bluuebunny/crossref_metadata_2025 huggingface dataset:
Possible involvement of cytoskeletal reorganization in PKC‐induced cerebroarterial constriction
Authors: EL‐Yazbi Ahmed Fawzy
Published: June 2021

Several hypotheses have been proposed to account for force production evoked by PKC activation in the resistance circulation based on pharmacological experiments with intact and permeabilized vessel preparations. These include: an increase in calcium influx, an increase in calcium sensitization via phosphorylation of CPI‐17, and thin filament regulation via phosphorylation of calponin and/or caldesmon. However, biochemical evidence of a role for these phosphoproteins has not been forthcoming. Here, rat middle cerebral arteries were used to investigate the molecular mechanism responsible for PKC‐mediated constriction evoked by 5‐HT or direct activation with phorbol dibutyrate (PDBu). A highly sensitive 3‐step western blotting method was used to detect changes in protein phosphorylation. PDBu caused a much larger contraction compared to that evoked by a maximal concentration of 5‐HT, but a similar increase in LC20 phosphorylation. PKC inhibition with GF109302X abolished the 5‐HT‐induced constriction with minimal change in pLC20 content, indicating a role for a non‐LC20‐dependent mechanism(s). Phosphorylation of calponin and caldesmon were not affected by direct PKC activation. These findings suggest a possible contribution of actin cytoskeletal rearrangement to force generation evoked by PKC activation in resistance arteries. (CIHR MOP‐97988, CIHR & AHFMR Fellowships)
Interaction between sympathomimetic amines, ephedrine, tyramine and norepinephrine at uptake 1 in sympathetic nerves and the role of nitric oxide
Authors: Simaan Joseph A.
Published: June 2021

Ephedrine (E) is alpha and beta adrenoceptor sympathomimetic agonist that acts partly directly on receptors and partly by liberating norepinephrine (NE) from vesicular stores. Repeated administration leads to tolerance. Since E enters uptake 1 in sympathetic nerve terminals to liberate NE, its interaction with tyramine (T) indirect‐ acting and NE direct‐ acting sympathomimetics and the role of nitric oxide (NO) in E tolerance and this interaction, were explored in anesthetized rats. Repeated single doses of E (2 mg/kg) produced tolerance in mean blood pressure from 28+/−4 to 3+/−1 mmHg after 9+/−0.8 doses (cumulative 18.1+/−1.8 mg/kg). Blockade of the synthesis of NO with nitro‐L‐arginine (NA15mg/kg) and restoration of the induced rise in pressure to control with an infusion of nitroglycerin (NG), a NO donor, partly reversed the tolerance by 8+/−1 mmHg, (285+/−62%). Tyramine (T 0.025– 0.2 mg) but not NE (0.05–0.2 ug) given before and after E tolerance, exhibited cross tolerance of 84–89% and partial recovery after NA and NG by 109–386 %. It is concluded that E enters uptake 1 to release NE and with repeated doses blocks its own entry and that of T, a process partly dependent on fresh synthesis of NO. The rest of tolerance could be due to retention of E by uptake 1 and not due to changes in receptors and post‐ receptor pathways since the effect of NE was preserved. Supported by the American University of Beirut.
Pioglitazone Ameliorates Cyclosporine‐Induced Attenuation Of Carbachol Renovascular Vasodilations: Role Of PPARγ/Nitric Oxide Synthase Signaling
Authors: El‐Mas Mahmoud M , El‐Gowelli Hanan M , Saad Evan I , Abd‐Elrahman Khaled S , Abdel‐Galil Abdel‐Galil A
Published: June 2021

We previously reported that cyclosporine A (CSA) impairs reno‐vascular reactivity. This study investigated the role of peroxisome proliferator‐activated receptor‐gamma (PPARγ)/NOS signaling in the CSA‐induced attenuation of endothelium‐dependent vasodilations in phenylephrine‐preconstricted perfused kidneys of rats. Bolus injection of carbachol (4 μmol) reduced the renal perfusion pressure (RPP) with a peak depressor effect observed at 2 min. CSA (5 μM) infusion attenuated carbachol vasodilations. Similar inhibitory effects on carbachol vasodilations were evident after infusion of NG‐nitro‐L‐arginine methyl ester (L‐NAME, NOS inhibitor) or GW9662 (PPARγ antagonist). The CSA‐evoked attenuation of carbachol vasodilation was abolished in presence of L‐NAME in contrast to no effect for GW9662. Pioglitazone, PPARγ agonist, abolished the attenuation of these responses elicited by CSA; an effect that was not manifest in presence of GW9662. The specificity of the interaction with carbachol was verified by the lack of effect of CSA on vasodilations caused by papaverine. It is concluded that the interruption of PPARγ/NOS signaling mediates, at least partly, the inhibitory effect of CSA on renovascular responses to carbachol. Further, functional NOS is necessary for the amelioration by pioglitazone of the CSA‐carbachol interaction.
Adjusting Serum Biomarker of Vitamin D Status for Within‐person Variation
Authors: Sempos Christopher , Carriquirry Alicia , Bailey Regan , Joseph Maria , Durazo Ramon , Yetley Beth , Dawson‐Hughes Bess , Dwyer Johanna , Picciano Mary Frances
Published: June 2021

