bluuebunny/crossref_metadata_2025 huggingface dataset:
An interactive, case study computer module for teaching medical genetics using genomic sequence analysis tools.
Authors: Munzenmaier Diane H. , Worthey Elizabeth A. , Jacob Howard J.
Published: June 2021

Aim

The sequencing of the human genome has issued the promise of individualized medicine for the near future. Technology has accelerated to a point where it is clear that genomic sequence will be a basic component of medical care before today's medical students complete their residencies. This interactive computer laboratory has been designed to allow the student to be involved in practical and ethical decision making based on symptom presentation, family history, and genetic test results and to use genomic sequence tools to aid them in their diagnoses.

Methods

Cases are discussed in small groups as students use genetic test results and genome sequence as well as online tools and resources to diagnose, determine carrier status, and make decisions regarding treatment and testing of family members. Prenatal diagnosis, preimplantation genetic diagnosis, prophylactic screening, and ethics are also addressed. Genetic disorders represented in these case studies cover a wide range of types of genetic variation and presentation and increase in complexity through the module.

Conclusion

This teaching module provides a link between genetics lectures and the clinic. Taught during the medical physiology course in the second semester of the first year, it attempts to integrate recently acquired knowledge in anatomy, biochemistry, neuroscience, physiology, and genetics with clinical genetics testing and diagnosis.
Mitochondrial DNA damage in vascular endothelial cells exposed to shear stress
Authors: Galbraith Valerie K , Giedt Randy J , Panduri Vijji , Van Houten Bennett , Alevriadou B Rita
Published: June 2021

Superoxide radicals (O2=), hydrogen peroxide (H2O2) and other reactive oxygen species (ROS) produced by endothelial cells (ECs) play an important role in cell (dys)function. We recently showed that, during cultured EC exposure to shear stress, nitric oxide (NO), produced by nitric oxide synthase, diffuses to the mitochondria and reacts with O2−, produced by the electron transport chain (ETC), resulting in peroxynitrite (ONOO−) formation and inhibition of the ETC complex activities. EC incubation with H2O2 or ONOO− is known to induce mitochondrial DNA (mtDNA) damage. Hence, we hypothesized that the endogenous shear‐induced ROS may cause mtDNA damage. DNA damage was assessed by a quantitative PCR assay that relies on the principle that oxidative DNA lesions inhibit DNA polymerases. DNA damage was quantified by comparing the relative efficiency of amplification of large DNA fragments (12.2 kb for DNA polymerase β gene; 8.9 kb for mtDNA) and normalizing it to the amplification of smaller fragments. EC exposure to shear stress (10 dynes/cm2) for 1 h caused a 2‐fold increase in mtDNA damage (1.1±0.4 lesions/10 kb) compared to nuclear. At 6 h, there was a complete loss of nuclear DNA damage, but mtDNA damage persisted. Shear followed by incubation with H2O2 had an additive effect on mtDNA damage. Our goal is to examine the time/dose dependency of mtDNA damage on shear stress, and reveal how the DNA damage affects cell function. Funding by NIH HL91417.
A fractal analysis of human fetal pituitary blood vessels
Authors: Chirculescu Andy Remus Marius , Staicu Cristian , Coculescu Mihai Gr , Chirculescu Mihaela , Morris John F.
Published: June 2021

Fractal analysis allows a non‐Euclidian evaluation of the tissue‐blood vessels ratio and distribution. The fractal dimension varies from 1 to 2. Di Ieva et al have shown that fractal dimension is a good quantitator for microvasculature of normal (2007) and adenomatous (2008) pituitaries.

We present here a fractal analysis study of pituitary vasculature in human fetuses of 9‐24 weeks obtained from terminations with ethical approval, performed on digital images taken from toluidine blue‐stained semithin sections and fluorescence microscopy after lectin binding or propidium iodide.

Both light microscopy after toluidine blue and fluorescence microscopy are suitable for fractal analysis; fluorescence in red gave the best results. The fractal indices varied from 1.41 to 1.86, progressively decreasing from 9 to 24 weeks; the distribution of individual values varied following a logarithmic curve, thus demonstrating that in each case, there is a limit, over which the vessels fractal dimension does not increase; the ratio cells/blood vessels varies from 1.15 (9‐10 weeks) to 0.91 (18 to 24 weeks).

These data show that fractal analysis is suitable for the study of dynamic changes in blood vessels in the human fetal pituitary, during ontogenesis, giving a quantitative image of its progressive development and time‐related evolution, complementary to the data derived from morphometric vascular density measurements.
Regulation of Embryonic Cardiac Wall Growth and Vascularization by FGF‐2
Authors: Nanka Ondrej , Petrovova Elena , Fikrle Michal , deAlmeida Angela , Sedmera David
Published: June 2021

The aim of this study was to assess the effect of FGF‐2 signaling on growth of chick embryonic ventricular wall and its vascularization. In the first set of experiments, left ventricular wall of embryonic day 7 (E7) chick embryos was injected with either GFP‐expressing replication‐deficient adenovirus alone or in combination with adenoviral vector encoding human FGF‐2 gene. In the second set of experiments, FGF signaling was blocked in E8 embryos using FGFR1 receptor tyrosine kinase inhibitor SU 5402. Cell proliferation was measured in both cases using BrdU incorporation and immunohistochemistry on E9.

Morphological examination of adenovirus‐injected hearts revealed no difference in normal myocardial architecture, but increased levels of myocyte proliferation in hearts injected with FGF‐2 adenovirus. There was no difference in capillary density or coronary artery anomalies. Blocking of FGF signaling with SU 5402 led to hemorrhages in the areas of developing vasculature in the tips of limbs and epicardium. However, the rate of myocyte proliferation was unchanged.

