bluuebunny/crossref_metadata_2025 huggingface dataset:
Oxidative stress response triggered by the lipid drug edelfosine indicates lipid peroxidation mediates its cytotoxic effect in yeast
Authors: Bitew Teshager , Zaremberg Vanina
Published: June 2021

Edelfosine (1‐O‐octadecyl‐2‐O‐methyl‐rac‐glycero‐3‐phosphocholine) is the prototype of a family of lipid drugs that interferes with cellular membrane function and selectively induces apoptosis in tumor cells. It has been difficult to further optimize its efficacy as its cellular target(s) has yet to be elucidated. Previously, we have validated S. cerevisiae as model system to study the biochemistry and mode of action of Edelfosine. In this study, we have found that microM concentrations of the antioxidant Vitamin E (alpha‐tocopherol) protected yeast cells from Edelfosine cytotoxicity. We have assessed the possibility of Vitamin E correcting the membrane curvature introduced by Edelfosine. Cells were then challenged with a methylated Vit E that has lost its antioxidant properties but could still be inserted into membranes. This compound could not protect cells from Edelfosine, indicating lipid peroxidation mediates its cytotoxic effect. Furthermore, a genetic approach using strains lacking genes known to protect yeast from oxidative stress identified a small set of mutants lacking phospholipid hydroperoxide glutathione peroxidases (GPX1, GPX2 and GPX3) and the two master transcription factors, YAP1 and SKN7, required for oxidative stress tolerance, as hypersensitive to the drug. Altogether these results strongly support lipid peroxidation mediates Edelfosine effect.
Architecture of the PKA RIIβ Holoenzyme
Authors: Brown Simon , Copps Jeffrey , Smith‐Nguyen Eric , Blumenthal Donald K. , Taylor Susan S.
Published: June 2021

How the dimeric PKA regulatory (RI and RII) subunits assemble into an inactive tetrameric complex with the catalytic subunits is very isoform specific. By solving structures of holoenzyme complexes, we now appreciate the global conformational changes that take place in each regulatory subunit as they release cAMP and wrap themselves around the catalytic subunit. By using monomeric deletion mutants we can map the novel isoform‐specific interactions of the inhibition sites in the RI and RII subunits. In parallel we have used small angle X‐ray scattering (SAXS) and small angle neutron scattering (SANS) to map the global architectures of the different conformational states. To define the shape and conformational dynamics of the RIIβ subunit, we have engineered a dimeric mutant, RIIβ(1‐280), that lacks the second cAMP binding domain. In contrast to the RIIα subunit, which forms a highly asymmetric complex, and RIα, where the tetramer is Y‐shaped, RIIβ undergoes a major conformational change. While the free RIIβ subunit forms an extended dimer, the RIIβ holoenzyme folds into a compact, globular conformation. Based on SAXS data, the properties required for this change are included within the RIIβ(1‐280) deletion mutant, and most likely relate to the linker that is disordered in the free RIIβ dimer. [Supported by NIH GM34921 to SST]
Correcting metabolic deficits with allogeneic bone marrow stem cells in the cat
Authors: Vulliet Richard , Halloran Mitch , Tallon Kelli , Lyons Leslie , Rogers Quinton , Fascetti Andrea
Published: June 2021

We investigated the potential of allogeneic lipoprotein lipase (LpL) +/+ expressing bone marrow stromal cells (MSCs) to correct the metabolic deficiency in LpL‐/‐ cats. Bone marrow was harvested from LpL+/+ cats and fifty million MSCs injected into LpL‐/‐ cats twice using two week intervals. Plasma turbidity, triglycerides, cholesterol and lipase activity were monitored prior to and following the injection of the MSCs. Injection of MSCs resulted in a decrease in the plasma lipid profile three to five days following administration. Following the first injection, there was a modest decrease in plasma turbidity and lipids that correlated with a slight increase in LpL activity. The second injection resulted in a much larger increase in LpL activity and a greater decrease in plasma turbidity and lipids. Injection of allogeneic MSCs corrects the LpL‐/‐ genetic defect and transiently returns the plasma lipid profile to near normal in a dose dependent manner. The enhanced response following the second injection suggests an accumulation of LpL+/+ MSCs might occur. Using this dose and injection protocol, the bi‐phasic duration of therapeutic action appears to last approximately three months. This is a very powerful and robust animal model for studying cytokinetics and cytotherapeutics.