Adjusting for within‐person variability (Sw2) is an important strategy in estimating prevalence for nutritional biomarkers. Using replicate biomarker data from the NHANES III (NH3) we computed the Sw2 for serum 25‐hydroxyvitamin D (25OHD) & serum total cholesterol (TC) for the DRI age & sex groups. Overall the ratio of within to between‐person ( Sb2) variation for 25OHD was about 0.2, a low value, while that for TC was very similar, i.e. ≈ 0.25. Age‐Sex specific Sw2 estimates for those biomarkers were then applied to the corresponding groups to adjust NH3 & NHANES 2001–2006 (NH01‐06) data for ages 12 +y. Adjusting for Sw2 had an impact on the estimates of the % below cut‐points for 25(OH)D but not for TC. For 25(OH)D the % < 27.5 nmol/L before & after adjustment in NH3 & NH01‐06 were <4% to 1% & about 7% to <4%, respectively. The decrease in % < than selected cut‐points after adjustment for Sw2 diminished steadily with increasing level of 25(OH)D so that approximately no difference was found for the % < 50 nmol/L & the % < 75nmol/L increased after adjustment. The results indicate that regardless of the size of the Sw2, it is important to assess its impact on prevalence estimates in biomarkers of nutritional status.
Improved Antioxidant And Lipid Profiles Underlie The Protective Effect Of Pioglitazone Against Cyclosporine‐Induced Endothelium Dysfunction In Isolated Rat Aortas
Authors: El‐Mas Mahmoud M , El‐Gowelli Hanan M , Saad Evan I , Abd‐Elrahman Khaled S , Abdel‐Galil Abdel‐Galil A
Published: June 2021

This study reports on potential protective effect of pioglitazone against functional, biochemical, and histological vascular derangements caused by cyclosporine (CSA) in rats. CSA (20 mg/kg/day, s.c., for 14 days) impaired aortic vasorelaxations induced by carbachol. The EC50 and Emax of carbachol were'/significantly increased and decreased, respectively, by CSA. These effects of CSA were abolished upon concurrent exposure to pioglitazone (2.5 mg/kg/day). In contrast, aortic responses to sodium nitroprusside, an endothelium‐independent vasorelaxant, were not altered by CSA. The possibility that alterations in the antioxidant and/or lipid profile contributed to the CSA‐pioglitazone interaction was investigated. CSA caused deterioration of the aortic antioxidant capacity as suggested by the decreases in superoxide dismutase activity and increases in malondialdyde and nitrite/nitrate levels. Further, CSA increased serum LDL and LDL/HDL ratio, effects that were also abrogated by simultaneous administration of pioglitazone. Histologically, CSA caused focal disruption in the endothelial lining of the aorta and this effect disappeared in presence of pioglitazone. These findings highlight a protective effect for pioglitazone against the vasculotoxic action of CSA and perhaps underlying oxidative and dyslipidemic effects.
Antiarrhythmic drug‐induced modulation of Kv channel surface density in cardiomyocytes
Authors: Schumacher Sarah Marie , Zhang Lian , Martens Jeffrey R
Published: June 2021

Conventional antiarrhythmic drugs target the ion permeability of channels. This therapeutic strategy, however, is limited by proarrhythmia in the ventricles due to a lack of ion channel selectivity and overlap in channel expression. Given the atrial specific expression of Kv1.5, and its known alterations in atrial fibrillation (AF), this channel has emerged as a primary target in academic and industrial research. In this study, treatment of HL‐1 cells expressing Kv1.5‐GFP with the class I antiarrhythmic agent quinidine resulted in a dose‐ and time‐dependent internalization of Kv1.5, concomitant to channel block, that was subunit‐dependent and stereospecific. Alanine‐scanning mutagenesis of Kv1.5 channel protein revealed partial, but not complete, overlap in the binding sites required for block and internalization. Additionally, structure‐activity relationship studies demonstrated key components of the drug molecule that are required for alterations in channel trafficking. Furthermore, we have expanded these studies to include analysis of homomeric versus heteromeric assembled channels. Together these data indicate that the pharmacophores for pore block and channel internalization may be separable. This highlights the potential for new compounds that selectively alter the membrane stability of Kv1.5 as a means to gain atrial selectivity in the treatment of AF. Supported by NIH HL0270973, JRM.
No Distinct Address and Message Domains Involved in Endothelin‐1/ETA‐mediated Arterial Contractions
Authors: Compeer Matthijs G , Meens Merlijn JPMT , Hackeng Tilman M , De Mey Jo GR
Published: June 2021

Objective

Many peptidergic GPCR agonists contain distinct binding (address) and activating (message) domains. We evaluated whether this is also the case for the vasomotor effects of calcitonin gene‐related peptide (CGRP) and endothelin‐1 (ET‐1).

Methods

Contractile and relaxing effects of commercial peptides and locally synthesized loop‐fragments were recorded in 2nd order rat mesenteric arteries by wire‐myography under conditions excluding the endothelium and the sensory‐motor nerves.

Results

In arteries contracted by 16 nM ET‐1, 0.1–100nM CGRP1‐37 caused relaxations that were antagonized by 1μM CGRP8‐37; neither effect was modified by 1μM CGRP1‐7. 1–16 nM ET‐1(1–21) caused ETA‐mediated contractions that were neither mimicked nor antagonized by 1μM Ala1,3,11,15ET‐1 (1–21), ET‐1(11–21), ET(16–21) or ET‐1(1–15). Intact ET‐3(1–21) caused contractions of similar amplitude with 30 times lower potency.

Conclusion

While distinct domains of CGRP take part in its binding and vasodilator effects, both the intact N‐terminal loop and the C‐terminal tail of ET‐1 are required for high affinity binding and activation of vasoconstrictor ETA‐receptors.

This study was performed within the frame of TIPharma project T2‐301.
Hydrogen Sulfide Stimulates Cyclic AMP Formation In Mammalian Retina Through the Adenylyl Cyclase Pathway
Authors: Njie‐Mbye Ya Fatou , Kulkarni Madhura , Opere Catherine A , Ohia Sunny E
Published: June 2021

We tested the hypothesis that Hydrogen sulfide (H2S) stimulates the formation of cyclic AMP (cAMP) in neural retina of pigs and cows via activation of the adenylyl cyclase pathway.

Methods

Isolated bovine and porcine retina were exposed to H2S donors, NaHS, Na2S, the cyclic nucleotide phosphodiesterase (PDE) inhibitor, IBMX and/or the diterpene activator of adenylate cyclase, forskolin and prepared for cAMP content by EIA.