We conclude that up‐regulation of FGF‐2 signaling has a positive effect on growth of the early fetal heart, while its inhibition impacts mainly vasculogenesis, pointing to functional redundancy in paracrine control of myocyte proliferation in the developing heart.

Supported by MSMT VZ 0021620806, LC 06061, AS CR AVOZ50450515.

Grant Funding Source

MSMT VZ 0021620806
Antitumoral effect of melatonin by G‐protein mediated toll‐like receptor (TLR) activation through autophagic cell death in gynecologic cancer cells
Authors: Hong Yonggeun , Won Jinyoung , Lee Youngjeon
Published: June 2021

Toll‐like receptors(TLRs) induce innate immune responses that are essential for host defenses against invading microbial pathogens. Toll‐like receptor 4(TLR‐4) mediates many biological effects of lipopolysaccharide(LPS), which has antitumoral effects on tumor cells. However, the cellular mechanism of these antitumoral effects remains unknown. Here, we speculated that stimulation of TLR‐4 signaling by G‐protein activation plays important roles, which gives signal to stimulate of autophagic cell death in genecologic tumor, not to lead the activation of NF‐kB and inflammatory pathway. The role of TLR‐4 in the antitumoral effect of LPS was assessed in primary cultured cervical, endometrial and angioma cells. Cancer cells were transiently transfected with small G‐protein encoding plasmids, and then treated with LPS. Immunofluorescence microscopy, western blotting, and RT‐PCR were performed. LPS activated TLR‐4 and caused activation of autophagosome formation. However, when cells transfected with G‐protein, we observed inhibition of inflammatory cytokines pathway. These effects were induced by overexpression of G‐protein and lead cell death pathway such as apoptosis. These studies suggest that activation of G‐protein mediated TLR‐4 signaling induces autophagic cell death, not to lead necrotic cell death.

Supported by KRF‐E00344, BioGreen21 Program (#20070401034006) of Rural Development Administration, South Korea.
Unbiased discovery of in vivo enzyme activities by metabolomic profiling of gene‐deleted mice: proof‐of‐principle using xanthine oxidoreductase
Authors: Gross Steven S , Goligorsky Michael S , Ratliff Brian , Park H‐C , Chen Qiuying
Published: June 2021

A lofty goal of metabolomics is untargeted discovery of disease‐associated and drug‐induced changes in the expression of diverse small molecules in biological samples. Despite the enormous potential of metabolic profiling for translational biomedicine, effective analytical and informatic platforms remain to be established. This study sought to determine whether an untargeted metabolomics approach, utilizing accurate mass LC‐MS, feature profiling and unsupervised learning could effectively "discover" a purine metabolism defect in mice where xanthine oxidoreductase (XOR) is genetically‐deleted or pharmacologically‐inhibited. Untargeted profiling of XOR deficient mice conclusively revealed a marked decrease in the XOR product ureate (as predicted) in plasma and urine, as well as increases in purine metabolites upstream of XOR. Statistical methods, including principal component analysis and categorical clustering, demonstrated a remarkably similar metabolite profile in XOR‐inhibited and xor‐/‐ mice. Further, these methods revealed clear differences between the metabolite profiles of XOR deficient mice vs. xor+/+ and xor+/‐ mice. Together, the present studies provide proof‐of‐principle for the efficacy of untargeted metabolite profiling to discover altered biochemical pathways in diseases and in response to drug treatments.
Mutation of eukaryotic initiation factor‐2 α at serine 51 inhibits foam cell formation
Authors: Yang Hong , Zhao Yanfeng , Guo Zhongmao
Published: June 2021

We previously reported that ApoE‐deficient, ApoB48‐containing (E?/B48) lipoprotein‐induced foam cell formation was associated with an increased phosphorylation of eukaryotic initiation factor‐2α (eIF‐2α), a hallmark feature of endoplasmic reticulum stress. The objective of this study is to examine the impact of a nonphosphorylatable eIF‐2α mutant on E?/B48 lipoprotein‐induced changes in gene expression and foam cell formation. We observed that incubation of mouse peritoneal macrophages (MPMs) with E?/B48 lipoproteins reduced the protein levels of lysosomal hydrolases and cholesterol transporters, and enhanced eIF‐2α phosphorylation. Transfection of MPMs with an eIF‐2α mutant, in which serine 51 was replaced by alanine, alleviated the suppressive effect of E?/B48 lipoproteins on lysosomal hydrolase and cholesterol transporter expression, increased macrophage degradation of E?/B48 lipoproteins, enhanced cholesterol efflux, and suppressed foam cell formation. These observations suggest that induction of eIF‐2α phosphorylation is a mechanism by which ApoE‐deficient lipoproteins transform macrophages into foam cells. NIH grant support: K01HL076623 and RR003032 (H.Y.), R01ES014471 (Z.G.).
Rpb1 Sumoylation in Response to UV Radiation or Transcriptional Impairment in Yeast
Authors: Li Shisheng , Chen Xuefeng , Ding Baojin , LeJeune Danielle
Published: June 2021

It has been well known that in response to DNA damage that blocks transcription elongation, Rpb1, the largest subunit of RNA polymerase II, is ubiquitylated and subsequently degraded in mammalian and yeast cells. Here we show that in response to UV radiation or impairment of transcription elongation, Rpb1 is also covalently modified by the Small‐Ubiquitin‐like Modifier (SUMO) in yeast cells. Ubc9, an E2 SUMO conjugase, and Siz1, an E3 SUMO ligase, play important roles in Rpb1 sumoylation. K1487, which is located in the acidic linker region between the C‐terminal domain and the globular domain of Rpb1, is the major sumoylation site. Rpb1 sumoylation is not affected by its ubiquitylation, and vice versa, indicating that the two processes do not crosstalk. Abolishment of Rpb1 sumoylation at K1487 does not affect transcription elongation or transcription coupled repair (TCR) of UV‐induced DNA damage. However, deficiency in TCR enhances UV‐induced Rpb1 sumoylation, presumably due to the persistence of transcription‐blocking DNA lesions in the transcribed strand of a gene. Remarkably, abolishment of Rpb1 sumoylation at K1487 enhances UV‐induced phosphorylation of Rad53, especially in TCR‐deficient cells, suggesting that the sumoylation may play a role in restraining the DNA damage checkpoint response caused by transcription‐blocking lesions.
NADPH oxidase 4 (NOX4) derived ROS mediate fibroblast to myofibroblast transdifferentiation in the diseased prostatic stroma
Authors: Sampson Natalie , Plas Eugen , Berger Peter
Published: June 2021

