change the band pattern with the R5 antibody. We conclude that in IMCD cells NKCC1 is a phospho‐protein and that hypertonicity affects the mobility or phosphorylation state of the NKCC1 phosphoprotein.
Molecular Cloning, Tissue Distribution and Ontogenetic Expression of Sodium Proton Exchanger (NHE2) mRNA in the Small Intestine of Pigs
Authors: Zou Shigeng , Feng Dingyuan , Huang Zhiyi , Wang Xiuqi , Zuo Jianjun , Zhi Aimin , Zhou Xiangyan
Published: June 2021

Molecular cloning, tissue distribution and ontogenetic regulation of sodium/proton exchanger isoform 2 (NHE2) mRNA expression were evaluated in the pig small intestine during postnatal development. The 2872‐bp porcine full cDNA sequence of the NHE2 (EF672046) cloned in this study showed 80% and 70% homology with known human and mouse gene sequence, respectively. Hydrophobic prediction suggests 13 putative membrane‐spanning domains within porcine NHE2. The porcine NHE2 mRNA was detected in brain, liver, kidney, heart, lung, small intestine and muscle. The small intestine had the highest NHE2 mRNA abundance and the brain, lung and liver had the lowest NHE2 mRNA abundance (P < 0.05). Along the longitudinal axis, the duodenum had the highest NHE2 mRNA abundance and the ileum and colon had the lowest NHE2 mRNA abundance (P < 0.05). The NHE2 mRNA level was increased from the day 1 to day 26 in the duodenum (P < 0.05) and dropped dramatically on day 30 (P < 0.05). There is no difference between day 1 and day 7 (P > 0.05). After day 30, the NHE2 mRNA level stayed at the same except on day 90 (P > 0.05). The mRNA expression of NHE2 was not only differentially regulated by age but also differentially distributed along the small intestine of piglets at early stages and growing stages of life, which may contribute to changes in NHE activity.
Chronic inhibition of 11beta‐hydroxysteroid dehydrogenase activity reduces cardiovascular comorbidities in corpulent and stroke‐prone SHR
Authors: Schnackenberg Christine G. , Costell Melissa H. , Eybye Marianne E. , Cui Jianqi , Maniscalco Kristeen , Bentley Ross , Willette Robert N. , Yue Tian‐Li , Doe Chris P.
Published: June 2021

Changes in the secretion or metabolism of cortisol are associated with human hypertension and metabolic disorders. 11beta‐hydroxysteroid dehydrogenase 1 (11β‐HSD1) converts inactive glucocorticoid cortisone to active form cortisol within tissues. We tested the hypothesis that 11β‐HSD1 contributes to the hypertension, insulin resistance, dyslipidemia, and end organ damage in SHR‐SP or obese SHR‐cp rats. Cardiovascular comorbidities were measured before and during 3 weeks of vehicle or compound 11 (10 mg/kg/d) administration in SHR‐cp. In association with significantly decreasing 11β‐HSD1 activity in liver and adipose of obese SHR‐cp, compound 11 significantly reduced hyperglycemia, hyperinsulinemia, hypertriglyceridemia, and hypertension without affecting microalbuminuria or creatinine clearance. In a separate study, cardiovascular comorbidities were measured before and during 8 weeks of high salt and high fat intake in the presence or absence of compound 11 (24 ppm) administration in SHR‐SP. In association with significantly decreasing 11β‐HSD1 activity in liver of SHR‐SP, compound 11 reduced hypertension, plasma LDL and triglycerides, left ventricular wall thickness and hypertrophy, and microalbuminuria. These studies suggest that 11β‐HSD1 plays an important role in the pathogenesis of hypertension, metabolic disorders, and end organ damage.
GABAergic transmission in the A5 area modulates sympatho‐respiratory response to hypoxia and breathing pattern variability in rats
Authors: Dhingra Rishi R , Fishman Mikkel , Hsieh Yee‐Hsee , Loparo Kenneth A , Dick Thomas E
Published: June 2021