Results

The PDE inhibitor, IBMX (2 mM) caused a two‐three fold increase in basal cAMP levels in bovine and porcine retina respectively. The combination of both IBMX and forskolin (10 μM) produced a synergistic increase (p < 0.001) in cAMP levels over basal. NaHS (10 nM‐1 μM), and Na2S (10 nM‐100 μM) increased cAMP in both tissues. For instance, 100 nM NaHS caused an increase of 20% in bovine retina whilst 1 μM NaHS produced a 15% increase in porcine retina. In bovine and porcine retina, the effects caused by forskolin on cAMP production were not potentiated by addition of low or high concentrations of NaHS or Na2S.

Conclusions

Both NaHS and Na2S significantly increased cAMP levels in bovine and porcine retinae indicating that this nucleotide serves as a mediator of effects caused by H2S. Furthermore, effects produced by forskolin on cAMP formation were not additive with those elicited by H2S donors postulating that these agents may share a common mechanism in their action on the adenylyl cyclase pathway.
A dominant negative allele of Gs alpha blocks signaling through adenylyl cyclase
Authors: Rylaarsdam Robin Pals , Sultan Dahlia , Ghouse Amrien , Patel Pooja
Published: June 2021

McCune‐Albright Syndrome (MAS) is a human genetic disorder caused by a mutation that constitutively activates the Gs alpha subunit by abolishing GTP hydrolysis. Previous work in this laboratory modeled the MAS mutation in a yeast system, and identified an intragenic suppressor of the MAS mutation, which substituted two residues in the GTP‐binding site: L318P and D319V. To extend these studies, the human GNAS1 gene, encoding Gs alpha, was subjected to site‐directed mutagenesis. Constructs expressing the MAS mutation (R201H), the MAS mutation plus the mutations homologous to the yeast suppressors (R201H/F699P/D700V), the MAS mutation plus each of the amino acid changes in the yeast suppressor (R201H/F699P), (R201H/D700V), or the yeast suppressor mutation alone (F699P/D700V) were transfected into HEK293 cells, and basal and receptor‐stimulated cAMP levels were measured. R201H/F699P/D700V abolished the constitutive activity of the MAS mutation. Interestingly, F699P/D700V blocked the response to hCG. Cotransfection of F699P/D700V with R201H blocked the rise in basal cAMP. Thus, F669P/D700V acts as a dominant negative allele by competing with other G‐proteins for adenylyl cyclase, not the GPCR.
EV71 induces COX‐2 expression and PGE2 synthesis through c‐Src/EGFR/p42/p44 MAPK/CREB in SK‐N‐SH cells
Authors: Tung Wei‐Hsuan , Yang Chuen‐Mao
Published: June 2021

EV71 has been shown to induce cyclooxygenase‐2 (COX‐2) expression through MAPKs, NF‐κB, and AP‐1 in SK‐N‐SH cells. CREB has also been shown to be implicated in the expression of COX‐2. Here, we reported that EV71 induced COX‐2 expression and PGE2 production, which were inhibited by pretreatment with the inhibitor of PKA (H89) and transfection with CREB siRNA. In addition, EV71‐stimulated c‐Src and EGFR phosphorylation and COX‐2 expression were attenuated by pretreatment with the inhibitors of c‐Src (PP1), EGFR (AG1478), or EGFR neutralized antibody. EV71‐stimulated Akt and p42/p44 MAPK phosphorylation were diminished by pretreatment with AG1478. Morever, we found that EV71 regulated CREB activation in a p42/p44 MAPK‐dependent manner. EV71‐induced COX‐2 expression was also blocked by pretreatment with the inhibitor of p300 (GR343) or transfection with p300 siRNA. EV71‐stimulated the association of CREB and p300 with COX‐2 promoter region was further confirmed by immunoprecipitation and chromatin immunoprecipitation assays. These results demonstrated that EV71‐induced CREB phosphorylation was mediated through the c‐Src/EGFR/p42/p44 MAPK pathway, in turn initiated the activation of p300, and ultimately induced COX‐2 expression and PGE2 production in SK‐N‐SH cells.
Mechanistic exposure‐response model of combination rituximab chemotherapy in murine lymphoma xenografts
Authors: Mager Donald E. , Harrold John M.
Published: June 2021

Objectives

To develop a model for describing rituximab effects (CD20 antibody), alone and in combination with fenretinide or rhApo2L, in murine Ramos cell xenografts.

Methods

Pharmacokinetic (PK) and tumor growth data were extracted from the literature. Drug exposure driving functions were derived, and rituximab dynamics were set proportional to antibody‐dependent cell‐mediated cytotoxicity (ADCC) and complement‐dependent cytotoxicity (CDC) driven by CD20 occupancy. Combination regimen effects reflected changes in Bcl‐xL (rhApo2L) and NFÎºB (fenretinide) expression, and a series of transit compartments were evaluated for capturing signal transduction. All model parameter estimation and simulations were performed in MATLAB.

Results

Compartmental models well described fenretinide, rhApo2L, and rituximab PK. Efficacy and potency parameters of CDC and ADCC signals were estimated with good precision. Fenretinide and rhApo2L combination regimens required modified cell kill terms modulated by Fas induction and decreased Bcl‐xL expression. For validation, the model based on single agents was able to predict combination therapy synergism.

Conclusion

A mechanism‐based model of rituximab combination chemotherapy has been developed which is capable of predicting observed synergistic responses.