Benign prostatic hyperplasia (BPH) and prostate cancer (PCa), two of the most common proliferative disorders affecting elderly males, are associated with stromal remodeling in particular fibroblast to myofibroblast transdifferentiation, which promotes disease progression via elevated secretion of mitogenic cytokines and growth factors. Microarrays of primary prostatic stromal fibroblasts (PrSCs) undergoing transdifferentiation revealed a pro‐oxidant shift in intracellular redox homeostasis with upregulation of NOX4 and downregulation of reactive oxygen species (ROS) scavenging enzymes. Consistently, transdifferentiation was associated with elevated ROS production, which temporally correlated with NOX4 induction prior to induction of transdifferentiation markers, αsmooth muscle cell actin (α‐SMA) and Insulin‐like growth factor binding protein 3 (IGFBP3). Inhibition via the antioxidant selenite or NOX4 knockdown indicated an essential role of NOX4‐derived ROS in driving transdifferentiation. Time course assays showed biphasic ERK phosphorylation with late phase phosphorylation being ROS dependent. ROS inhibition prior to late phase ERK phosphorylation attenuated not only ERK phosphorylation but also inhibited α‐SMA induction. Consistently, an ERK inhibitor prevented IGFBP3 induction. These data provide first evidence that NOX4‐derived ROS drives stromal remodeling associated with prostatic disease, at least in part via redox‐regulation of ERK phosphorylation and indicate potential therapeutic benefit of NOX4 inhibition.
Specific Lysine Sites in Histone H3 Contribute To Spurious Transcription
Authors: Peng Weimin , Li Kunwu , Kurdistani Siavash K
Published: June 2021

Transcriptional co‐factors including histone modifying enzymes regulate gene expression at both initiation and elongation stages of transcription. Recent studies demonstrate that Set2‐catalyzed histone H3K36 methylation serves as a signal for recruitment of histone deacetylase Rpd3 Small complex that decreases histone acetylation in coding regions and inhibits aberrant initiation of transcription from cryptic promoters within the coding region of certain genes. In this study, we investigated the possible roles of individual lysine sites in histones H3 and H4 in spurious transcription initiation by Northern blotting, chromatin immunoprecipitation and quantitative RT‐PCR. Our results indicate that H3K4 methylation, H3K14 and H3K23 acetylation specifically contribute to aberrant transcription initiation in the absence of Set2. H3K4 mutation synergistically enhances the suppression of cryptic transcription initiation by H3K14 and H3K23 mutations in set2 deletion. In addition, the functions of these three lysine sites in H3 in transcription initiation are regulated by a number of proteins including Ccr4‐Not complex, Set1 and Spt3 that participate in regulation of H3K4 methylation and histone H3 acetylation, suggesting a complex regulating network exists in cryptic transcription initiation. Our study reveals new roles for these three histone lysines in H3.
Plasma and interstitial fluid (ISF) bioavailability of green tea catechins
Authors: Gerber Sophie , Myracle Angela , Peters Catrina , Ferruzzi Mario , Janle Elsa
Published: June 2021

Green tea (GT) catechins are antioxidants which have many potential health benefits. Their bioavailability has previously been evaluated only by plasma pharmacokinetics, but target tissue concentrations are likely more important to delivery of specific health benefits. The aim of this study was to demonstrate the relationship between plasma and ISF pharmacokinetics of catechins from GT. Sprague Dawley rats were implanted with ultrafiltrate probes, for sampling of ISF, and femoral catheters. Rats were dosed by oral gavage with 50 mg of GT extract and 10 mg ascorbic acid. The ascorbic acid was added to increase bioavailability. Blood samples were collected at baseline and 0.5, 1, 2, 4, 6, and 8 hours. ISF was collected two hours before gavage and every two hours post gavage for 16 hours. Catechins in the plasma and ISF were extracted with 0.01 % butylated hydroxytoluene in ethyl acetate and analyzed by HPLC. The results showed that epigallocatechin peaked in the plasma between one and two hours and epicatechin peaked in the plasma at half‐an‐hour. Both catechins peaked in ISF about two hours post gavage. This data suggests that plasma and ISF pharmacokinetics may not be the same for all catechins. Some catechins may provide benefits to target tissues for a longer duration of time than indicated by plasma pharmacokinetics. Supported by NIH, Office of Dietary Supplements and NCCAM Grant P50 AT 00477.
Resveratrol and quercetin target microRNA 27a in their anti‐cancer effects in colon cancer cells
Authors: Del Follo Armando , Noratto Giuliana D. , Mertens‐Talcott Susanne
Published: June 2021

