bluuebunny/crossref_metadata_2025 huggingface dataset:
Regulator of G‐Protein Signaling 2 Blunts Cellular Viability Mediated By A Stabilized Lysophosphatidic Acid Analogue In the Presence of Chemotherapy In An Ovarian Cancer Cell Model of Chemoresistance
Authors: Altman Molly Koertel , Callihan Phillip , Ali Mourad , Hooks Shelley , Murph Mandi
Published: June 2021

Lysophosphatidic acid (LPA) enhances growth, survival, and proliferation in numerous models. The receptors for LPA are implicated in oncogenic signaling and represent a group of therapeutic targets in cancer. Regulator of G protein Signaling 2 (RGS2) is a GTPase activating protein (GAP) that turns off G protein‐coupled receptor (GPCR) signaling. Previously, we demonstrated that reduced RGS gene expression develops with the occurrence of drug resistance in ovarian cancer cell lines. Here, we investigate whether the LPA receptors are also involved in mediating viability as a result of enhanced GPCR signaling. To test this idea we used the metabolically stabilized LPA analogue L‐sn‐ 1‐O‐oleoyl‐2‐O‐methylglyceryl‐3‐phosphothionate (2S‐OMPT) as a potent agonist for LPA receptors at a concentration (10 μM) sufficient to activate multiple LPA receptors. We hypothesized that if LPA signaling was not involved, then OMPT would override any contribution of RGS2 on modulating the viability response in the presence of chemotherapy. We generated an inducible stable model of RGS2‐expressing HeyA8‐MDR cells. After 48 h OMPT pre‐treatment, HeyA8‐MDR‐RGS2‐inducible cells were exposed to cisplatin for 24 h and evaluated for viability. All conditions pretreated with OMPT demonstrated enhanced viability even in the presence of chemotherapy; however, ovarian cancer cells that were pretreated with OMPT and cultured in tet‐free medium to induce RGS2 expression showed a significant blunting of viability. Research funded by the NCI, NIH, and GCC.
Epigenetic regulation of regulators of G‐protein signaling RGS10 and RGS17 expression in chemoresistant ovarian cancer cells
Authors: Ali Mourad Wagdy , Callihan Phillip , Altman Molly , Murph Mandi , Hooks Shelley
Published: June 2021

The development of chemoresistance represents a critical therapeutic challenge in epithelial ovarian carcinoma. Receptor‐stimulated survival pathways are amplified in chemoresistant cells. RGS proteins suppress GPCR stimulated signaling. We have recently reported RGS2, 10, and 17 are downregulated in models of acquired chemoresistance in ovarian cancer. Further, RGS transcript expression is decreased following short term exposure to chemotherapeutic drugs. This suggests a model in which RGS expression is reduced following exposure to chemotherapeutic drugs, thus indirectly amplifying GPCR mediated survival signaling. The current study investigates mechanisms for suppressed RGS transcript expression in chemoresistant ovarian cancer cells. Our analysis shows that RGS2, 10, and 17 gene promoters have high GC content, suggesting that DNA methylation could negatively regulate expression of these RGS transcripts. Treatment with 5‐azacytidine, an inhibitor of DNA methyltransferase increased expression of RGS2, 10 and 17. We measured DNA methylation across the promoter regions of these RGS genes, and compared frequency of methylation at specific CpG sites in normal and chemoresistant cell lines and following acute exposure to chemotherapy. This research has been funded by NIH, Elsa Pardee Foundation and Georgia Cancer Coalition.
Behavioral and metabolic changes in mice lacking Gβ5‐R7 complex
Authors: Wang Qiang , Levay Konstantin , Slepak Vladlen
Published: June 2021

Gβ5 is a unique G protein beta subunit that dimerizes with R7 family RGS proteins instead of Gγ. Gβ5:R7 association is essential for stability of their complex and so the knockout of Gβ5 causes degradation of the entire Gβ5‐R7 family. Here we analyze the phenotypes of Gβ5 knockout (KO) and heterozygotes (HET) animals. The KO mice are lean, resistant to high fat diet, hyperactive and have lower serum insulin. The HETs are also slightly more active than the WT, but surprisingly, display signs of metabolic syndrome: late‐onset obesity, insulin resistance and liver steatosis. Thus, even a partial reduction in Gβ5‐R7 level causes distinct phenotypes. The expression of Gβ5‐R7 complexes was thought to be restricted to the nervous system. To understand the mechanisms involved in the etiology of Gβ5 KO and HET phenotypes, we re‐examined expression of Gβ5 complex in peripheral tissues. We found that it is present in glands, including adrenal and pancreas, with subsequent analysis of endocrine cells with ablated Gβ5 provides novel insights into the role of this complex.

This study was supported by U.S. National Institutes of Health (NIH) grants GM 060019 (V.Z.S).
Factors regulating the subcellular localization of Activators of G‐protein Signaling 3: The role of serine/threonine residues in the G‐protein regulatory domain
Authors: Kelesoglu Fatih Mehmet , Oner Sukru Sadik , Ma Dzwokai , Lanier Stephen M
Published: June 2021