This research was support by the Center for Companion Animal Research at U C Davis and the Winn Feline Research Foundation.
Comparison of conjugated linoleic acid (CLA) and conjugated nonadecadienoic acid (CNA) with regard to lipid metabolism in mice
Authors: Park Yooheon , Rhee Siyeon , Park Yeonhwa
Published: June 2021

It has been reported that conjugated nonadecadienoic acid (CNA) exhibits modulation of body fat with mechanisms that are similar to those of CLA. In this current investigation, we compared the effects of CNA and CLA on various feeding period, in particular studying lipid metabolism from white adipose tissue. One hundred thirty 10 week‐old male CD‐1 mice were fed a semi‐purified powdered diet with 20% total fat based on the AIN‐93 diet. After a 2‐week adaptation period, mice were randomly assigned into control and 2 treatment groups (0.5 w/w% CLA and 0.1 w/w% CNA). Relative mRNA expression of peroxisome proliferator‐activated receptor‐γ (PPAR‐γ), acetyl‐CoA carboxylase (ACC), fatty acid synthase (FAS), hormone‐sensitive lipase (HSL), and glucose transporter 4 (GLUT4) were determined at week 0, 2, 4, 8, and 12 by real time PCR from the white adipose tissue. The effect of CNA treatment on total adipose tissue mass was less effective than CLA over time. HSL mRNA expression was significantly increased by CNA at week 8 and 12, while CLA had no effect at any time point. PPAR‐γ expression was significantly decreased by both CNA and CLA after week 2 and 4, respectively, and the decreased expression of PPAR‐γ was maintained for 12 weeks. FAS expression was enhanced by CNA during week 4 and 8 and by CLA at all time points. ACC expression was increased by CNA longer than 8 weeks, while CLA increased it at all time points. GLUT4 expression was decreased at week 8 by CNA but not by CLA. In conclusion, CNA reduces body fat mass similar to CLA. Although CNA shares mechanisms with CLA on body fat control and lipid metabolism, there were differences in the effects on lipid metabolism between CNA and CLA over time. Supported by AHA SDG and ILSI North America
THE EFFECT OF RESVERATROL AND ZINC ON INTRACELLULAR ZINC STATUS IN NHPrE CELLS
Authors: ZHANG JUN JUN , WU MIN , SCHOENE NORBERTA W. , CHENG WEN‐HSING , WANG THOMAS T. Y. , ALSHATWI ALI , ALSAIF MOHAMMED , LEI KAI Y.
Published: June 2021

To evaluate the influence of resveratrol on cellular zinc status, normal human prostate epithelial (NHPrE) cells were treated in 6 levels of resveratrol (0, 0.5, 1, 2.5, 5 and 10 µM) and 4 levels of zinc [0, 4, 16 and 32 µM or zinc deficient (ZD), zinc normal (ZN), zinc adequate (ZA) and zinc supplemented (ZS), respectively]. Among each zinc treatment, a progressive reduction in cell growth or increase in cellular total zinc was observed with increases of resveratrol from 2.5, 5 and 10 µM or from 5 and 10 µM, respectively. In ZS cells, a much higher increase in cellular total zinc was observed as early as 1 µM resveratrol. A flow cytometry study revealed that the resveratrol (10 µM) induced G2/M arrest was responsible for the depressed cell growth. Data from an in vitro experiment using zinquin, as an indicator of intracellular free Zn(II) status, demonstrated complex formation between resveratrol and zinc ion. Zinquin ethyl ester fluorescence spectrofluorimetry and microscope imaging revealed that intracellular labile free zinc decreased in ZD and ZN NHPrE cells but increased in high zinc (ZA and ZS) cells. Furthermore, increases in cellular zinc status induced enhanced levels of reactive oxygen species (ROS) as well as senescence detected by morphological and histochemical changes in cells treated with 2.5 or 10 µM resveratrol, especially in ZA and ZS cells. Thus, increases in free labile zinc may induce ROS and senescence.