This research was funded by the Center for Protein Therapeutics at the University at Buffalo
Danazol in the treatment of thrombocytopenia associated with the use of peg‐interferon alfa 2a and Ribavirin in patients with chronic hepatitis C
Authors: Gómez‐Galicia Diana , Cabrera‐Alvarez Guillermo , Rodriguez‐Fragoso Lourdes , Reyes‐Esparza Jorge
Published: June 2021

The aim of this study was to assess the effectiveness of danazol in thrombocytopenia associated with the use of peg‐interferon alfa2a y ribavirin (IFN + RIBA) in patients with chronic hepatitis C (HCV). A clinical trial open, prospective, with historical control in patients with chronic HCV infection (confirmed by PCR) was carried out. We included patients who had platelets less than 90,000/mm3 and were under treatment with IFN + RIBA. Danazol (300 – 600 mg/day) was administered by a minimum of six months and until the end of treatment with IFN + RIBA. The monitoring was performed by a monthly analysis of platelets and liver function tests. The viral load was done at the beginning and at the end of treatment. 47 patients initiated danazol therapy along with IFN + RIBA but only 42 completed it, and 28 had a sustained viral response. Treatment with danazol, not only resulted in a detention in the decline in the platelets, but also there was a slight increase of 3 000 plaquetas/mm3 per month. It was also noted a reduction in ALT levels, but did not reach the normal values. We found an increase in levels of fibrinogen too which correlates with the increase in the platelets. Similarly, it was noted that the increase in the number of platelets correlated with a decrease in the activity of ALT. In conclusion, danazol could be used along with the IFN + RIBA for treating the thrombocytopenia in patients with HCV.
Inhibition of NRF2 in cancer cells enhances apoptotic response and suppresses cell growth:a potential involvement of the p53 signaling
Authors: Kim Tae‐hyoung , You Aram , Manandhar Sarala , Nam Chang‐won , Kang Su‐Jin
Published: June 2021

The integrated regulation of oxidative stress‐response transcription factors could be critical for animals to promote the survival of normal cells and to facilitate the removal of abnormal cells with inherited genetic damage. In the current study, we have investigated the effect of NRF2, a cell survival‐promoting factor, on the p53 signaling. In NRF2 inhibited ovarian cancer cells, the growth arrest in G0/G1 phase and the loss of tumorigenecity in mice were accompanied by the increase in p53 transcription activity as well as its target gene expression such as p21 and Gadd45a. In NRF2‐deleted murine embryonic fibroblasts (MEFs), the accumulation of p53 protein together with the suppression of murine double minute 2 (MDM2) expression was observed although accumulated p53 protein might not be functional. Our further promoter analysis showed that NRF2 can be involved in the regulation of basal expression of MDM2 through the proximal ARE in its promoter. Collectively, our results suggest that a novel evidence that the inhibition of NRF2 may stimulate the p53 signaling, which can result in cell growth limitation and facilitation of apoptotic response upon oxidative stress.
Radiosensitizing effects of xanthohumol on human cancer cells
Authors: Heo Se‐Woong , Kang Youra , Park Min‐A , Kang Keon Wook , Kim Jung‐Ae
Published: June 2021

Xanthohumol, the principal flavonoid in hops, was investigated for its radiosensitizing activity on human cancer cells (human breast cancer MCF‐7 and ardiamucin‐resistant MCF‐7 (MCF‐7/ADR) cells, and human colon cancer HT‐29 and HCT116 cells). Cell survival was measured by the MTT assay 2 days after 10 Gy of ionizing radiation. Cells expressing mutant p53 (MCF‐7/ADR and HT29) are more resistant to the ionizing radiation than wild‐type p53 cells (MCF‐7 and HCT116). However, pretreatment of the cells with xanthohumol (10 μM) for 24 h prior to the radiation significantly enhanced radiosensitivity of all the cells, irrespective of p53 status. Some studies showed that there is persistent activation of signal transducer and activator of transcription 3 (STAT3) in a number of human solid tumors including colon cancer. In addition, MCF‐7/ADR showed persistent activation of STAT3. Xanthohumol significantly suppressed the expression of STAT3. In the cells co‐treated with xanthohumol and radiation could have the therapeutic potential as a radiosensitizer in human cancer cells overexpressing STAT3.
Histone Deacetylases Play a Critical Role in Regulating IFNα‐mediated Anti‐HCV Gene Expression
Authors: Mathews Stephanie , Ademosu Folasade , Joshi‐Barve Swati , McClain Craig , Barve Shirish
Published: June 2021

Hepatitis C virus (HCV) is a major cause of chronic liver disease, which is a huge burden on the United States healthcare system. The FDA‐approved standard of treatment is pegylated interferon‐alpha (IFNα) in combination with ribavirin; however up to 60% of patients do not respond to this therapy. An understanding of the regulatory mechanisms that play an essential role in the expression of IFNα anti‐HCV genes may lead to the development of therapeutic options that increase the effectiveness of IFNα. The present work was carried out to examine the potential role of histone deacetylases (HDACs) in the regulation of IFNα‐stimulated anti‐HCV genes.

Two HDAC inhibitors (HDACi) were used to treat human hepatoma cells or primary human hepatocytes prior to stimulation with IFNα. HDACi pretreatment inhibited induction of several IFN‐mediated antiviral genes, as analyzed by real‐time PCR. Additionally, HDACi led to retention of phospho‐STAT1 in the cytoplasm potentially due to its altered acetylation, and corresponded to inhibited antiviral protein expression. Immunoprecipitation analysis of lysates from IFNα‐stimulated cells identified HDAC1 binding to STAT1 in the nucleus and this interaction was inhibited by HDACi. Taken together, the data from these experiments reveal a unique requirement for HDACs in regulating IFNα‐mediated anti‐HCV gene expression in hepatocytes.
Ethanol Inhibits Lipid Raft‐Dependent TCR Signaling and IL‐2 Expression: Potential Mechanism of Alcohol‐Induced Immune Suppression
Authors: Joshi‐Barve Swati , Ghare Smita , Patil Madhuvanti , Hote Prachi , Suttles Jill , McClain Craig , Barve Shirish
Published: June 2021

Alcohol abuse has long‐term deleterious effects on the immune system, and results in reduced function of CD4+T lymphocytes, which regulate both innate and adaptive immunity. T lymphocyte activation via T‐cell receptor (TCR) involves the plasma membrane lipid raft‐dependent aggregation of proteins into the immunological signalosome, which in turn, triggers a signaling cascade resulting in interleukin‐2 (IL‐2) production. IL‐2 is a critical cytokine that regulates the proliferation/clonal expansion of activated T‐cells, and is essential for an effective immune response.