Activators of G‐protein Signaling (AGS) proteins contain one to four G‐protein regulatory motifs (GPR) that serve as docking sites for Gαi/o/t. The GPR‐Gα signaling module is regulated by guanine nucleotide exchange factors and provides unexpected signaling diversity for the “G‐switch”. AGS3, which contains seven tetratricopeptide repeats (TPR) and four GPR motifs, is involved in a wide range of biological functions including asymmetric cell division, autophagy and addictive behavior. Interaction of AGS3 with Gαi or FRMPD1 stabilizes AGS3 at the cell cortex, whereas disruption of the TPR motif stabilizes the protein in the aggresome pathway. We asked if AGS3 trafficking was regulated by phosphorylation. As a first approach to this issue, we determined the subcellular distribution of a mutant GFP‐tagged AGS3 (AGS3‐GFP‐PM1) in which 24 S/T residues in the GPR domain of the protein were changed to alanine to eliminate S/T phosphorylation in this region. GFP tagged AGS3 WT, PM1 and TPR mutants were expressed in HEK‐293 cells and the subcellular distribution determined by immunofluorescence microscopy. In contrast to wild type AGS3, both the PM1 and TPR mutants distributed to preaggresomal structures in the cytosol. These data suggest that phosphorylation of AGS3 may play a regulatory role in its trafficking within the cell. Dysregulation of the subcellular location of AGS3 may contribute to pathologies involving altered protein processing through the aggresomal and autophagic pathways.
The molecular mechanism of betacellulin induced corneal epithelial wound healing
Authors: Peterson Joanne L. , Phelps Eric D. , Ceresa Brian P.
Published: June 2021

The epidermal growth factor receptor (EGFR) has been shown to be necessary and sufficient for mediating corneal epithelial homeostasis and wound healing. Experimentally, activation of EGFR by epidermal growth factor (EGF), increases healing in a corneal wound; however, therapeutic use of EGF does not improve corneal healing in patients. This study looks at wound healing elicited by other EGFR ligands, specifically betacellulin (BTC). Using human telomerase‐immortalized corneal epithelial cells (hTCEpi) in an in vitro wound healing assay, BTC was shown to be more efficacious than EGF during wound closure. Since BTC can also stimulate ErbB4, an EGFR family member, the efficacy of BTC may be due to activation of this receptor population. However, mRNA and protein levels were assessed in both hTCEpi and primary human corneal epithelial cells and revealed the absence of ErbB4. Differences in ligand binding properties could also explain BTC efficacy. Using radiolabeled ligands to assess binding sites, ligand affinity, pH sensitivity and competition revealed differences between BTC and EGF in these areas. We conclude that the increased efficacy seen with BTC over EGF is not from stimulation of ErbB4 but is, at least in part, from differences in binding properties. Funding: NIH 1R01GM092874 and P20 RR 017703.
Differential effect of low and standard dose of conjugate equine estrogen treatment in mesenteric venular response to angiotensin II from ovariectomized spontaneously hypertensive rats
Authors: de Araujo Priscila Xavier , Ceravolo Graziela Scalianti , Costa Tiago Januário , DoSantos Rosangela Aparecida , Oliveira Maria Aparecida , Akamine Eliana Hirome , de Cássia Tostes Rita , Fortes Zuleica Bruno , Carvalho Maria Helena Catelli
Published: June 2021

Controversy still exists whether estrogen hormonal therapy confers cardiovascular benefit or harm. The potential harm caused by standard dosages of estrogen including coronary heart disease and venous thromboembolism may be mitigated by use of lower doses. The aim of this study was evaluate the venular effect of conjugate equine estrogen (CEE) treatment, in ovariectomized (OVX) SHR, at standard therapeutic (SD) vs low dose (LD). For that, isolated mesenteric venular bed was perfused with angiotensin II (Ang II) in a concentration‐ dependent manner. Venular hyper reactivity to Ang II (maximal response), augmented ROS generation, reduced SOD, catalase activity and NO bioavailability were found in OVX and SD vs. control rats in physiological estrous and LD. On the other hand, the reduced MVB response to Ang II in LD rats was related to increased endothelial NO synthase activity, reduced ROS generation and increased Ang II AT2 receptor expression. We suggest that CEE at a low dose has a protective effect in OVX mesenteric venular bed. The standard therapeutic dose might increase the risk for venular disease by inducing alterations in venular reactivity.
In vivo administration of LPS reduces dexmedetomidine‐induced contraction in isolated rat aortae
Authors: Manio Michael Magtoto , Man Ricky Y.K. , Vanhoutte Paul M , Ng Jacobus K.F.
Published: June 2021

The vascular effects of dexmedetomidine during sepsis are largely unknown. The present experiments were designed to determine ex vivo responses to dexmedetomidine in arteries of animals after short‐ or long‐term exposure to lipopolysaccharide (LPS).

Eight to ten weeks old male SD rats received different doses of LPS or normal saline in vivo two or 72 hours prior to sacrifice. Arterial blood pressure, heart rate and plasma cytokine levels were measured before isolating their aortae. Rings, with or without endothelium, were suspended in organ chambers, for isometric tension recording. In the absence or presence of pharmacological inhibitors [ L‐NAME, indomethacin, TRAM‐34, UCL1684 and iberiotoxin ].

LPS injections increased the plasma levels of TNF‐á and IL‐6 and reduced arterial blood pressure. They dose‐dependently attenuated dexmedetomidine‐induced contractions whether given two or 72 hours prior to sacrifice. However, contractions were increased in preparations without endothelium, and to a lesser extent in rings with endothelium after 72 hours. In the presence of TRAM ‐34, but not UCL1684, dexmedetomidine‐induced contractions were enhanced on rings with endothelium, while they were reduced by iberiotoxin.