Ventrolateral pontine neurons (A5 area) receive inputs from cardiovascular‐ and respiratory‐related sources, but appear to only modulate the sympatho‐respiratory response to hypoxia. We previously reported that the GABA(A) antagonist bicuculline (BIC) increased the average amplitude of splanchnic sympathetic nerve activity (sSNA) during the second half of expiration and accentuated the prolongation of expiratory duration during the hypoxic ventilatory decline, but had no effect during basal conditions. To test the hypothesis that inhibition of neurons in the A5 area affects patterning during basal conditions, we recorded PNA and sSNA before and after bilateral microinjections of BIC into the A5 area of anesthetized, paralyzed, ventilated, vagotomized and thoracotomized adult male rats (Sprague Dawley). We analyzed the respiratory pattern by systematically applying a set of time‐series analytical tools. During basal conditions after BIC, the co‐efficient of variation (CV) was not significantly altered. However, nonlinear properties of the breathing pattern (Mutual Information, Sample Entropy of the Intervals and Sample Entropy of the Successive Differences) were ablated after BIC. These observations suggest that GABAergic transmission in the A5 area affects breathing pattern variability even during basal conditions. (HL‐080318)
Effects of exercise on expression of skeletal muscle acyl‐CoA synthetase isoform gene expression in obese African‐American and Caucasian women
Authors: Woodlief Tracey Leigh , Sweazey Melanie A. , File Courtney , Hickner Robert C. , Neufer P. Darrell , Cortright Ronald N.
Published: June 2021

Reduced skeletal muscle (SKM) mitochondrial fatty acid oxidation (FAO) in African‐American women (AAW) may be due to reduced activity of Acyl‐CoA Synthetase‐long form (ACSL). However, endurance exercise training (EET) has been shown to increase SKM FAO in both AAW and Caucasian women (CW). The purpose was to determine the metabolic flexibility of SKM to expand the activity of ACSL in response to EET in obese (BMI 35–40 kg/m2) AAW and CW women. Following a 12 hr fast, biopsies from AAW and CW were obtained from either the rectus abdominus or vastus lateralis before and 4 h after acute exercise in the sedentary and trained (10 d and 8 wk of cycle ergometry) condition. Assays for total ACSL activity, protein content, and gene expression of ACSL 1, 3, 4, 5, and 6 isoforms were assessed. In rectus abdominus, lower (P<0.05) ACSL‐5 protein content was observed in obese AAW vs. CW. Prior to EET, total ACSL activity in vastus lateralis was lower in both obese groups compared to leans, although activity was lower in obese AAW. 10 days of EET did not result in elevated ACSL activity in either obese AAW or CW. However, following 8 wk of EET, both groups demonstrated increased enzyme activity. In response to acute exercise, ACSL‐1 and ‐3 isoform gene expression rose solely in CW. In the trained, rested state, ACSL‐1 and ‐3 isoform expression was elevated in obese CW only (P < 0.05). Results support ACSL as a potential molecular mechanism to explain reduced SKM mitochondrial oxidative capacity in AAW although the increase in FAO in AAW after EET can not be explained by ACSL isoform gene expression.

Supported by NIH DK065183‐01 and DK075880‐01
EFFECTS OF NORMOXIC AND HYPOXIC BREATHING ON TISSUE PO2 IN THE HYPOTHALAMIC PVN: IMPLICATIONS FOR HYPOXIC ACTIVATION OF SYMPATHETIC NERVE ACTIVITY (SNA).
Authors: Chen Qing‐Hui , Andrade Mary A , Calderon Alfredo S , Mifflin Steven W , Toney Glenn M
Published: June 2021