The present work examines the mechanisms underlying ethanol‐induced dysfunction of CD4+T lymphocytes, and demonstrates for the first time, that physiologically relevant concentrations of ethanol inhibit lipid raft‐dependent TCR signaling and decrease production of IL‐2 in both primary and cultured human CD4+T lymphocytes. Specifically, ethanol substantially down‐regulates lipid raft colocalization and activation/phosphorylation of Lck, ZAP70 and LAT, resulting in a marked decrease in DNA‐binding activity of NFAT and IL‐2 mRNA and protein. Importantly, inhibition of ethanol metabolism prevented these inhibitory effects. Overall, our findings have clinical relevance in alcohol abuse‐associated immune dysfunction, especially in conditions such as HIV and HCV infection where alcohol abuse is a known comorbidity.
The effect of obesity on drug metabolism in African‐American children
Authors: Chiney Manoj , Lee Johnson L'Aurelle Anna
Published: June 2021

It is well established that oxidative enzyme activity differs between obese and healthy weight adults. However, the effect of obesity on drug metabolism in children is poorly understood. Therefore this study examined whether obese and healthy weight children vary with respect to phase I enzyme activity. In vivo CYP 1A2, 3A and 2D6 enzyme activity was assessed in African‐American children between the ages of 6–10yrs by measuring administered dextromethorphan (0.5mg/kg; max=30 mg) and caffeine (11.5mg). Cumulative 8hr metabolic urine ratios (MR) of dextromethorphan/dextrorphan and dextromethorphan/3‐methoxymorphinan were used to determine CYP 2D6 and 3A activity, respectively. Caffeine oral salivary clearance (Dose/AUC0–8) was used to determine CYP 1A2 activity. Nine healthy weight and 6 obese children were evaluated. The median (range) of CYP 3A MR was 2.08 (0.54–7.36) vs. 9.21 (1.24–19.92) in healthy weight and obese children, respectively. CYP 2D6 MR was 0.0378 (0.0018–0.2144) in healthy weight children and 0.1839 (0.0077–0.9657) in obese children. Obese children trended toward a higher oral clearance of caffeine with a median (range) of 0.0064 (0.0021–0.0192) L/hr vs. 0.0040 (0.0013–0.0066) L/hr. These preliminary results suggest that there may be a phenotypic difference in CYP 3A, 2D6, and 1A2 enzyme activity between healthy weight and obese children. Research supported by K12HD052187‐01.
Mitochondria‐Targeted Antioxidant Peptide Promotes ATP Recovery and Reduces Renal Ischemia‐Reperfusion Injury
Authors: Cheng Feng‐Ying , Liu Shaoyi , Darrah Shaun , Zhao Zhihong , Soong Yi , Wu Dunli , Szeto Hazel H
Published: June 2021

SS‐31 is a mitochondria‐targeted small peptide that can inhibit mitochondrial permeability transition (MPT) and scavenge reactive oxygen species. The purpose of this study is to determine whether SS‐31, by protecting mitochondrial function and reducing oxidative damage during renal ischemia‐reperfusion (IR) injury, can reduce tubular cell death, inflammation, and promote epithelial regeneration. Male Sprague‐Dawley rats underwent bilateral renal ischemia for 45min. SS‐31 or saline was administered subcutaneously before ischemia as well as upon and after reperfusion. By treatment with SS‐31, animals show improved mitochondrial respiration and ATP production. Overall cell death, oxidative stress, and inflammation are reduced and renal function is improved. Furthermore, early signs of tubular regeneration are observed in SS‐31 treated group. SS‐31 represents a putative first‐in‐class therapeutic agent that can reduce renal IR injury and accelerate renal recovery. Our results support the hypothesis that MPT and oxidative stress underlie the inflammation and fibrosis induced by renal IR injury.
Determination of fractional breakdown rate of slow turnover proteins
Authors: Holm Lars , O'Rourke Bruce , Ebenstein David , Toth Michael J , Kjaer Michael , Matthews Dwight E
Published: June 2021

OBJECTIVE

To develop and test a method to measure fractional breakdown rate (FBR) of proteins using deuterium exchange from water.

DESIGN

4‐wks old rats weight 77±1g (mean±SE) were grown for 42 days while drinking 8% 2H2O to label free alanine that is incorporated into body proteins. After the labeling period, the rats were given unlabeled water for 21 days to remove 2H from both body water and free alanine. At this point, rats were sacrificed at 21, 22, 23, and 25 days to follow the rate of decrease in protein bound 2H‐alanine with time, which represents FBR.

RESULTS

The decrease in protein bound 2H‐alanine enrichments over 4 days were modeled to determine the FBR of mixed liver protein (5.00±1.50%/d), heart left ventricle myofibrillar (5.30±0.88%/d) and collagen protein (3.64±0.61%/d), extensor digitorum longus myofibrillar protein (3.27±0.66%/d), soleus myofibrillar (3.35±0.79%/d) and collagen protein (2.99±0.66%/d), patella tendon collagen protein (0.66±0.28%/d), femur bone head collagen (0.93±0.71%/d) and shaft collagen protein (0.46±0.37%/d).

CONCLUSION

The measured FBR values were slightly lower than the fractional synthesis rates (FSR) reported by others from similar tissues/protein fractions, which was expected as the rats were still growing. This approach using 2H2O and 2H‐alanine labeling determines FBR in slow turnover proteins.