The present data indicate that in vivo exposure to LPS differentially attenuates the contractile responses to dexmedetomidine, an action which may involve, BKCA .
Underlying mechanisms of tert‐butyl hydroperoxide induced vascular cell dysfunction and senescence in rats: in vivo and in vitro studies
Authors: Yeh Yueh-Chiao , Liu Tsun-Jui , Lo Jun-Chin , Wang Li-Chuan , Peng Chu-Ying , Wang Ren-Ching
Published: June 2021

Endothelial dysfunction remains one of the leading causes for the pathogenesis of cardiovascular diseases. tert‐butyl hydroperoxide (t‐BHP), a short‐chain lipid hydroperoxide analog, has been referred to cause cell injury in different systems. The study aimed to elucidate the effects of t‐BHP on oxidation‐induce apoptosis and cellular senescence and its possible mechanisms in rat aorta. Primary cultured endothelial cells were treated with vehicle or t‐BHP. Dose‐dependent manner of t‐BHP significantly reduced cell viability, augmented cell toxicity, increased DNA damage, initiated cell cycle arrest, provoked positive senescence‐associated beta‐galactosidase staining and apoptotic cells, as well as diminished the telomerase activity, precipitated the cell cycle and apoptosis sinaling regulatory proteins. Similarly, male rats at 6‐week of age were administrated with vehicle or t‐BHP and t‐BHP‐treated rats were characterized by luminal expansion and thickening of intima and media in thoracic aorta, induction of endothelium damage, and increase of aortic apoptosis and senescence‐positive staining. This in vivo and in vitro study showed t‐BHP impaired vascular cell survival at least partially through inhibition of cell cycle regulatory proteins as well as activation of intracellular signal of the apoptotic cascade and cellular senescence related signaling pathways.
Lipocalin‐2 mediates linoleic acid‐induced endothelial dysfunction
Authors: Song Erfei , Fan Pengcheng , Xu Aimin , Vanhoutte Paul M. , Wang Yu
Published: June 2021

Lipocalin‐2, a 25kDa glycoprotein secreted by adipocytes, mediates endothelial dysfunction induced by dietary obesity in mice. The mechanisms underlying the deteriorating effect of lipocalin‐2 on endothelial function are not fully understood. The present experiments were designed to explore the role of lipocalin‐ 2 in endothelial dysfunction caused by fatty acids. Wild type (WT) and lipocalin‐2 knock‐out (LKO) mice fed with standard chow were injected intraperitoneally with lipocalin‐2, alone or in combination with palmitic, oleic or linoleic acid. Carotid arteries were collected to measure endothelium‐dependent contractions (EDC) to increasing concentrations of acetylcholine (in the presence of L‐NAME). In WT mice, EDC were enhanced significantly by linoleic, but not palmitic or oleic acid, and/or lipocalin‐2. On the other hand, LKO mice were irresponsive to either linoleic acid or lipocalin‐2 given alone. However, combined administration of linoleic acid andlipocalin‐2 in LKO mice resulted in EDC of similar magnitude as in WT mice treated with the combination. Thus, lipocalin‐2 permits the deteriorating effect of linoleic acid on endothelial function. Supported by the Hong Kong Research Grant Council
Impact of substrate stiffness and hypoxia on endothelial spreading and contratility
Authors: Kuhr Frank K , Schraufnagle Dean E. , Christman John W , Yuan Jason X.-J. , Levitan Irena
Published: June 2021

Hypoxia is known to promote proliferation of endothelial cells (ECs) and facilitate angiogenesis to restore a functional vasculature. However, conditions such as pulmonary fibrosis and scleroderma that result in an increase in tissue stiffness may impair endothelial function and inhibit angiogenesis. Little, however, is known about the sensitivity of lung microvascular endothelial cells to substrate stiffness. Here we show that in contrast to aortic endothelial cells that spread more on stiff substrates, human microvascular endothelial cells from the lung (HMVEC‐L) spread more and proliferate better on softer versus stiffer substrates (0.5 mg/ml compared to 1.5mg/ml type I collagen) in three‐dimensional collagen gels. We also show that HMVEC‐L contractility, the ability of the cells to generate force, in both soft and stiff gels was significantly facilitated by hypoxic conditions (3% O2), as evidenced by gel‐contraction assays. The contractile phenotype is not due to an increase in proliferation as the cell number in both hypoxic and normoxic conditions were similar after 48 hrs. We propose that hypoxia‐induced increase in endothelial contractility contributes to the angiogenic response to hypoxic conditions but that it might be compromised by an increase in tissue stiffness. Supported by T32 HL 82547‐5, HL083298,
P‐Rex1 is critical for vascular hyper‐permeability and edema in the lungs
Authors: Naikawadi Ram Prasad , Cheng Ni , Vogel Stephen M , Wu Dianqing , Malik Asrar B , Ye Richard D
Published: June 2021

The primary objective of this study was to investigate a role for P‐Rex1 (PIP3 dependent Rac exchanger 1) a guanine nucleotide exchange factor for Rac, previously known for G protein‐coupled receptor signaling, in mediating TNF‐α‐induced vascular hyper‐permeability. Using gene deletion and knockdown approaches, we investigated the potential role of P‐Rex1 in TNF‐α‐induced lung vascular injury. P‐Rex1, previously found in neutrophils and neurons, is also expressed in endothelial cells. In cultured human lung microvascular endothelial cells (HLMVECs), small interference (si) RNA‐mediated knockdown of P‐Rex1 prevented TNF‐α‐induced intercellular gap formation, Rac activation and reactive oxygen species (ROS) production. TNF‐α stimulated P‐Rex1 membrane translocation and the resulting Rac activation in a PI3K dependent manner. P‐Rex1 knockout mice were also refractory to TNF‐α‐induced lung vascular hyper‐permeability and edema. Both, in vivo and in vitro, absence of P‐Rex1 resulted in significantly less ICAM‐1 induction and neutrophil transendothelial migration. These results demonstrate for the first time that P‐Rex1 is activated downstream of TNF‐α, which is not a GPCR. We conclude that P‐Rex1 is a novel mediator of endothelial barrier disruption and therefore could be a potential therapeutic target in the control of vascular permeability and neutrophil infiltration to inflammatory tissues.
Enhanced 5‐HT‐induced contraction of the rat pulmonary artery in hyperthyroidism involved activation of calcium‐activated chloride channels
Authors: Oriowo Mabayoje , OOmmen Elsie , Khan Islam
Published: June 2021