Carbon fiber electrodes were implanted (3–14 days) in the PVN of rats (n=4) for amperometric recording of tissue pO2. Conscious rats were then placed in a plexiglass chamber and ambient pO2 was varied from normoxic (21%) to hypoxic (10%) levels every 3 minutes. Tissue pO2 averaged 13 ± 2 mmHg in the PVN during normoxic breathing and fell to 3 ± 1 mmHg during hypoxic breathing, during which an increase of renal SNA was observed. The same rats were then anesthetized and identical cycles of normoxic/hypoxic ventilation were performed and produced nearly identical changes of PVN tissue pO2. Effects of physiological variations of tissue pO2 on the activity of RVLM‐projecting pre‐sympathetic PVN neurons were investigated in brain slices. Slices were perfused with aCSF gassed (O2/N2/5%CO2 mixtures) to produce the same levels of tissue pO2 recorded in the PVN in vivo. In whole‐cell voltage‐clamp recordings (Vhold: −80 mV), tissue hypoxia caused an inward shift of holding current (17 ± 3 pA, n=4). Identical experiments in rats exposed to intermittent hypoxia for 7 days, showed that tissue hypoxia induced larger inward shifts of holding current (37 ± 4 pA, n=4). We conclude that tissue pO2 in the PVN varies in phase with normoxic and hypoxic breathing/ventilation and that the level of tissue hypoxia occurring in the PVN in vivo is sufficient to acutely activate currents capable of exciting sympathetic‐regulatory PVN neurons. Support: HL086804 (SWM), HL071645 (GMT)
Leptin signaling in the nucleus tractus solitarii increases sympathetic outflow to the kidney.
Authors: Agassandian Khristofor , Morgan Donald A. , Cassell Martin D. , Rahmouni Kamal , Mark Allyn
Published: June 2021

Leptin receptors (Ob‐Rb) are located in the nucleus tractus solitarii (NTS). Leptin into the NTS decreases food intake and body weight. We have demonstrated that intra‐arcuate leptin increases sympathetic nerve activity (SNA) to brown adipose tissue (BAT) and kidney. We tested the hypothesis that activation of leptin receptors in the NTS affects SNA. In anesthetized Sprague‐Dawley rats, we microinjected leptin 0.25 or 0.5 ug or saline 50 nl into the commissural NTS while recording simultaneously SNA to BAT and kidney. Localization of the site of injection was confirmed in each experiment. Microinjection of leptin into the commissural NTS produced a dose‐related increase in renal SNA (+93 +/− 32% with 0.5 ug, P=0.04 vs. saline), but did not increase SNA to BAT (−17+/− 9%). Cooling of the rats increased SNA to BAT demonstrating the responsiveness of BAT SNA in our preparation. NTS leptin did not increase SNA in Zucker obese rats with mutations in the Ob‐Rb indicating that the increase in renal SNA in the Sprague Dawley rats is receptor mediated. Leptin injection into the rostral NTS did not affect BAT or renal SNA. In conclusion, microinjection of leptin into the commissural NTS increases SNA to kidney but not to BAT. This occurs through an Ob‐Rb mediated mechanism. Leptin produces a different pattern of sympathetic responses when administered into the NTS than it does into the arcuate nucleus.
Vascular endothelial growth factor decreases blood pressure in hypertensive pregnant rats
Authors: Gilbert Jeffrey Stephen , Karumanchi S Ananth , Fournier Lillian , Cockrell Kathy , Granger Joey P
Published: June 2021