The polyphenolics resveratrol and quercetin in combination previously were shown to exert synergistic anticarcinogenic effects. The objective was to investigate anticancer properties and underlying mechanisms of resveratrol and quercetin in combination in colorectal adenocarcinoma cells (HT‐29). Cell viability was determined after 24, 48, 72 and 96h, and IC50 values were 62, 24, 21 and 14 mg/L (combined concentration of resveratrol and quercetin in 1:1 ratio), respectively. Reactive oxygen species (ROS) increased up to 2.25‐fold at 5‐30mg/L and mRNA of the cell cycle regulator p53 and the apoptotic enzyme caspase‐3 were increased by up to 2‐fold and 2.5‐fold within a concentration range of 20‐60 mg/L after 24h, respectively. mRNA of zinc‐finger protein ZBTB10 a putative suppressor of the oncogenic specificity protein Sp‐1, was significantly increased by 3.5‐fold at 30 mg/L, coinciding with a significant decrease in Sp‐1mRNA levels. miRNA27a, which in previous studies was determined to be a downregulator of ZBTB10, was upregulated by 2.5‐fold, indicating the involvement of this miRNA in the down‐regulation of Sp1 protein and potentially the induction of apoptosis. In conclusion, the combination of resveratrol and quercetin was highly effective in the reduction of cell viability in colon cancer cells, employing apoptosis and the inhibition of microRNA27a as underlying mechanism.

Grant Funding Source

Texas A&M University
Effect of long‐term green tea ingestion on cellular signaling systems related to oxidative stress and survival in the aging rat hippocampal formation
Authors: Assunção Marco , Santos‐Marques Maria João , Carvalho Félix , Andrade José Paulo
Published: June 2021

We previously found that prolonged consumption of green tea (GT), a rich source of antioxidant polyphenols, protected proteins and lipids against oxidation and reduced lipofuscin deposition in the hippocampal formation and improved spatial memory during aging. Therefore, we investigated if GT treatment could interfere with age‐associated changes of cellular signaling systems related to oxidative stress and survival in the same brain region. Five male Wistar rats aged 19 months were fed with GT since 12 months of age (GT‐19M) and results compared to age‐matched controls (C‐19M). A third group of rats was evaluated at 12 months of age (C‐12M) to provide baseline data. In hippocampal tissue, the levels of nuclear factor kappa B (NF‐κB) and cyclic AMP response element binding (CREB) protein involved in oxidative stress response were quantified as well as the brain‐derived neurotrophic factor (BDNF) and the anti‐apoptotic protein Bcl‐2. GT increased the levels of CREB, BDNF and Bcl‐2, but not NF‐κB, relatively to C‐19M rats. Interestingly, most of the effects mediated by GT ingestion corresponded to amelioration of age‐related modifications. We conclude that long‐term GT consumption induced changes in transcription factors leading to neuroprotection that can be correlated to the high levels of BDNF and Bcl‐2 and the earlier described cognitive improvements. Granted by Unit 121/94 and SFRH/BD/19497/2004 from FCT.
Coconut oil enhancement of conjugated linoleic acid‐induced body fat loss and lipolysis in mice
Authors: Ippagunta SiriManasa , Barnes Kimberly M.
Published: June 2021

Dietary conjugated linoleic acid (CLA) causes a body fat loss that is enhanced when mice are fed coconut oil (CO) compared to soy oil (SO). The objectives were to determine if CLA feeding altered adipose tissue lipolysis or the expression of perilipin. Male mice (n=80; 3 wk old) were fed 7% SO or CO diets for 6 wk then 0 or 0.5% CLA for 12 d. A body fat index was calculated: (retroperitoneal+epididymal (EPI) fat pads)*100/body weight. Lipolysis was determined by NEFA and glycerol release from EPI explants. The relative expression of perilipin and phosphorylated perilipin (P‐perilipin) were determined by western blotting. The body fat index was reduced by both CLA (P<0.05) and CO (P<0.001) but there was no interaction. NEFA release was increased by CLA in CO‐fed mice (2.94 vs 8.63 µmol/g; P<0.05) but not in SO‐fed mice (1.76 vs 2.26 µmol/g tissue). Glycerol release was not affected by CO or CLA. Total perilipin was not altered by diet but P‐perilipin tended to be increased by CLA in SO‐fed mice and decreased by CLA in CO‐fed mice (P=0.08). This may indicate that the CLA‐stimulated lipolysis in CO‐fed mice is on the decline since P‐perilipin is associated with increased lipase activity.

Grant Funding Source

None
Mallory Denk Body Formation is Associated with an Increase of the Expression of TNF and IFNg Receptor
Authors: Oliva Joan , Bardag‐Gorce Fawzia , Li Jun , French Barbara A , French Samuel W
Published: June 2021

Mallory‐Denk body (MDB) formation is observed in the liver cells of alcoholic patients, as well as in other non‐alcoholic diseases. Feeding mice the drug DDC for 10 weeks induces MDB formation. Several factors, including proteasome inhibition, are hypothesized to be the cause of MDB formation. Inhibition of the proteasome by PS‐341 in vivo, increased the number and size of MDBs, in this mouse model. The expression of LMP2, LMP7 and MECL‐1 are induced by DDC feeding. The overexpression of these 3 subunits of the proteasome indicates a switch from the 26S proteasome to the immunoproteasome. As with FAT10, a protein induced by DDC feeding, these proteins are up regulated by TNFa and IFNg. FAT10, LMP2, LMP7, TNFa are localized in the MHC Class I on chromosome 6, in the mouse. By Real‐Time RT‐PCR, the expression of TNFRSF21 and IFNgR1/IFNgR2 are induced by DDC feeding. SAMe, but not Betaine, prevents this up regulation of TNFRSF21 induced by DDC. The receptor for IFNg is a heterodimer composed of IFNgR1 and IFNgR2. The up regulation of these genes is partially recovered after 1 month of drug withdrawal. In conclusion, these results support the concept that, under inflammatory stimulation, the proteasome population is switched from the 26S proteasome to the immunoproteasome. This results in the accumulation of altered and oxidized proteins, and thus protein aggresomes such as MDB form.
Cyclooxygenase‐2 prevents Fas‐induced acute liver failure in mice
Authors: Li Guiying , Huang Shuo , Wu Tong
Published: June 2021