Experimentally‐induced hyperthyroidism in rodents is associated with pulmonary hypertension. The main objective of this study was to investigate reactivity of the pulmonary artery to 5‐HT and the possible underlying signaling pathway in artery segments from thyroxine‐treated rats. 5‐HT concentration‐dependently contracted artery segments from control and thyroxine‐treated rats. The maximum response was significantly greater in artery segments from thyroxine‐treated rats but there was no change in pD2 values (5.04±0.19 and 5.34±0.14, in control and thyroxine treated rats, respectively). BW 723C86, a 5‐HT2B‐receptor agonist and CP 93129, a selective 5‐HT1B‐receptor agonist had no effect on pulmonary artery segments from both groups. There was no significant difference in the level of expression of 5‐HT2A‐receptor protein between the two groups. Ketanserin antagonized 5‐HT‐induced contractions in both groups with equal potency. Nifedipine (10−6M) inhibited 5‐HT‐induced contractions in artery segments from control and thyroxine‐treated rats and was more effective against 5‐HT‐induced contraction in artery segments for thyroxine‐treated rats. The calcium‐activated chloride channel blocker, niflumic acid (10−4M) also inhibited 5‐HT‐induced contractions in artery segments from control and thyroxine‐treated rats and was more effective against 5‐HT‐induced contraction in artery segments for thyroxine‐treated rats. It was concluded that hyperthyroidism enhanced 5‐HT‐induced contractions of the rat pulmonary artery via activation of calcium‐activated chloride channels.
Insulinotropic Effects of Novel I1‐Imidazoline Agonist S43126
Authors: Edwards Lincoln Paul , Tesfai Jerusalem , Crane Louis , Baziard-Mouysset Genevieve
Published: June 2021

The I1‐imidazoline receptor is a novel drug target for hypertension and insulin resistance associated with Type II diabetes. We hypothesize that the novel I1‐imidazoline agonist S43126, act centrally to lower blood pressure and enhance glucose uptake by causing insulin release and phosphorylation of protein kinase B (PKB). Blood pressure measurements were made following cumulative injection of S43126 (5 nmol) and moxonidine (5nmol) into the brain of SHR. ELISA was used to measure insulin release from Min 6 cells under conditions of high (16.8mM), or low (2.8mM) glucose, following treatment with varying doses of S43126 [10‐8M to 10‐5M], for various times (5–60mins), in the presence or absence of inhibitors. Our results showed that both S43126 and moxonidine lowered arterial pressure by 20 mmHg and 45 mmHg respectively. S43126 [10‐5M] stimulated insulin secretion under elevated glucose concentration compared to basal. In addition, insulin secretion and Ca2+ influx mediated by S43126 [10‐5M] were decreased by efaroxan and nifedipine. Furthermore, S43126 [10‐5M] increased [Ca2+]i and 45Ca2+ uptake in a time and dose‐dependent manner. S43126 also caused increased protein expression of Imidazoline Receptor Antisera‐Selected (IRAS) protein, as well as increased phosphorylation of both ERK1/2 and PKB, in a dose‐dependent manner. These data indicate that S43126 may be useful in treating metabolic diseases.
Decynium‐22 enhances SSRI‐induced antidepressant effects in mice: Uncovering new targets to treat depression
Authors: Apple Deana M , Horton Rebecca E , Baganz Nicole L , Cano Sonia , Owens W Anthony , Vitela Melissa , Gould Georgianna G , Koek Wouter , Daws Lynette C
Published: June 2021

A major problem in the treatment of depression is that many patients do not experience therapeutic benefit. The most commonly prescribed antidepressant drugs are the selective serotonin (5‐HT) reuptake inhibitors (SSRIs), which act by blocking the high‐affinity 5‐HT transporter (SERT). The increase in extracellular 5‐HT produced by SSRIs is thought to be critical to initiate downstream events needed for therapeutic effects. A potential explanation for their limited therapeutic efficacy is the recently characterized presence of low‐affinity, high‐capacity transporters for 5‐HT in brain, (e.g. organic cation transporters [OCTs]), which may limit the ability of SSRIs to increase extracellular 5‐HT. Decynium‐22 (D‐22) is an OCT blocker, and using this compound we uncovered a significant role for OCTs in 5‐HT uptake in SERT knockout mice, raising the possibility that pharmacological inactivation of OCTs might enhance the neurochemical and behavioral effects of SSRIs. Here we show that [3H]D‐22 binding sites are richly expressed in mouse hippocampus, and that D‐22 enhances the effects of the SSRI fluvoxamine to inhibit 5‐HT clearance and to produce antidepressant‐like activity. Our findings provide a mechanistic basis for poor therapeutic outcome following treatment with SSRIs, and point to D‐22‐sensitive transporters as novel targets for new antidepressant drugs with improved therapeutic potential. Supported by National Institutes of Health Grants R01 MH064489, R03 MH086708, Morrison Trust, and a NARSAD Independent Investigator Award.
Pyrazoloquinazolinecarboxilate analogues inhibit nerve growth factor in vitro
Authors: Eibl Joseph Karl , Strasser Bridget , Ross Gregory M
Published: June 2021