We have recently shown that placental ischemia in the pregnant rat results in hypertension and elevated concentrations of circulating soluble fms‐like tyrosine kinase‐1 (sFlt‐1), a naturally occurring vascular endothelial growth factor (VEGF) antagonist that is over‐expressed in preeclampsia. We hypothesized that chronic VEGF administration during the last trimester of pregnancy would attenuate the hypertension associated with placental ischemia in our well characterized model of reduced uterine perfusion pressure (RUPP). VEGF (80 ug/kg/day) was administered for five days via osmotic mini‐pump placed intraperitoneal. Arterial pressure (AP) and tissues were obtained on gestation day 19 from RUPP+VEGF, RUPP and normal pregnant (NP) dams. VEGF administration attenuated the hypertension produced by RUPP (115 ± 1 vs. 135 ± 3 vs. 104 ± 1 mmHg; P < 0.001) but did not normalize AP to NP levels (mmHg; P < 0.01). VEGF ameliorated the fetal (1.9 ± 0.2 vs. 1.8 ± 0.1 vs.2.2 ± 0.1; P < 0.05) and placental (0.46 ± 0.12 vs. 0.36 ± 0.07 vs.0.57 ± 0.03; P < 0.05) growth restriction observed in RUPP compared to NP dams. These data demonstrate that VEGF may be an effective treatment for reducing high blood pressure associated with placental ischemia. Further, these results indicate that VEGF supplementation may also promote fetal and placental growth despite reductions in uterine perfusion pressure.
Analysis of Transgenic Mice Containing the Cystatin S Promoter
Authors: Shaw Phyllis A. , Li Jinhua , Luo Jian
Published: June 2021

Phylogenetic “footprinting” of the cystatin S gene suggests that DNA‐binding sequences for specific functions are clustered in the 5′‐flanking region. The 1.9kb promoter has 3 types of transcription factor binding “modules”, including a hormone module containing three regions found in the 5′‐flanking sequence of all known salivary gland specific genes. To determine which of the cis‐elements are functional and responsible for tissue specific regulation of the cystatin S gene, we generated two transgenic mouse models. One, the 1.9kb/GFP expressing transgene, contains the hormone module and the salivary gland‐specific elements. We examined the 1.9kb promoter driven expression of GFP mRNA during development of the submandibular, sublingual, and parotid glands, and liver, heart, kidney, muscle, spleen, brain, and lung, using real time RT‐PCR analysis of total RNA. The cystatin S 1.9kb promoter regulates GFP mRNA expression in all the tissues examined during development, albeit at a low, perhaps basal, level in some. Submandibular and sublingual glands exhibit the highest levels of GFP mRNA at 10, 15 and 25 days of development. The spleen, heart, muscle and lung have the highest level of GFP mRNA at day 10. The brain shows the highest level of GFP mRNA at 25 days; the parotid glands, liver and kidney show the highest level of GFP mRNA at 35 days. We are currently examining GFP protein levels in the same tissues during development of the 1.9kb/GFP transgenic mice to determine if tissue‐ and cell type‐specific regulation is at the protein level rather than the mRNA level.
IT TECHNOLOGY ENABLES SAME‐DAY PAP TEST
Authors: Markovic Olivera , Markovic Nenad Svetomir
Published: June 2021

INTRODUCTION

The MarkPap® test, a new improved biomarker‐based assay for cytological cervical cancer screening was introduced to the Experimental Biology audience in 2004. In clinical trials, this test was found to be more sensitive and with less false negatives than the control Pap test. The biomarker is amenable for digital imaging and we have developed a prototype of a device to exploit on this characteristic and to explore whether human participation could be efficiently replaced with electronic communication technology for the purpose of mass cervical cancer screening.

METHOD

We developed a prototype of a device‐‐composed of biomarker‐based cytology and digital imaging included into an IT communication network‐serving, at the input side, the providers of specimen image files and, at the output site, the image evaluators (result providers). We tested our IT communication protocol, and began developing a diagnostic protocol.

RESULTS

The striking improvement was achieved with the time reduction between specimen arriving in the laboratory and providing the final results from a remote site. The time for specimen staining, screening, image and demographic data collection, filing a series of typical abnormal images into case folders, forwarding those folders to the central processing unit, alerting the examiners, login, reviewing the folders, consulting other experts if necessary, writing case reports and reporting the results back, could be achieved for approximately two hours per evaluation of an abnormal slide.