This study was designed to investigate the role of cyclooxygenase‐2 (COX‐2) in Fas‐induced hepatocyte apoptosis in vivo. We generated transgenic mice with targeted expression of COX‐2 in the liver by using the albumin promoter‐enhancer driven vector. The COX‐2 transgenic (Tg), COX‐2 knockout (KO), and wild type mice were treated with the anti‐Fas antibody Jo2 (0.5 ìg/g body weight) for 4‐6 hours and the extent of liver injury was assessed by histopathology, serum transaminases, TUNEL staining and caspase activation. The COX‐2 Tg mice showed resistance to Fas‐induced liver injury when compared to the wild type mice, as reflected by the lower ALT and AST levels, less liver damage and less hepatocyte apoptosis (p<0.01). In contrast, the COX‐2 KO mice showed significantly higher serum ALT and AST levels, more prominent hepatocyte apoptosis, and higher levels of caspase‐8, 9, 3 activities than the wild type mice (p<0.01). The liver tissues from the COX‐2 Tg mice express higher levels of EGFR and several key molecules of the downstream Akt signaling pathway when compared to the wild type mice. Pretreatment with the COX‐2 inhibitor (NS‐398) or the EGFR inhibitor (AG1478) exacerbated Jo2‐mediated liver injury and hepatocyte apoptosis. These results demonstrate that COX‐2 prevents Fas‐induced hepatocyte apoptosis and liver failure in vivo and the effect is mediated at least in part through EGFR/Akt.
Antagonism of benzodiazepines by flumazenil in rats discriminating midazolam: Schild analyses
Authors: Gerak Lisa R
Published: June 2021

The pharmacological selectivity of drug discrimination experiments is often exploited to determine mechanism of action of drugs; however, some classes of drugs, such as positive GABAA modulators, have similar discriminative stimulus effects despite actions at distinct sites. By studying drugs in combination and using quantitative pharmacological analyses, actions at different modulatory sites on GABAA receptors can be elucidated. In the current study, the ability of flumazenil to attenuate the discriminative stimulus effects of the benzodiazepines midazolam and flunitrazepam and the neuroactive steroid pregnanolone was studied in 6 rats discriminating midazolam. Midazolam, flunitrazepam and pregnanolone produced >80% midazolam‐lever responding; flumazenil dose‐dependently antagonized the effects of flunitrazepam and midazolam. Schild analyses for flumazenil studied in combination with flunitrazepam yielded a slope that did not deviate from unity and a pA2 value of 5.87. A dose of 5.6 mg/kg flumazenil, which shifted the flunitrazepam dose‐effect curve 30‐fold to the right, did not attenuate the discriminative stimulus effects of pregnanolone. Because these data conform to theory in a quantifiable manner, drug combination studies in rats can be used to further evaluate interactions between drugs acting at different sites on GABAA receptors. Supported by USPHS grant DA09157.
Protective Effect of BG9928, an Adenosine Receptor Antagonist, against Cisplatin‐Induced Acute Renal Injury in Rats: Comparison with Furosemide
Authors: Whalley Eric , Gill Alan , Wortham Kathleen , Costa Don , Davis Wendell , Ticho Barry
Published: June 2021

Cisplatin (CIS),is nephrotoxic due to renal vasoconstriction and decreased GFR, effects believed to involve of adenosine mediated activation of adenosine A1 receptors (A1Rs). We studied the effect of the A1R antagonist BG9928 (BG) and furosemide (FUR) in CIS‐induced acute renal injury in rats.

BG was given p.o. at 1mg/kg bid from d0‐1 or d0‐6, FUR at 30 mg/kg p.o. d0‐5 to female SD rats (n=10) pretreated with CIS at 5 or 5.5 mg/kg i.v. Venous blood samples were taken for measurement of creatinine (CRE) and urea nitrogen (BUN) over 13 days.

Data was similar for CIS at 5 and 5.5 mg/kg. CIS, 5 mg/kg gave significant elevations in CRE (3.7±0.41 mg/dL), BUN (146.2±18.3 mg/dL) (normal levels: 0.7 ± 0.26 mg/dL CRE & 16.9±2.8 mg/dL BUN) and reduction in body weight.BG d0‐1 or d0‐6 resulted in significant effects, including limiting accumulation of CRE and BUN to 60% of control, accelerating body weight gain and improved renal histopathology. FUR significantly elevated CRE(5.7±1.3 mg/dL), BUN (207±52 mg/dL) and reduced body weight. All FUR animals died on d7.

These data support the involvement of A1Rs in CIS‐induced acute renal injury in rats. BG9928 may be useful in the prevention/treatment of renal failure induced by agents like CIS and in other conditions where activation of A1Rs by adenosine in the kidney occurs.
A novel inhibitor of fatty acid amide hydrolase, the enzyme responsible for the hydrolysis of the endocannabinoid anandamide
Authors: Fowler Christopher John , Björklund Emmelie , Congiu Cenzo , Onnis Valentina
Published: June 2021