NGF is known to regulate the development and survival of select populations of neurons via its binding/activation of the TrkA and p75NTR receptors. However, NGF dysregulation can result in debilitating pathologies. Thus, the identification of small molecules which inhibit NGF signaling have significant therapeutic potential. PD 90780, Ro 08‐2750, and ALE 0540 are small molecules that have been reported to bind and inhibit NGF activity. Importantly, the docking site of these compounds is hypothesized to occur at the loop I/IV cleft of NGF ‐ a region which is required for efficient and selective binding of a neurotrophin to its receptor(s). Molecular modeling predicts these molecules share conserved molecular features and have the ability to bind and modify the molecular topology of NGF. In order to understand the binding mechanism, we synthesized a pyrazoloquinazolinecarboxilate (PQC) analogue series and tested each compound in an NGF‐dependent PC12 cell differentiation assay. In vitro data confirms that the PQC analogues functionally inhibit NGF's agonist effects on PC12 cell differentiation. The results of this study provide evidence to refine the docking mode of PQC‐like compounds for the purposes of inhibiting NGF in vitro. In addition, we identified series analogue PQC 083 which is markedly better than previously described NGF antagonists.
A Novel Apparatus for Conducting Passive Avoidance Procedures in the Zebrafish
Authors: Herrera Gerald M. , Patel Ami U. , Ashline Jake , Simpson Robin , Weber Daniel N.
Published: June 2021

We developed a novel apparatus for studying passive avoidance in the zebrafish as a model of learning & memory and aversive conditioning. It consists of a tank in which the fish can swim back and forth. Photobeams detect the fish on either side of the tank. One side of the tank is shaded while the other side is lit. A mild shock is delivered to the shaded compartment when a fish enters that zone. The system tracks zebrafish as small as 4 mm wide. In our passive avoidance procedure, fish habituated to the tank are trained by being placed into the lit compartment, and the delay in crossing to the shaded side is recorded. Once a fish swims to the shaded side, it receives a mild shock. This process is repeated up to 10 trials, or until a fish fails to cross after 180 sec. The following day, testing occurs by measuring the delay to cross from the lit to the shaded side. During training, delay to cross was 35 ± 10 sec during trials 1–5. During trials 6–10, delay to cross increased to 111 ± 18 sec (n=4), indicating acquisition of the avoidance response. On average, training sessions ended after 8 ± 1 trials. Testing sessions the following day ended sooner than the training sessions, as the fish failed to cross after an average of 2 ± 0 trials. The average delay to cross during testing sessions was 100 ± 31 sec. In conclusion, this apparatus can be used to examine both acquisition and recall of memories formed during aversive conditioning in the zebrafish.
Spatial memory impairment in mice with immunotoxic lesions of the cholinergic basal forebrain
Authors: Wrenn Craige Charles , Schaapveld Caitlin Marie , Bennett Nick , Bansal Sonia , Willoughby Christy , Peterson Joel , Krogh Kelly , Wolfe Brady , Hidding Jennifer
Published: June 2021

Degeneration of the cholinergic basal forebrain (CBF) is a feature of Alzheimer's disease (AD) that correlates with dementia severity. This correlation has motivated investigators to develop animal models of AD by lesioning the CBF. We tested the hypothesis that mu‐p75‐saporin creates cholinergic lesions in mice and that these lesions result in deficits in spatial cognition. C57BL/6J mice received intraventricular injection of either a high (4 μg) or low (2 μg) dose of mu‐p75‐saporin and were tested in the Morris water maze. Mice receiving 4 μg showed increased latency to find the hidden platform during training. This increased latency was confounded by motor deficits because impairments in swim speed were observed. In a probe trial, mice receiving 4 μg swam a greater average distance from the platform location than saline‐treated mice. Mice receiving 2 μg showed increased latency to find the platform with no confounding effect on swim speed. In a probe trial, mice receiving 2 μg swam with similar proximity to the platform location as saline‐treated mice. Staining of the brains for acetylcholinesterase, p75, and parvalbumin showed that the treated animals had specific lesions of the CBF. In summary, features of Alzheimer's disease can be modeled in mice using mu‐p75‐ saporin, but higher doses produce confounds in motor ability.
Lactobacillus rhamnosus GG supplementation attenuates chronic‐alcohol induced liver injury by inhibiting hepatocyte apoptosis
Authors: Wang Chunhong , Wang Yuhua , Liu Yanlong , McClain Craig J , Feng Wenke
Published: June 2021

Previous studies have demonstrated that chronic alcohol consumption induces liver injury, which is associated with ethanol metabolism‐mediated oxidative stress and apoptotic cell death, in human and experimental animals. As probiotic supplementation is known to reduce the formation of reactive oxygen species (ROS) and reactive nitrogen species (RNS) in the intestine and the liver in the mice chronically fed alcohol, the present study was aimed to investigate whether probiotic supplementation could inhibit alcohol‐induced hepatic apoptosis in mice. C57BJ/6N mice were fed the Lieber deCarli diet containing 5% alcohol with or without Lactobacillus rhamnosus GG (LGG) for 4 weeks. LGG supplementation completely inhibited the chronic alcohol consumption‐increased hepatic apoptosis measured by TUNEL assay along with ROS and RNS production. The anti‐apoptotic effect of LGG on the liver of the alcohol‐treated mice was further confirmed by measuring Caspase 3 activation, and the protein levels of anti‐/pro‐apoptotic proteins, such as Bcl‐2 and Bax, and death signal proteins, such as FasR and FasL. These results indicate that LGG treatment reduces alcohol‐induced hepatic apoptotic cell death by multiple mechanisms, and provides a mechanistic insight for utilization of probiotics for the treatment of ALD.
Matrix metalloproteinase (MMP)‐9 and VEGF gene interaction models and MMP‐9 plasma levels in preeclampsia and gestational hypertension
Authors: Luizon Marcelo Rizzatti , Sandrim Valeria C. , Palei Ana Carolina T. , Lacchini Riccardo , Cavalli Ricardo C. , Duarte Geraldo , Tanus-Santos Jose Eduardo
Published: June 2021