CONCLUSION

This result indicates that using IT technology for telecytopathology‐based cervical cancer screening could transform the current practice into a same‐day pap test‐a huge improvement in time and cost of labor.
NEUROGENESIS OF AIRWAY PROTECTIVE BEHAVIORS IN THE CAT: COUGH AND PHARYNGEAL SWALLOW
Authors: Bolser Donald C. , Rose Melanie J. , Pitts Teresa E. , Davenport Paul W. , Baekey David M. , Segers Lauren S. , Nuding Sarah C. , Lindsey Bruce G. , Shannon Roger , Gestreau Christian , Morris Kendall F.
Published: June 2021

Neurons in the ventrolateral medulla involved in generating the motor pattern for breathing also have roles in the production of cough and swallowing behaviors. The reconfiguration of brainstem neural networks into selective behavioral control assemblies (BCAs) that govern different motor patterns required to optimize airway protection is not well understood. We challenged anesthetized cats with simultaneous cough and swallow‐producing stimuli to examine temporal relationships between these behaviors when co‐expressed. We also recorded from ventrolateral medullary respiratory neurons during cough and swallow. Swallows occurred during repetitive cough and were restricted to the cough E2 phase. The cough E2 phase was longer when a swallow occurred (3.7±1.5 s control, 5.7±1.5 s with swallow, p<0.002). Prolonged apnea occurred during repetitive swallow and was associated with increased tonic discharge of a subset of expiratory decrementing (E‐Dec) and silence of some inspiratory (I) neurons. Other E‐Dec and I neurons had phasic increases in discharge rate during swallow. The results support: a) an important role of E‐Dec neurons in coordinating cough and swallow, and b) complex coordination of these behaviors to ensure airway protection, presumably mediated by elements common to both cough and swallow BCAs. Supported by HL89104, HL89071, and NS19814.
Acute reverse microdialysis (MD) of atropine into the pontine respiratory group (PRG) of goats at night has no affect on breathing, but does tend to increase NREM and REM sleep
Authors: Bonis Joshua , Neumueller Suzanne , Krause Katie , Kiner Tom , Smith Al , Qian Baogang , Pan Lawrence , Forster Hubert
Published: June 2021

Acetylcholine is an important neurotransmitter in the PRG (Respir. Physiol. Neurobiol. 2004 Nov 15;143(2‐3):235‐49). Tonic pontine influences may have increased importance during sleep to stabilize and prevent large fluctuations in breathing. Abnormalities in this tonic facilitation, such as attenuation of cholinergic modulation (Respir. Physiol. Neurobiol. 131:135‐144, 2002.), may contribute to sleep‐disordered breathing. Accordingly, we chronically implanted microtubules into the PRG of 7 goats to assess the response of MD of atropine (50mM) into the PRG at night. MD of atropine at night did not have a significant effect on pulmonary ventilation (VI), breathing frequency, or tidal volume compared to MD of mock cerebral spinal fluid (mCSF). However, a normal state‐dependent respiratory depression was observed, where VI during REM < VI during NREM < VI during wakefulness. The percent of NREM and REM sleep tended to increase from the first night of ipsilateral MD, to the second night of contralateral MD, and to the third night of bilateral MD. We conclude that MD of atropine into the PRG at night does not affect breathing, but does tend to increase both NREM and REM sleep. Supported by the Department of Veterans Affairs and NIH 25739.
Central terminations of the afferent nerves regulating cough in guinea pigs
Authors: Canning Brendan James , Mori Nanako
Published: June 2021