Inhibitors of fatty acid amide hydrolase (FAAH) have shown utility in models of inflammatory pain, and in some cases in models of neuropathic pain. The non‐steroidal anti‐inflammatory drug ibuprofen is a weak inhibitor of FAAH (IC50 value ~200 µM, Fowler et al., J Pharmacol Exp Ther 283 [1997] 729‐34), but can be used as a template for the design of more potent compounds. In this respect we have reported an analogue of ibuprofen, ibu‐am5, that retains the COX inhibitory properties of ibuprofen, but is a more potent FAAH inhibitor (IC50 value 5 µM, Holt et al., Eur J Pharmacol 565 [2007] 26‐36). Here we have investigated a series of 28 analogues of acetaminophen, ibuprofen and flufenamic acid for their FAAH inhibitory properties. The most potent compound, TPE22 (4‐acetamidophenyl 2‐(4‐(2‐(trifluoromethyl)pyridin‐4‐ylamino)phenyl)propanoate) inhibited rat brain FAAH in the submicromolar range. The inhibition, which was of mixed type, with Ki(slope) and Ki(intercept) values of 160 nM and 1.9 µM, respectively, was not increased upon preincubation of the compound with FAAH. Thus, TPE22 is a potent novel inhibitor of FAAH.
Design and development of novel non‐peptide agonists at natriuretic peptide receptor‐C
Authors: Hobbs Adrian J , Gane Paul , Lumsden Natalie , Nobles Muriel , Rebstock Anne‐Sophie , Simone Michela , Villar Inmaculada , Worthington Roberta , Xiao Fang , Ahluwalia Amrita , Selwood David
Published: June 2021

Endothelium‐derived C‐type natriuretic peptide (CNP) possesses several cytoprotective functions that might be key in preventing cardiovascular disease (Ahluwalia & Hobbs [2005] Trends. Pharmacol. Sci., 26, 162). Since many of the vasoprotective effects of CNP are mediated via natriuretic peptide receptor (NPR)‐C, this receptor might represent a novel therapeutic target.

Thus, we have undertaken a multi‐disciplinary drug development programme, incorporating computer modelling, chemical synthesis and cell‐based biological screens to generate novel small molecule mimetics of CNP (i.e. NPR‐C agonists). Using the NPR‐C crystal structure (He et al. [2001] Science, 293, 1657) as a starting point, we have (a) tested fragments of CNP to elucidate which amino acids make fundamental contact with NPR‐C; (b) modified the NPR‐C antagonist AP‐811, retaining the non‐peptide region and using pharmacophore searches to replace the peptide component; (c) manipulated the secondary conformation of CNP to replace key functional groups with spatially‐similar molecular structures.

These strategies have led to the identification of a series of novel, selective, non‐peptide NPR‐C agonists that have sub‐micromolar potency to reverse vasoconstrictor‐induced Ca2+ flux in rat isolated vascular smooth muscle cells and relax rat isolated mesenteric arteries in vitro. Such molecules represent an excellent starting point from which to develop NPR‐C agonists for the potential treatment of cardiovascular disease.

Supported by The Wellcome Trust
SCH 530348 Is a Novel Oral Antiplatelet Agent Selective for Protease‐Activated Receptor‐1 (PAR‐1) Receptor Subtype Without Partial Agonist Activity
Authors: Chintala Madhu , Kurowski Stan , Zhai Ying , Lachowicz Jean
Published: June 2021

Objectives

Existing antiplatelet therapies target the thromboxane A2 and P2Y12 ADP platelet activation pathways, but do not affect an important pathway triggered by binding of thrombin to the platelet PAR‐1 receptor. The present study evaluated SCH 530348, a novel thrombin receptor antagonist (TRA) selective for PAR‐1, on human platelet function.

Methods

The effect of SCH 530348 on platelet aggregation in human platelet‐rich plasma was assessed by optical aggregometry in the presence of various agonists. The effect of SCH 530348 on basal and PAR‐1 agonist peptide‐induced expression of P‐selectin, a marker of platelet activation, was also determined.

Results

SCH 530348 inhibited platelet aggregation induced by the PAR‐1 agonist peptide and α‐thrombin in a dose‐related manner, without affecting platelet aggregation in response to the PAR‐4 agonist peptide, ADP, collagen, and thromboxane mimetic. SCH 530348 effectively inhibited PAR‐1 agonist peptide‐induced P‐selectin expression, but did not affect the basal expression of P‐selectin.

Conclusions

SCH 530348 selectively inhibits PAR‐1 on human platelets, with no partial agonist activity. SCH 530348 may incrementally reduce pathologic platelet activation not targeted by existing antiplatelet agents, and thus potentially further reduce the risk of ischemic events. This hypothesis is being tested in large clinical trials.
JSIM modeling raises questions about calcium‐independent contractions of porcine pulmonary artery (PPA)
Authors: Gerber Claire , Badenhop Sarah Beth , Kumar Ashwin V , Alimin Marcela , Pelaez Nancy J
Published: June 2021

Vascular smooth muscle uses both extracellular calcium [Ca2+]o and intracellular stores of calcium to signal a strong, sustained contraction. There is also evidence of calcium‐independent regulation. This study investigated the contraction of PPA in the absence of [Ca2+]o with the prediction that high potassium depolarization would induce an increase of intracellular calcium [Ca2+]i from [Ca2+]o and the sarcoplasmic reticulum (SR). When PPA was repeatedly stimulated to contract in 0 [Ca2+]o Krebs solution with EGTA, it was expected that SR calcium would become depleted. Isometric force was measured in isolated PPA tissue, and the resulting rate constants and magnitude of force development were modeled using JSIM Physiome Modeling Software. After repeated contraction in the absence of [Ca2+]o a fast rate process was missing compared to contractions in the presence of [Ca2+]o, but the magnitude of force did not decline. Only the ability to sustain force and to remain relaxed became impaired. Physiome modeling with JSIM will be used to explore alternative contractile mechanisms to explain these results.
The molecule mechanisms of L‐arginine inhibit pulmonary hypertension
Authors: Ou Jingsong , Wei Wei , Huang Dade , Ou Zhijun , Luo Wei , Luo Zhaoliu , Deng Weibing , Cheng Wenguang
Published: June 2021