Grant Funding Source

UNIV. MARYLAND
Optimally ripened kimchi increased antimutagenicity and anticancer effects in HT‐29 human colon carcinoma cells
Authors: Park Kun‐Young , Kim Boh‐Kyung , Bak Soon‐Sun , Han Gwi‐Jung , Lee Sun‐Mi
Published: June 2021

The antimutagenic and anticancer activities of kimchi during fermentation period were investigated under in vitro and cellular system using HT‐29 human colon carcinoma cells. The methanol extract of fresh kimchi (pH 5.8), optimally ripened kimchi (Opt‐R kimchi, pH 4.1) and over ripened kimchi (pH 3.7) fermented at 5°C were prepared. Among the kimchi samples, Opt‐R kimchi showed the strongest inhibitory activities against the mutagenicities induced by aflatoxin B1 and N‐methyl‐N′‐nitro‐N‐nitrosoguanidine in Salmonella typhimurium TA100. Kimchis inhibited the survival or growth of HT‐29 cells in MTT assay and growth inhibition test. Opt‐R kimchi exhibited the strongest antiproliferative effect against the cancer cells. Kimchis also induced apoptosis by DAPI staining assay. Opt‐R kimchi showed the increased ratio of apoptotic bodies among the kimchi samples. The anti‐proliferative effect of kimchi, especially Opt‐R kimchi, was associated with a marked induction of p53 and Cdk inhibitor p21WAF1/CIP. Opt‐R kimchi induced apoptosis in HT‐29 cells was associated with the activation of caspase‐9, decrease of anti‐apoptotic Bcl‐2 as well as Bcl‐XL expression, and increased expression of Bax levels. The present study suggests that optimally ripened kimchi would have increased antimutagenic and antiproliferative potentials during the fermentation.
Effects of low and high carbohydrate meals on postprandial plasma lipid concentrations
Authors: Bergman Sarah , Kumagai Melissa , Smith Julie , Gillingham Melanie , Stadler Diane
Published: June 2021

Background

Chronic postprandial hyperlipidemia is associated with increased risk of cardiovascular disease (CVD). Very low carbohydrate (VLC) diets are a popular choice for weight‐loss, but acute effects of VLC diets on postprandial plasma fatty acids have not been tested.

Methods

Four healthy, normal weight adults participated in a randomized crossover feeding study. Subjects consumed a standard diet [51% carbohydrate (CHO), 14% protein, 35% fat] for 3 days. On day 4 participants consumed either VLC (4%) or high CHO [HC (58%)] meals. Subjects then repeated standard diet and alternate test meals. Pre‐ and postprandial concentrations of plasma triglycerides (TG) and individual fatty acids were analyzed by modified Wahlefeld method and GC/MS, respectively. Differences between diets were compared by paired t‐tests.

Results

TG were significantly higher on the VLC diet compared to the HC diet (Area under the curve for TG: VLC 1217±420 and HC1033±369 mg·h/dL, p=0.03). Saturated fatty acids (SFA) were significantly higher 4 hours after a VLC meal (VLC 19960±1094 vs HC 12193±906 µmol/L, p=0.020). There was no difference in postprandial omega‐6 or omega‐3 fatty acid concentrations between diets.

Conclusions

Plasma‐TG and SFA were higher after the VLC meals when compared to the HC meals. Chronic elevation of postprandial lipids may contribute to the development of CVD. Supported by the Graduate Programs in Human Nutrition, PHS Grant UL1 RR024140, and National Center for Complementary and Alternative Medicine (R21 AT002753‐01).
Unsaturated fatty acids diminish elevated adipocyte fatty acid binding protein (aP2) expression by lipopolysaccharide in macrophages
Authors: Park Young‐Ki , Lee Ji‐Young
Published: June 2021