Preeclampsia (PE) is a major contributor to maternal mortality worldwide but its etiology remains unclear. Although epistasis is suggested to be an important contributor to complex diseases, only a few studies have addressed this issue in PE. We detect interaction models predictive of PE (n=122) and gestational hypertension (GH, n=107) between polymorphisms in the promoter region of MMP‐9 (C‐1562T) and VEGF (G‐634C) genes using robust multifactor dimensionality reduction (RMDR) method. This best model of interaction included the ‐1562CT genotype and the ‐ 1562T allele was previously associated with increased MMP‐9 expression. However, previous findings showed no significant differences in plasma MMP‐9 levels in women with PE when single genotypes or MMP‐9 haplotypes were considered. Therefore, we investigate if gene‐gene interactions were associated with MMP‐9 levels in PE and GH. We observed only a marginally association finding (P=0.055) in GH. Indeed, the origin of plasma MMP‐9 levels is unknown, and it is possible that fetal MMP‐9 genotypes, tissue inhibitors of MMPs (TIMPs) and environmental factors may also contribute to the plasma MMP‐9 levels. We characterized interactions among genes related to angiogenesis and endothelial dysfunction seen in PE. Moreover, our study also provides further insights in the pathophysiology of PE and GH. Research support: CNPq, FAPESP and CAPES
A study of the prevalence of Orthostatic Hypotension (OH) and risk of falls among older patients presenting to the Community Health and Psychiatry Health Center (CHPHC
Authors: Smith Latoya Tameka , Gossell-Williams Maxine
Published: June 2021

Orthostatic Hypotension (OH) is the reduction of systolic/diastolic blood pressure of at least 20/10mm/Hg respectively. The objective was to evaluate the prevalence of OH and risk of falls among older patients on antihypertensive or antidepressant medication. A cross‐sectional study was conducted with patients ≥60 years. Patients were excluded if they were unable to stand or have Parkinson's disease. Sitting and standing blood pressure was measured at 3 minutes intervals. The Time Up & Go (TUG) test and the Tinetti Performance‐Oriented Mobility Assessment (POMA) Tool were used to assess the risk of falls. Seventy cases were identified with 8.6% being males and 91.4% being females. Twenty percent were OH positive, with these patients being significantly older (mean age ± sd: 80.14 ± 6.61 years versus 71.84 ± 8.65 years; p = 0.002). OH positive patients had a significantly higher TUG scores (± sd: 24.14 ± 11.46 seconds versus 16.70 ± 3.61 seconds; p = 0.006) and a significantly lower POMA scores (± sd: 19.50 ± 6.25 versus 24.20 ± 12.81; p = 0.004). POMA and TUG scores had a significant strong negative correlation (Pearson’ r = −0.709; p = 0.000). Age with TUG and POMA scores had a significantly low correlation (Pearson's r = 0.398; p = 0.001 and −0.401; p = 0.001 respectively).Findings suggest that 2 in every 10 elderly patients are likely to experience OH; they are older and are at a greater risk for falls. Funding: UWI Graduate Studies
Functional Characterization of Cytochromes P450 2B from Woodrats
Authors: Wilderman P Ross , Jang Hyun-Hee , Angermeier Elizabeth , Malenke Jael R. , Dearing M. Denise , Halpert James R.
Published: June 2021

Much of the structure‐function information on cytochromes P450 (P450) 2B is from a limited number of pharmacologically relevant species. Recent data suggest that desert woodrats (Neotoma lepida) have multiple P450 2B enzymes with greater than 85% identity to P450 2B1. Populations of N. lepida consuming creosote (Larrea tridentata) or juniper (Juniperus osteosperma) have differential expression of P450s 2B. Following previous protocols, we modified, expressed, and purified one P450 2B from one woodrat fed a creosote diet and one fed a juniper diet. (−)‐a‐pinene, β‐myrcene, and d‐limonene, which are common in juniper and previously reported to be inhibitors of P450 2B1, show very high affinity (<50 nM) for both woodrat enzymes. The woodrat enzymes show similar activity to P450 2B1 with 7‐ethoxy‐4‐ trifluoromethyl‐coumarin (7‐EFC), but significant differences in activity and product profile are seen in ticlopidine metabolism. Residue 262 shows a lysine – arginine difference between the two enzymes. This residue has demonstrated functional differences in the human P450 2B6, and the known human genetic polymorphism K262R produces significant changes in ligand interactions. Differences in P450 2B structure in woodrats may confer functional differences in the metabolism of dietary toxins. (Supported by ES003619 and T32‐DK07233).
Cytochrome P450 1A2 forms catalytically relevant homomeric complexes
Authors: Connick John Patrick , Reed James R. , Cheng Dongmei , Cawley George F. , Backes Wayne L.
Published: June 2021

Our laboratory and others have shown that one P450 enzyme can affect the catalytic function of other P450s. The goal of this study was to determine if CYP1A2 forms functionally relevant homomeric complexes. The catalytic activity of CYP1A2 was measured in reconstituted systems containing CYP1A2 and varying levels of NADPH‐cytochrome P450 reductase (CPR). Interestingly, a sigmoidal response was observed with respect to [CPR] instead of the hyperbolic curve expected for simple Michaelis‐Menten kinetics – the sigmoidicity increased with increasing [CYP1A2]. A physical interaction between CYP1A2 molecules was verified using bioluminescence resonance energy transfer (BRET). Varying ratios of Renilla luciferase‐ and GFP2‐tagged CYP1A2 were cotransfected into HEK293T cells and energy transfer from luciferase to GFP2 was measured. Cotransfection of the tagged CYP1A2 proteins with increasing amounts of unlabeled CYP1A2 decreased the BRET response, indicating a specific physical interaction. Taken together, these results show that CYP1A2 forms CYP1A2•CYP1A2 complexes that exhibit altered catalytic activity.
Application of Kinetic Isotope Effects and Molecular Modeling In Understanding the Mechanistic Complexities of CYP4F12
Authors: Foti Robert S
Published: June 2021