L‐arginine(L‐arg) has been shown to attenuate pulmonary hypertension (PH). Monocrotaline can induce PH. To investigate the molecule mechanism by which L‐arg attenuates PH, SD rats were injected intraperitoneally with Monocrotaline once with or without L‐arg 3 for weeks. The right ventricular systolic pressure (RVSP) was measured. Nitric oxide (NO) concentration in plasma was assayed with chemical colorimetry. Superoxide anion (O2▸−) generation in lung was determined by hydroethidine. Pulmonary arteries wall thickness was observed by histology. The expressions of endothial nitric oxide synthase (eNOS) and heat shock protein 90 (HSP90) and their association in lung were determined by immunoprecipitation and western blot. Monocrotaline increased RVSP and pulmonary arteries wall thickness with attenuating NO concentration in plasma, enhancing O2▸− generation in lung, decreasing eNOS experssion and the association between eNOS and HSP90 in lung. L‐arg can partially decrease the RVSP, inhibit pulmonary arteries wall thickness, restore NO level in plasma, block O2▸− generation in lung, restore eNOS expression and the association between eNOS and HSP90 in lung in Monocrotaline‐treated SD rats. Our findings illustrated part of molecular mechanisms of L‐arg preventing PH. Such findinds may provide an opportunity for exploring the pathogenesis and preventive and therapeutic measures for PH.
Role of Endogenous Bone Marrow Derived Mesenchymal Stem Cells in Lung Injury
Authors: rojas mauricio , Xu Jianguo , Brigham Kenneth L , Torres Edilson , Mora Ana L
Published: June 2021

Several groups had reported the protective effect of infused Bone Marrow Derived Mesenchymal Stem Cells (BMDMSC) but there is no direct evidence that endogenous BMDMSC have an active role during the injury process. We propose the use of parabiosis to study cell trafficking. Parabiosis, the surgical attachment of two distinguishable mice, establishes a common circulation between the parabionts and therefore creates chimeras without immunosuppression. Two C57BL/6 mice, one GFP+ and the other GFP‐, were surgically connected creating a common circulation. After recovery, the GFP‐ mouse was treated with a single dose of bleomycin or saline intratracheally. Three, 7 and 14 days later, the lungs from the GFP‐ mouse were harvested, cell suspensions made and flow cytometry analysis performed to determine the expression of GFP+ BMDMSC in the lung. We observed an increase in CD45+ GFP cells that peak at day 14 opposite to what is observed with the CD45‐ that peak at day 3. BMDMSC were approximately 0.05% in the control mouse lung. After bleomycin treatment, the levels BMDMSC significantly increased at days 7 (2.0 fold, p<0.05) and 14 (6.5 fold, p<0.01). In addition we observed a decrease in the severity in the lung injury that can be explained by the additive effect of having two bone marrows to supply BMDMSC. We conclude that mobilization of BMDMSC is a sequential set of events during the process of repair
Expression of Psoriasin in Normal and CF Airway Epithelial Cell Lines
Authors: Li Tiesong , Marsh Shauna , Cowley Elizabeth A
Published: June 2021

Psoriasin is a low molecular weight, Ca2+binding protein with antimicrobial activity. Using RT‐PCR and immunoblotting, we determined that psoriasin is expressed in a number of human airway epithelial cells lines. We next investigated physiological and pathophysiological mediators we hypothesised might influence psoriasin expression, specifically comparing the normal human bronchial epithelial cell line 16HBE14o‐ to the CF bronchial epithelial cell line CFBE41o‐. Psoriasin was expressed at much lower levels in the CFBE41o‐ cells compared to non‐CF cells. Treatment with the cytokine IL‐22 significantly upregulated psoriasin expression in the 16HBE14o‐ cells, while there was no effect on the CFBE41o‐ cells. Treatment with the pro‐inflammatory cytokines IFNγ, TNF‐α and IL‐1β also effected psoriasin expression: IFNγ and TNF‐α increased expression in both cell lines, while IL‐1β increased expression in the CFBE41o‐ cells only. Exposure to the oxidant stresser t‐butylhydroperoxide decreased psoriasin expression in normals with no effect on the CF cell line. Finally, inhibition of the CFTR Cl‐ channel also affected psoriasin expression. This is the first evidence of psoriasin expression in human airway epithelia, and suggests that differential responses exist between normal and CF airway cells, potentially contributing to their innate antimicrobial defence. Supported by the Canadian CF Foundation.
Resistance arteries and aorta from Angiotensin II hypertensive mice do not exhibit decreased relaxation responses to Angeli's Salt, a nitroxyl anion donor
Authors: Wynne Brandi M. , Labazi Hicham , Webb R. Clinton
Published: June 2021

The endothelium is an important regulator of vascular tone, releasing various vasoactive substances, including nitric oxide (NO), a primary vasodilating agent. NO can exist in three different redox states; the uncharged NO, the oxidized state, nitrosonium cation (NO+), and the reduced state, nitroxyl anion (NO−). This reduced form, NO−, has recently become an emerging candidate in vascular smooth muscle regulation. We hypothesized that relaxation to the nitroxyl anion donor, Angeli's Salt (AS) in resistance arteries and aorta will be decreased in vessels from Ang II mice. Ang II (90ng/min) or sham mice were killed and first order mesenteric arteries and aorta were isolated. Concentration‐response curves (CRC) to AS were performed in phenylephrine contracted vessels in the presence of L‐NAME (10−4M), ODQ (3x10−6) or high K+ (14mM or 30mM). Removal of the endothelium did not decrease the relaxation response to AS. The relaxation responses to AS were similar in Ang II and sham mice. Also, no differences were observed between hypertensive and normotensive vessels with NOS inhibition. However, Ang II vessels exhibited a decreased relaxation with sGC inhibition, and increased relaxation in the presence of high K+ (14mM). These data suggest that Ang II vessels may have increased sGC activity/expression and that K+ channel activation via the nitroxyl anion is increased in Ang II hypertensive mice.
Restoration of sarcolemmal nNOS is essential to normalize α‐adrenoceptor control of muscle blood flow in transgenic mdx mice
Authors: Thomas Gail D , Lai Yi , Duan Dongsheng
Published: June 2021