The vagal afferents regulating cough in anesthetized guinea pigs (GPs) are acid sensitive mechanoreceptors that are differentiated from airway/ lung C‐fibers and rapidly adapting receptors (RARs) by their conduction velocity and insensitivity to capsaicin and changes in airway luminal pressure (J Physiol, v557, 543‐558, 2004). We hypothesized that the 'cough receptors' would have unique patterns of termination in nTS. We found a location rostral and lateral to obex at which a combination of NMDA (AP‐5) and nonNMDA (CNQX) glutamate receptor antagonists could be microinjected bilaterally at doses that nearly abolished coughing evoked by citric acid challenge (10±2 and 2±1 coughs in control and treated GPs, respectively; n=6‐10) but was without effect on basal respiratory rate or tachypnea evoked by either iv. histamine (RAR‐selective) or bradykinin (C‐fiber selective). Microinjecting these drugs into adjacent nTS locations (1‐2mm distal) had no effect on cough (n=12). Neural tracing studies confirmed the selective termination of the cough receptors in this nTS location. Unique patterns of CNS termination of these vagal afferents may facilitiate their selective regulation of airway reflexes.
Dietary carbohydrate interacts with dietary fat to influence leptin responsiveness in rats
Authors: Haring Samantha Jean , Harris Ruth BS
Published: June 2021

Previously we showed that a 60% kcal fructose, and 10% kcal fat diet induces leptin resistance in rats. All rats fed the 60% kcal fructose diet were thinner than those fed a diet with 17% fructose and 10% fat (LFruc/LF), therefore, we tested leptin responsiveness in rats fed either a 40% kcal fructose and 30% kcal fat (MFruc/HF) or the LFruc/LF diet. An i.p injection of 2.0 mg/kg leptin inhibited food intake and weight gain of both MFruc/HF and LFruc/LF rats after days 7, 14, and 21 on diet. On day 36, leptin inhibited 14‐hour energy intake only in LFruc/LF fed rats. To determine whether changes in leptin responsiveness were due to fructose specifically or increased dietary monosaccharide, a second study tested leptin responsiveness in rats fed a 50% kcal glucose and 30% kcal fat diet (HGluc/HF), MFruc/HF or LFruc/HF diet. After 10.5 weeks, i.p. injections of 2.0 mg/kg leptin inhibited 14 hour weight gain and food intake in MFruc/HF and HGluc/HF groups but not LFruc/HF rats. These data suggest that dietary fat and carbohydrate have independent effects on leptin responsiveness and monosaccharides may reverse dietary fat‐induced leptin resistance. Supported by NIH grant DK053903.
Role of the Renin Angiotensin System on Bone Marrow‐Derived Stem Cell Function and its Impact on Skeletal Muscle Angiogenesis After Electrical Stimulation
Authors: Resende Micheline M , Stodola Timothy J. , Greene Andrew S.
Published: June 2021

Autologous bone marrow cell (BMC) transplantation has been shown as a potential approach to treat ischemic disease. However, BMC dysfunction has been reported in several conditions, leading to poor cell engraftment and failure in tissue revascularization. We have previously shown that skeletal muscle angiogenesis induced by electrical stimulation (ES) is impaired in the SS/Mcwi rats and this effect seems to be related to an impaired regulation in the renin angiotensin system (RAS). The present study explored the role of the RAS on BMC function and its impact on skeletal muscle angiogenesis induced by ES. SS/Mcwi rats were randomized to receive BMC from: SS/Mcwi; SS‐13BN/Mcwi; SS/Mcwi rats infused with saline or ANGII (3 ng.kg‐1.min‐1). BMC were injected in the stimulated tibialis anterior muscle of SS/Mcwi rats. After 7 days of ES, unstimulated and stimulated muscles were harvested and the vessel density was evaluated. BMC isolated from SS/Mcwi or SS/Mcwi rats infused with saline failed to restore angiogenesis induced by ES. However, cells isolated from SS‐13BN/Mcwi consomic rats as well as from SS/Mcwi rats infused with ANGII showed a significant increase in vessel density after 7 days of ES. This study suggests that an impaired RAS regulation may be associated with a significant BMC dysfunction and have profound effects on the angiogenesis response induced by ES in SS/Mcwi rats. (Support NIH HL‐29587, N01 HV‐28182).