To compare the relative promiscuities of CYP isoforms in regard to inhibition and catalysis, our group has demonstrated a potentially unique ligand profile for CYP4F12. The objective of the work presented here is to explain the inhibitory and catalytic characteristics of CYP4F12 using kinetic isotope effects and molecular modeling along with various other in vitro and in silico tools. Calculation of promiscuity scores for CYP4F12 by a panel of 60 compounds demonstrated that while CYP4F12 displays a susceptibility towards inhibition that is in line with the majority of CYP isoforms, it has a decreased ability to metabolize the same compounds. Furthermore, experiments carried out with d3‐astemizole revealed a significant isotope effect on the O‐demethylation of astemizole by CYP4F12 resulting in a decreased clearance, whereas the same isotope effect was not observed for CYP3A4. A homology model of CYP4F12 was constructed and used to compare the active site characteristics of CYP4F12 and CYP3A4. Preliminary results indicate that while the volume of the CYP4F12 active site is such that it can accommodate many of the same ligands as CYP3A4, the architecture of the active site results in a more constrained ability of the ligands to achieve catalytically favorable orientations.
RNA‐Seq identifies novel alternative transcripts of cytochrome P450s in human hepatocytes
Authors: Li Dan , Peng Lai , Lee In-Hee , Li Jennifer , Visvanathan Mahesh , Zhong Xiao-bo
Published: June 2021

Most human genes can produce alternative transcripts by transcriptional regulation or RNA processing, which in many cases encode functionally distinct proteins. The cytochrome P450 enzymes (P450s) catalyze the oxidation of lipids, steroid hormones, drugs, and toxic chemicals. The purpose of this study was to identify novel transcripts of P450 genes and characterize their expression and induction in human hepatocytes. RNA sequencing (RNA‐Seq) was used to identify novel transcripts of P450s in human hepatocytes or HepaRG cells after treatment with rifampicin or phenobarbital. In 30 P450 genes with the expression levels >10 (FPKM: fragments per kilobase of exon per million fragments mapped) in human hepatocytes or HepaRG cells, RNA‐Seq identified 67 alternative transcription events, including exon skipping, intron retention, alternative splicing, and polyadenylation. Some events were reported in literature, but most were novel. Several novel events were confirmed by PCR or rapid amplification of cDNA ends plus DNA sequencing. Furthermore, differential expression of alternative transcripts was observed in some P450 genes after drug treatment.

Conclusion

RNA‐Seq identified novel transcripts for P450 genes. Alternative transcription variants of P450 genes had differential expression during drug treatments, which may serve as a potential mechanism for the differential enzyme activity in human liver.
Trials and tribulations ‐ FDA regulatory support for academic sponsor‐investigators
Authors: Seymour E Mitchell , Weatherwax Kevin
Published: June 2021

Clinical research conducted at academic medical centers significantly contributes to discovery and development of drugs and devices and to the repurposing of FDA‐approved products. Many such studies require FDA oversight through the Investigational New Drug (IND) or Investigational Device Exemption (IDE) designation. Investigator‐initiated research requires the investigator to serve as a sponsor‐investigator and the “holder” of an IND/IDE. This role involves complex submissions to and interactions with the FDA, but investigators may be poorly prepared or resourced to meet these additional obligations. An increasing number of academic medical centers offer regulatory support for sponsor‐investigators. Presently, we describe one high‐volume regulatory support program at a large medical center. Several critical challenges occur in the initial steps of investigator collaboration – gap analysis, protocol refinement, and industry interaction. Select case studies reveal both specific challenges and successful approaches. FDA regulatory compliance is challenging for under‐resourced sponsor‐investigators. Given the complexity of FDA regulatory requirements, early support for sponsor‐investigators can accelerate study execution and completion and is a growth asset to the academic research enterprise.
Epigenetic Alterations caused by Decreased Cellular Methylation Potential Induce Fas‐FasL‐mediated Apoptotic Death in Leukemic T Lymphocytes
Authors: Patil Madhuvanti , Ghare Smita , Gobejishvili Leila , Joshi-Barve Swati , McClain Craig , Barve Shirish
Published: June 2021

The transmethylation (TM) pathway is up regulated in proliferating T cells and transformed T leukemic cells but not in resting T cells, making it an ideal target to eliminate leukemic T cells. The TM pathway involves methyltransferase‐mediated conversion of S‐adenosylmethionine (SAM) to S‐adenosylhomocysteine (SAH). SAH is a potent feedback inhibitor of methyltransferases and is hydrolyzed by S‐adenosylhomocysteine hydrolase (SAHH), which determines the SAM: SAH ratio, known as the cellular methylation potential.

The role of cellular methylation potential in the regulation of cell death in T leukemic cell lines was examined by inhibiting SAHH using pharmacologic SAHH inhibitors and RNAi. We demonstrate that a decrease in cellular methylation potential induces apoptosis in leukemic T lymphocytes. SAHH inhibitor treatment increases expression of FasL protein and activates the Fas‐FasL signaling pathway. Additionally, we observed an up regulation of FasL mRNA expression in leukemic T cells. ChIP analysis of histone H3 modifications at the FasL promoter showed increased transcriptional activation marks, namely acetylation at lysine 9 and 14, trimethylation at lysine 4 and increased recruitment of RNA Polymerase II. Overall, the data indicate the mechanistic role of cellular methylation potential in transcriptional regulation of FasL gene resulting in enhanced leukemic T cell death.
Targeting nuclear receptor‐mediated cell growth and death in pediatric solid malignancies
Authors: Beard Jordan A , Chen Taosheng
Published: June 2021