Expression of aP2 in macrophages and its role in pro‐inflammation have been reported. To evaluate the effect of fatty acids on the expression of aP2 in macrophages, murine RAW 264.7 macrophages were treated with individual fatty acid, including 14:0, 16:0, 16:1, 18:1, 18:2, 18:3, 20:4, 20:5 and 22:6 (100 μM), complexed with BSA (2.5:1 of fatty acid to BSA molar ratio) for 12 hours. After the fatty acid incubation, lipopolysaccharide (LPS, 100 ng/ml) was added to the media for additional 18 hours. Quantitative realtime PCR analysis was performed to measure aP2 mRNA abundance. LPS significantly elevated aP2 mRNA abundance by ~8‐fold. The increased aP2 expression by LPS was abolished by all the unsaturated fatty acids tested whereas saturated fatty acids did not elicit the same response. Western blot analysis also confirmed that the elevated aP2 protein level by LPS was diminished by unsaturated fatty acids. Concomitantly, unsaturated fatty acids reduced the expression of pro‐inflammatory genes, including tumor necrosis factor α, cyclooxygenase‐2, interleukin‐1β and interleukin‐6, in LPS‐activated macrophages. The results suggest that anti‐inflammatory property of unsaturated fatty acids could be mediated, at least in part, by down‐regulating aP2 expression in macrophages.
Weight Gain of Latino Infants at 6 and 12 months of Age: Maternal Depression and other Risk Factors
Authors: Wojcicki Janet Maia , Holbrook Katherine , Lustig Robert H , Caughey Aaron , Munoz Ricardo , Heyman Melvin B
Published: June 2021

Background

Rapid infant weight gain during the first 6 months is associated with future risk of obesity although lower weight at 1 year of age is associated with early adiposity rebound.

Objectives

To evaluate the relationship between maternal depressive symptoms and infant weight and 6 and 12 months of age.

Methods

We recruited Latina mothers prenatally and followed the infants to 12 months of age. Maternal depressive symptoms were assessed prenatally and postnatally. Logistic regression models were used to assess independent predictors for infant overweight at 12 months and excessive weight gain at 6 months of age.

Results

We enrolled 201 mother‐infant pairs. We had a high rate of infant overweight at 12 months (27.6%) and 38.5% experienced rapid infant weight gain at 6 months. In multivariate logistic regression models, independent predictors for infant overweight at 12 months was the absence of maternal depressive symptoms (odds ratio [OR], 0.17; 95% confidence interval [CI], 0.04‐0.78) and macrosomic birthweight (OR= 3.65; 95% CI, 1.03‐12.88). In multivariate logistic regression models, we found that depressive symptoms were associated with decreased risk for rapid weight gain (OR=0.27; 95%CI, 0.09‐0.77) as was increased infant birthweight (OR 0.997; 95%CI, 0.996‐0.998).

Conclusion

Our study found that maternal depressive symptoms were associated with reduced infant weight at 6 and 12 months of age.
Dietary Glutamic Acid Removal Reduced Growth of Weaning Rats in Protein Malnutrition
Authors: Sakai Ryosei , Kawamata Yasuko , Yoneda Junya , Torii Kunio , Chin Keigi
Published: June 2021

Protein nutrition is crucial for intestinal development and whole body growth of early life. Although glutamic acid (Glu) in the body derives generally from synthesis de novo, intestines require dietary input of Glu for energy production, alanine synthesis and so on. This study investigated the effects of Glu removal from foods on growth and intestinal morphology in weaning rats to discuss on essentiality of dietary Glu.

Weaning male SD rats were given any of the following 4 kinds of amino acid (AA) defined diets with normal or low AA (50% reduction) compositions or their Glu‐free compositions for 2‐3 weeks either in ad libitum or controlled feeding.