Grant support: NIH R01‐DK038429
The down regulation of ubiquitin‐proteasome proteolysis system in response to amino acids and insulin involves AMPK and mTOR pathways in rat liver hepatocytes
Authors: Chotechuang Nattida , Azzout‐Marniche Dalila , Bos Cécile , Chaumontet Catherine , Gaudichon Claire , Tomé Daniel
Published: June 2021

The purpose of this work was to examine whether changes in dietary protein level could elicit differential responses of tissue proteolysis and the pathway involved in this response. In rats fed a high protein diet (55%, HP) for 14 days, the liver is the main organ where adaptations occur, characterized by an increased protein pool and a strong meal‐induced inhibition of protein breakdown rate (−42%) as compared to the NP diet (14%). This was associated with a decrease of key‐proteins involved in ubiquitin‐proteasome and autophagy pathway gene expression and a decrease of hepatic ubiquitinated protein level. In hepatocytes, we demonstrated that the increase of amino acid (AA) concentration was sufficient to down regulate ubiquitin proteasome pathway but the inhibition was stronger in the presence of insulin. Interestingly, our results highlights that the inhibition of AMPK and the activation of mTOR transduction pathways are requested for the down regulation of protein ubiquitination in response to high amino acid and insulin concentration.
Antihistaminergic effect of a pure compound (GT) isolated from garden slug (Diplosolenodes occidentalis) on tracheal smooth muscle contraction
Authors: Jacob Audrey Shivrani , Simon Oswald Rudolph , Wheatle Delano Samuel , Ruddock Peter Lanton
Published: June 2021

Folklore claims of the therapeutic effect of garden slug (Diplosolenodes occidentalis) extract relieving brochoconstriction in asthmatic individuals were never validated scientifically. Preliminary findings confirmed this effect. The aim of this study was to isolate the compound associated with this effect. Purification of the crude garden slug extract was done by flash column chromatography and Thin Layer Chromatography methods. Structural details were confirmed mainly by Nuclear Magnetic Resonance (NMR) spectroscopic data. The pure compound (GT) was identified as a triglyceride of linolenic acid. In vitro pharmacological studies showed that GT acted in a dose‐dependent manner. Doses of GT, (3.61 x 10−6M), (7.20x10−6M), (1.4x310−5M), (2.9x10−5M) and (5.7x10−5M) given cumulatively produced statistically significant relaxation (p<0.05, n=8) of histamine‐ induced tracheal tension; non‐competitive antagonism was displayed. GT is an antagonist at H1 receptors since it produced relaxation in tracheal contractions induced by the H1 agonist histamine trifluoromethyl toluidide dimaleate (200μg).These results classify GT as an antihistamine which acts specifically as an H1 antagonist. This work was funded by Graduate Studies and Research section of The University of the West Indies
Lung Apoptotic Signalling and Oxidative Stress During Sub‐Chronic Administration of Doxorubicin to Wistar Rats
Authors: Machado Nuno Gabriel , Oliveira Paulo Jorge
Published: June 2021

Besides oxidative stress, mitochondrial dysfunction, including induction of the permeability transition pore (MPTP), inhibition of respiration and apoptosis has been implicated in Doxorubicin (DOX) cardiotoxicity. The aim of the present work was to determine if DOX causes an increase in selected apoptotic proteins and oxidative stress in the lung. Lungs from male Wistar rats subchronically treated with vehicle or DOX with seven weekly injections (2 mg/Kg) were collected and analyzed by Western Blotting for a) subunits of the mitochondrial respiratory chain b) selected proteins of the MPTP and c) selected pro and anti‐apoptotic proteins. Caspase 9‐ and 3‐like activities and protein carbonyls were also studied. After DOX treatment, no differences in protein carbonyls or caspase 9‐ and caspase 3‐like activities were observed. No alterations were also observed in proteins involved in the mitochondrial permeability transition pore, respiratory chain and in the apoptotic processes studied. The data demonstrate that sub‐chronic DOX treatment apparently does not trigger apoptosis or oxidative stress in the lungs. For this treatment schedule, we conclude that differences exist regarding the susceptibility of the heart and lung tissue for DOX treatment, although lung mitochondrial function must still be evaluated.

Supported by research grant PTDC/SAU‐OSM/104731/2008 from the Portuguese FCT (to PO).
Long term effects of gamma‐tocopherol nebulization in cutaneous burn and smoke inhalation with early excision and skin autografting in sheep
Authors: Yamamoto Yusuke , Enkhbaatar Perenlei , Kraft Edward R , Rehberg Sebas‐tian , Sousse Linda E , Traber Lillian D , Traber Daniel L
Published: June 2021

We have previously reported that airway nebulization of gamma tocopherol (g‐T) had a beneficial effect in ovine model of acute respiratory distress syndrome. We hypothesized that nebulization of g‐T into the airway will effectively improve long‐term pulmonary function and wound healing following acute lung injury.

Method

Ten ewes were subjected to 20%TBSA, third‐degree flame burn and smoke inhalation (36 breaths of cool cotton smoke) under deep anesthesia. One g of g‐T dissolved in 2.2 mL of ethanol was continuously delivered by customized aerosolization device for 48 hrs, starting 3 h post‐injury (Treated group, n=5). Control animals (n=5) were not nebulized. At 24 h post‐injury, escharectomy and skin autografting was performed in both groups. All animals were placed on ventilator and weaning from the ventilator was initiated if PaO2/FiO2 was above 250 at 48 h post‐injury. The sheep were monitored for 3 weeks.

Results

At 96 h post‐injury, all animals were weaned from ventilator. PaO2/FiO2 (mean±SD) was 505±42 in the treated and 489±47 in the control groups at 3 weeks. Lung bloodless wet‐to‐dry weight ratio (mean±SD) was 2.87±0.16 in the treated and 4.72±1.95 in the control groups (p<0.05). The percentage of the surviving graft area (mean±SD) was 84.2±7.5% in treated and 78.7±5.3% in the control groups.