Nur77, an orphan nuclear receptor, has been shown to be overexpressed in adult cancers when compared to normal cells, indicating a role in proliferation. Nur77 is also implicated in apoptosis. Nuclear‐accumulated Nur77 promotes cell survival, whereas cytoplasmic accumulation promotes cell death. We have found that Nur77 gene and protein are expressed at high levels in pediatric rhabdomyosarcoma cells when compared to human myoblasts, indicating a role in the malignant phenotype of these cells. Pediatric tumor models with higher Nur77 expression were more susceptible to cell death when treated with CD437, indicating potential selectivity to cancer cells. RNAi knockdown of Nur77 decreased the ability of CD437 to activate caspase‐3/7, indicating a Nur77‐dependent mechanism. Knockdown of Nur77 reduced the growth of osteosarcoma cells demonstrating a role in proliferation. Immunohistochemical analyses of tumor tissues show that Nur77 is expressed in clinical cases when compared to normal tissue, implicating Nur77 in the pathogenesis of these pediatric malignancies. These data suggest that the Nur77 pathway represents an ideal target for pediatric cancer therapy due to the differential expression in normal and cancer cells. High‐throughput and high‐content screening assays are currently being developed to identify compounds that induce Nur77‐mediated cell death in pediatric cancer models.
The Human Host Defense Peptide LL‐37 Induces Caspaseindependent Apoptosis Via Apoptosis‐Inducing Factor And Endonuclease G in Colon Cancer Cells
Authors: Ren Shunxiang , Cho Chi Hin
Published: June 2021

LL‐37, a human host defense peptide, plays pivotal roles in diverse biological processes, including natural immunity, inflammation and tissue repair. However, the function of this peptide in tumorigenesis remains unclear. In this study, the effect of LL‐37 on human colon cancer HCT116 cells was characterized. LL‐37 induced extensive DNA fragmentation, chromatin condensation and phosphatidylserine externalization without causing caspase activation. Moreover, these effects were not blocked by caspase inhibitors. LL‐37 induced apoptosis via downregulation of Bcl‐2 and upregulation of Bak and Bax in a p53‐dependent manner. In this connection, the pro‐apoptotic effect of LL‐37 was reversed by Bcl‐2 overexpression, genetic ablation of Bax, or p53 siRNA. LL‐ 37 also induced the upregulation and nuclear translocation of apoptosis‐inducing factor (AIF) and endonuclease G (EndoG). Knocking down of these genes by siRNAs rendered the cells resistant to LL‐37‐induced apoptosis. Above all, the pro‐apoptotic effect of LL‐37 was found to be mediated through a pertussis toxin‐sensitive Gi‐coupled receptor. Taken together, we demonstrated that LL‐37 induced caspase‐independent apoptosis via the p53‐Bcl‐2/Bak/Bax and AIF/EndoG pathways. These findings open up a novel therapeutic avenue using LL‐37 for the treatment of colon cancer.

Grant support: Research Fund for the Control of Infectious Diseases, Food and Health Bureau, Hong Kong, China.
Influence of Zedoary Oil on Cell Cycle and Cathepsin K Expression in A549 Cell Line in vitro
Authors: Yang Changfu , Niu Jianzhao , Li yadong , Wang Xiaobo , Huang Chunfang , Tang Binghua , Zhao Peiwen , Wang Jifeng
Published: June 2021

Objective

To explore the inhibiting effects of Zedoary oil on the proliferation and cathepsin K expression in A549 cell line in culture.

Method

The proliferation of lung adenocarcinoma cell line A549 were determined by MTT assay. The cells cyclic retardation were examined by using flowcytometry. Cathepsin K expression of level were detected by using western blot.

Result

MTT showed that Zedoary oil had an inhibitory effect on the proliferation of A549 cells in 60~200 mg •L‐1. Flow cytometry indicated that Zedoary oil increased the percentage of G0/G1 phase cells and reduced the percentage of S phase cells. The results of western blot showed that the expression of cathepsin K was up‐regulated significangly in A549 cells in Zedoary oil groups compared with that in controls.

Conclusion

Zedoary oil can significantly inhibit the growth of A549 cells, blocking the cell cycle. The effects may due to the up‐regulation of cathepsin K. This work was supported by founds:2008DFA31970 and the 111 project B07007 of China. Dr. Niu Jianzhao is the responsibility author.
Role of SIRT1 in Resistance Exercise‐Induced Changes in Vascular Function
Authors: Servidio Lia R , Roberts Sally A. , Laycock Mary L. , Harris M. Brennan
Published: June 2021

SIRT1 has been identified as a regulator of eNOS and endothelial function and recently we have demonstrated that it plays a role in vascular adaptations to endurance exercise training. In this study, we investigated the effects SIRT1 inhibition on vascular reactivity in rat aortas following resistance exercise training programs of different durations. Male, Fisher 344 rats (3 mo old) were divided into three groups (n=10–12/group); sedentary (SED), 2‐day exercise (2DY), and 2‐weeks exercise (2WK). Resistance training consisted of climbing a 1 m wire ladder, at an 85° angle, 3d/wk for 2 days or 2 weeks with increasing weight added to the tail. Vascular reactivity was examined by constructing dose response curves to phenylephrine (PE, 10−8–10−5 M), sodium nitroprusside (SNP, 10−9–10−6 M), and acetylcholine (ACh, 10−9–10−6 M) in the presence or absence of salermide (30 μM). Salermide significantly (P<0.05) increased PE‐induced vasoconstriction and impaired ACh‐induced vasorelaxation in all groups. SNP‐induced vasorelaxation was not affected by salermide. In addition, 2 days of exercise resulted in significantly (P<0.05) greater PE‐induced vascocontriction and ACh‐induced vasorelaxation but had no affect on SNP‐induced relaxation. In conclusion, these data suggest that SIRT1 plays an important role in endothelium dependent vasodilation and that 2 days of resistance exercise training augments endothelial dependent vascular reactivity. Supported by the NIH R15 HL108428‐01A1 (MBH).