Neuronal nitric oxide synthase (nNOS) is abundantly expressed in skeletal muscle, where it is localized to the sarcolemma via the dystrophin glycoprotein complex (DGC). We previously reported that nNOS‐derived NO blunts α‐adrenergic vasoconstriction in contracting muscle and that this protective effect is absent in boys with muscular dystrophy and in mdx mice in which mutations in the dystrophin gene disrupt the DGC and displace nNOS. We therefore hypothesized that restoration of sarcolemmal nNOS is essential to rescue the normal contraction‐induced attenuation of α‐adrenergic vasoconstriction. In the resting hindlimbs of transgenic mdx mice expressing minidystrophin genes that restore the DGC with (ΔH2‐R15) or without (ΔH2‐R19) restoring nNOS, intraarterial norepinephrine evoked similar decreases in femoral blood flow and vascular conductance. These constrictor responses were markedly attenuated by 81±3% in the contracting hindlimbs of the ΔH2‐R15 transgenics (p<0.05 vs. rest; n=8), but by only 16±4% in the ΔH2‐R19 transgenics (p>0.05 vs. rest; n=6) and by 10±5% in transgene negative littermates (n=3). Minigene expression yielded similar results in mdx4cv mice, which have a different dystrophin mutation and genetic background than mdx mice. These data indicate that recovery of sarcolemmal nNOS is essential to restore normal blood flow regulation in mdx skeletal muscle. Supported by the NIH and MDA.
The immediate rise in leg blood flow during calf exercise is dependent on functional venous valves
Authors: Naadland Inger Helene , Wesche Jarlis , Sheriff Don , Toska Karin
Published: June 2021

The importance of the muscle pump in exercise hyperemia is incompletely understood. We have recently shown that a large hydrostatic column induced by head up tilt (HUT) increases the effect of the leg muscle pump on muscle perfusion in healthy subjects. In this study we investigated the effect of a larger hydrostatic column on muscle perfusion in patients with insufficient venous valves.

The transient and steady state rise in leg arterial blood flow (ultrasound Doppler) was investigated in 10 patients with venous insufficiency, both supine and 30º HUT.

Within ~15 s of the start of exercise, the rise in femoral flow was not significantly different between supine (0.21 L/min) and HUT (0.24 L/min). After 80 s of exercise, femoral flow was stable at 0.20 L/min and 0.22 L/min above rest in supine and HUT respectively (not significantly different).

These results indicate that there is no significant muscle pump effect on the immediate rise in muscle perfusion in patients with venous insufficiency.

This is in contrast to the findings in healthy subjects, where the transient rise in femoral flow was considerably larger in the tilted than in the supine position. The reduced immediate rise in blood flow observed in the present study indicates that the larger transient rise in femoral flow in healthy subjects was indeed dependent on functional venous valves, and thus caused by a leg muscle pump effect.

Research support: University of Oslo.
Following histamine perfusion onto Syrian hamster hippocampal slices, enhanced neural excitability was observed on a pathway projecting to the brainstem
Authors: Nguyen Yen Ha , Horowitz John M , Hamilton Jock S. , Horwitz Barbara A.
Published: June 2021

Previous studies suggest that signals from the hippocampus control the duration of a bout of hibernation. Specifically, histamine injected into the whole hippocampus lengthened each hibernation bout (Sallmen, et al., Brain Res. 2003, 966:317‐20). However, neural activity of the population of CA3 hippocampal neurons that project to the brainstem reticular activating system (RAS) has not been studied. We tested the hypothesis that histamine modulates CA3 region excitability. Using hippocampal slices from euthermic and hibernating Syrian hamsters, evoked responses were recorded before, during, and after histamine perfusion at 30oC. Histamine increased the slope of the second peak of the evoked response from ‐0.0366 + 0.010 to ‐0.0631 + 0.008, a 72.4% increase (P<0.05, 7 slices from 5 hamsters). Thus, in the presence of histamine, stimulation of the dentate granule cell axons elicited an enhanced oscillatory response in CA3 pyramidal cell axons projecting to the brainstem. These data are consistent with the hypothesis that histamine augments signals to brainstem RAS neurons and thus may prolong the duration of a hibernation bout. This study was supported by an UC Davis President's Undergraduate Fellowship award to YHN.
Effect of pyrene exposure on skin ionic conductance in Rana pipiens and Rana catesbeiana
Authors: Anderson Kristy A , Stabenau Erich K
Published: June 2021

Previous studies have demonstrated that a 2‐ringed polycyclic aromatic hydrocarbon (PAH) can alter ionic movement across frog skin (Blankemeyer and Hefler, 1990). However, no information is available on the effects of complex PAHs on ionic conductance across frog skin. In the present study, Rana pipiens were exposed to saturated pyrene levels, a 4‐ringed PAH commonly found in large concentrations in aquatic environments. Following a 7‐day exposure, frogs were anesthetized, pithed, and the abdominal skin mounted into Ussing chambers with frogs Ringers solution bathing each side of the tissue. I‐V relationships were generated following clamping of the skin at ‐40, ‐20, 0, 20, and 40 Volts. The data revealed that pyrene exposure significantly reduced the ionic conductance across the leopard frog skin. Using ion replacement studies on the mucosal and serosal sides of skin from control animals, it was determined that replacing mucosal Na+ and Cl− produced similar tissue conductance levels to that measured in pyrene‐exposed frogs without ion replacement. These data suggest that pyrene‐exposure significantly reduces Na+ and Cl− conductance in frog tissue. Limitations of the influx of these ions into the frogs may produce osmoregulatory limitations. It is noteworthy that similar effects were not observed in R. catesbeiana. These studies were funded, in part, by a Bradley University Research Excellence grant to EKS.