Conclusion

The nebulization of g‐T improved long‐term pulmonary oxygenation, reduced lung water content, and increased wound healing following acute lung injury in sheep. Delivery of g‐T into the lungs may be an effective approach in management of burn patients with concomitant lung injury.
Potentially useful interactions between theophylline and salbutamol on cytokine release in human monocytes
Authors: Ezeamuzie Charles , Shihab Puthiyaveetil
Published: June 2021

This study investigated the in vitro interaction between theophylline and salbutamol on cytokine generation from human monocytes and compared it with the interaction between dexamethasone and salbutamol. Purified monocytes pre‐treated with the drugs (alone or in combination) were stimulated with LPS for 24h and the released TNF‐α and IL‐6 determined by ELISA. Salbutamol (≥ 0.1μM) significantly inhibited the release of TNF‐α, but also significantly enhanced that of IL‐6. In contrast, theophylline (50μM), as well as dexamethasone (0.1μM) strongly inhibited the generation of both cytokines. When used in combination, the effects of theophylline and salbutamol were additive in inhibiting TNF‐α release but theophylline blocked the IL‐6‐enhancing effect of salbutamol. A similar effect was seen when dexamethasone was combined with salbutamol. These results show that β2‐agonists have opposing effects on the generation of TNF‐α and IL‐6, but that when combined with theophylline, the latter, like dexamethasone, was capable of augmenting the anti‐inflammatory effects of the β2‐agonists while preventing their pro‐inflammatory effect. Thus, theophylline may have a potentially useful steroid‐sparing effect.

Sponsored by Kuwait Foundation for Science (KFAS) grant # 2005‐130‐203
Peroxide‐Induced Production of Metabolites of Polyunsaturated Fatty Acids in Bovine Retina, in vitro
Authors: Opere Catherine Atieno , Jamil Jamal , Wright Ashley , Njie Ya Fatou , Ohia Sunny E
Published: June 2021

Neuroprostanes (nPs) are a series of isoprostane (IsoP)‐like compounds that are spontaneously formed in vivo and in vitro by free‐radical catalyzed peroxidation of the polyunsaturated fatty acid (PUFA), docosahexanoic acid. Although nPs are elevated in neurodegenerative conditions and in animal models of neuronal oxidative stress (Musiek et al., Brain Pathol. 15:149,2005), it is unknown whether nPs, like IsoPs, are endogenously produced in mammalian retina. We sought to measure endogenous production of the PUFA metabolites, A4‐nPs and F2‐IsoPs in bovine retinae, in vitro. Freshly isolated bovine retinae were exposed to hydrogen peroxide (H2O2) and cumene hydroperoxide (cuOOH) for up to 6h, in vitro. PUFA metabolites were measured by the stable isotope dilution methods employing GS/negative ion chemical ionization MS. Basal endogenous levels of A4‐nPs and F2‐IsoPs were found to be 35.0± 2.2 and 1.1± 0.001 ng/g tissue, respectively. Exposure of retina to H2O2 (1 mM) for 1h and 6h stimulated production of A4‐nP levels by 15% and 56% while bound F2‐IsoPs were elevated by 44% and 68%, respectively. Similarly, cuOOH (1 mM) enhanced (p<0.001) A4‐nP levels by 408% and 420% and F2‐IsoPs by 831% and 918% after 1h and 6h periods of treatment, respectively. In conclusion, basal levels of nPs exceed that of IsoPs in bovine retina. Furthermore, endogenous production of PUFA metabolites is enhanced by oxidant stress in retina.
Lymphatic Pump Manipulation Mobilizes Inflammatory Mediators into Lymphatic Circulation
Authors: Hodge Lisa M , Huff Jamie , Schander Artur , Williams Arthur , Downey Fred
Published: June 2021

Lymph stasis can result in edema and accumulation of particulate matter, exudates, toxins, and bacteria. This can lead to inflammation, impaired immune cell trafficking, tissue hypoxia, tissue fibrosis, and a variety of diseases. Previously, we demonstrated that osteopathic lymphatic pump treatment (LPT) significantly increased thoracic and mesenteric duct lymph flow. The purpose of this study was to determine if LPT would mobilize inflammatory mediators into the lymphatic circulation. Under anesthesia, thoracic or mesenteric lymph of dogs was collected at baseline (resting), during 4 min of LPT, and 10 min following LPT and the lymphatic concentrations of cytokines, chemokines, superoxide dismutase (SOD) and nitric oxide (NO) were measured. LPT significantly increased both thoracic and mesenteric lymph cytokine and chemokine concentrations when compared to their relative baseline concentrations. In addition, LPT increased lymphatic concentrations of SOD and NO. Ten minutes following cessation of LPT, both thoracic and mesenteric lymph cytokine, chemokine, NO and SOD concentrations were similar to baseline, suggesting their release is transient. This redistribution of inflammatory mediators during LPT may provide scientific rationale for the clinical use of LPT to enhance immunity and treat infection. Funding: NIH: U19 AT002023 (H. F.D), R01 AT004361 (L.M.H).
Sphingosine‐1‐phosphate and endothelin‐1 cause vasoconstriction of inner ear capillaries
Authors: Reimann Katrin , Scherer Elias Q. , Wier Withrow G. , Wangemann Philine
Published: June 2021

Hearing depends critically on cochlear blood flow. Overproduction of sphingosine‐1‐phosphate (S1P) and endothelin (ET‐1) may be associated with sudden and immune‐mediated hearing loss. We had shown that S1P and ET‐1 are vasoconstrictors of the spiral modiolar artery, which provides the main blood supply to the cochlea (Scherer et al. 2006, 2001) and that capillaries in the lateral wall of the cochlea are contractile (Wangemann & Liu 1996). Here the goal was to determine whether S1P and ET‐1 cause a vasoconstriction of the capillaries as a down‐stream mechanism to control regional blood flow in the cochlea. Capillaries were isolated, occluded on one end, opened on the other end, and visualized in vitro by laser‐scanning microscopy. Vasoconstriction was assumed when red blood cells moved toward the open end. S1P (10−7 to 10−5 M) and ET‐1 (10−10 to 10−8 M) induced dose‐dependent vasoconstrictions. These data demonstrate the S1P and ET‐1 are vasoconstrictors in the arterial and capillary microcirculation of the inner ear. Whether overproduction of S1P and/or ET‐1 contribute to sudden and immune‐mediated hearing loss remains to be determined. NIH‐R01‐DC04280 & HL073094.
Inter‐individual Differences in Arteriolar Tree Architecture in the Mouse Spinotrapezius May Suggest a Genetic Basis for Susceptibility to Ischemic Insult
Authors: Bruce Anthony , Mac Gabhann Feilim , Peirce Shayn M.
Published: June 2021

We have previously shown that the arteriolar network architecture of the murine spinotrapezius muscle and lattimus dorsi can be characterized as either highly interconnected or highly dendritic, depending on the strain of inbred mouse. We have also shown that there is a high degree of individual‐to‐individual variability in arteriolar network architecture in outbred CD‐1 mice. Specifically, Balb/c spinotrapezius muscle exhibits a microvascular architecture composed of unconnected dendritic trees, which we hypothesize are susceptible to ischemic injury. In contrast, C57BL/6 spinotrapezius muscle exhibits looped microvasculature architecture that may be protective against ischemic injury. Importantly, outbred CD‐1 mice exhibit either C57BL/6‐like or Balb/c‐like microvasculature architecture in the spinotrapezius muscle and respond to locally‐induced ischemic injury in a manner consistent with the respective inbred strain. We show that differences in microvasculature architecture appear as early as 10 days of age. It is common for CD‐1 littermates to possess either phenotype suggesting a genetic basis for architectural differences, and we have collected microarray data to probe this hypothesis. A method for inducing stable arteriole‐to‐arteriole connections could be very effective at minimizing the effect of ischemic events. Support provided by: NIH‐HL082838‐02 and NIH‐T32‐HL007284.
Oscillations in microvessel networks with RBC suspensions: Preliminary in vitro experimental results in a 3 node network
Authors: Carr Russell T , Maher Meaghan , Geddes John B
Published: June 2021

We have been exploring spontaneous oscillations caused by rheological properties of blood in the microcirculation. The range of parameters over which the oscillations occur in a simple 2 node network is too small to verify our predictions experimentally. The parameter range for oscillations is much greater for a three node topology. Three steady states often exist in 3 node networks. Of these three steady states, one is unstable, suggesting that flow through such a network may exhibit nonlinear bistability. One of the stable steady states has clockwise flow in one branch and the other stable steady state has counter clockwise flow in that branch. The computation predicts that the flow ratio at which the change in direction exhibits a hysteresis.

We have constructed a 3 node network of microchannels having 50 μm diameters. We conducted flow experiments using RBC suspensions and isotonic saline to test our bistability hysteresis prediction. Our model experiments did not reveal such a hysteresis but exhibited spontaneous oscillations. These fluctuations occur over a range of experimental conditions. We present video evidence of oscillations from these flow experiments. These are the first data we have obtained to test our predictions of microvascular fluctuations because of nonlinear dynamics.

Financial Support by UNH Research Experience Apprenticeship Program (REAP)
Distinct phenotypes after cell‐specific deletion of Cx40: Loss in endothelial cells impairs conduction in arterioles and loss in renin‐producing cells elevates blood pressure
Authors: Jobs Alexander , Kurtz Armin , de Wit Cor
Published: June 2021

The gap junction protein connexin40 (Cx40) is important in cardiovascular and renal physiology as concluded from mice globally deficient for Cx40. They exhibit hypertension, renin excess, and impaired conduction of dilations along the endothelium. However, the exact contribution of endothelial Cx40 vs. Cx40 expressed in renin‐producing cells is unknown. In mice carrying a cell‐specific deletion of Cx40 in endothelial (EC) or renin‐producing cells (RPC) arteriolar responses and blood pressure were measured in vivo.

Local stimulation of arterioles using ACh or bradykinin (Bk) elicited dilations that conducted rapidly without attenuation of the amplitude to upstream, remote sites (1200 μm) in mice carrying a floxed Cx40 gene (control). In contrast, remote dilations were significantly attenuated in EC‐Cx40ko (ACh by 28%, Bk by 32%) but not in RPC‐Cx40ko. However, local and remote dilations initiated by adenosine were comparable in all 3 groups of mice. Interestingly, awake EC‐Cx40ko were normotensive (EC‐Cx40ko: 115, control: 113 mmHg) whereas RPC‐Cx40ko exhibited a significantly elevated mean arterial pressure (138 mmHg) and heart weight.

These data demonstrate that EC‐specific deletion of Cx40 impairs conduction of endothelium‐dependent dilations, however, per se does not increase blood pressure. Conversely, elevated pressure in RPC‐Cx40ko does not affect such signal conduction along arterioles.
Prolyl hydroxylase inhibition attenuates hypoxia‐induced hyperpermeability via iNOS upregulation
Authors: Wood John Gibson , Moncure Michael , Thomas James H. , Holloway Naomi B. , Gonzalez Norberto C.
Published: June 2021

Acute systemic hypoxia (Hx) increases leukocyte adherence and vascular permeability in post‐capillary venules of rats. When animals are chronically hypoxic, the initial microvascular inflammation resolves in part due to upregulation of inducible nitric oxide synthase (iNOS). Inhibition of prolyl hydroxylase (PHD) has been shown to increase hypoxia‐inducible factor‐1 (HIF‐1) levels, which can increase iNOS expression.

Objective

This study evaluated whether inhibition of PHD attenuated Hx‐induced increased vascular permeability, and if upregulation of iNOS was involved.

Methods

Hx was produced by a reduction in inspired O2 from 21% to 10%. Intravital microscopy was used to measure vascular permeability in mesenteric venules of anesthetized rats. The PHD inhibitor EDHB was administered to rats 24 hrs prior to experiments.

Results

Hx increased vascular permeability markedly in vehicle‐treated rats but significantly less in EDHB‐treated rats. Superfusion of the mesentery with the iNOS inhibitor L‐NIL caused a significant increase in vascular permeability during Hx in EDHB‐ but not vehicle‐treated rats.

Conclusion

These results show that inhibition of PHD reduces Hx‐induced hyperpermeability in part through iNOS upregulation, and are consistent with a HIF‐1‐dependent mechanism.