Linear relation was seen between food intake and body weight gain in rats fed normal AA diet (r= 0.99). The relation in rats fed Glu‐free diets apparently different from that of normal AA; the growth was smaller for their food intakes as was in rats fed low AA diet, indicating growth reduction by Glu removal. This was confirmed by pair feeding study; feeding of the same amount of Glu‐free diet resulted in smaller growth, lower intestinal weight and shorter intestinal villus height. These changes caused by Glu removal were also evident in rats fed low AA diet. These results suggest nutritional importance of dietary Glu in general foods for normal growth including intestinal development of early life both in well‐nourished and protein malnourished states.
Differential expression of tolerance to hypothermia and mechanical analgesia in the guinea pig following chronic opioid versus cannabinoid exposure
Authors: Maguma Hercules T. , Taylor David A.
Published: June 2021

Studies using guinea pig longitudinal muscle‐myenteric plexus (LM/MP) have shown heterologous tolerance after chronic morphine (MOR) exposure and homologous tolerance after WIN55,212‐2 (WIN) exposure. Few studies have compared tolerance that develops to hypothermic and mechanical analgesic activity of opioids and cannabinoids with that of the LM/MP model. Tolerance was induced by MOR (7 day) or WIN (5 day) injection and assessed by challenge doses of WIN and MOR. Analgesia was assessed using paw pressure test and hypothermia by a rectal thermometer. Chronic MOR treatment produced tolerance to hypothermic and analgesic effects of MOR but not hypothermic effect of WIN. WIN treatment elicited tolerance to hypothermic effect of WIN and MOR, and analgesic effect of MOR. No analgesia was observed after WIN challenge, suggesting the paw pressure model may not be sensitive enough or WIN does not produce analgesia in the model. Results show that nature of tolerance in one model cannot be inferred to another even in the same species. Chronic MOR treatment produces heterologous tolerance in an in‐vitro model and homologous in vivo while WIN regimen elicits homologous tolerance in vitro but heterologous in vivo. This affirms the notion that nature of tolerance depends on model, drug, species, and regimen used. Data also suggest that multiple cellular effects may induce differential functional tolerance in different models.
Post‐Session Activation, but not Blockade, of Dopamine D2‐like Receptors Disrupts Instrumental Behavior
Authors: Bertz Jeremiah William , Woods James Henry
Published: June 2021

Rats will reduce their responding for flavored solutions if these solutions are paired with administration of the indirect dopamine receptor (DAR) agonist amphetamine or the nonselective direct DAR agonist apomorphine. The present studies sought to further characterize the effects of pairing DAR activation or blockade with instrumental behavior and its outcomes. Rats were trained in daily sessions to respond for access to unflavored tap water. Immediate post‐session administration of the D3‐preferring agonist quinpirole (QNP) or the D2‐preferring agonist sumanirole (SUM) produced progressive declines in responding across days that were characterized by orderly shifts of responding away from the ends of sessions. When control animals were given QNP or SUM on a quasirandom schedule that included an equal number of pre‐ and post‐session administrations, pre‐session administrations significantly decreased responding, whereas responding was unchanged by post‐session administrations. Thus, the progressive declines observed above result from learned associations between sessions and drug administration, not from drug accumulation. Similar results were obtained with food‐reinforced animals. In contrast to the effects of D2‐like DAR agonists, post‐session treatment with the D2‐like DAR antagonist haloperidol (HAL) did not disrupt water‐reinforced responding. These results are consistent with studies showing HAL fails to produce place‐ or taste‐avoidance, two other effects of associative learning. Supported by USPHS/NIDA grant R01 DA020669 and an NSF Graduate Research Fellowship.
Human Multidrug Resistance Protein 2 (MRP2/ABCC2) transports 3α, 6α, 7α, 12α‐(OH)4‐5β‐cholyl taurine (6α‐OH‐TC)
Authors: Megaraj Vandana , Kakiyama Genta , Iida Takashi , Vore Mary
Published: June 2021

Background and Aim

Genetic defects in the bile salt export pump (ABCB11) gene lead to fatal Type 2 progressive familial intrahepatic cholestasis (PFIC2). However, Abcb11 knockout (KO) mice exhibit only mild cholestasis and secrete large amounts of unusual tetrahydroxyl bile salts into bile (PNAS 98:2011). The formation of hydrophilic bile salts is postulated to detoxify the hydrophobic bile salts and prevent a severe cholestatic phenotype in the KO mice. An unidentified alternative canalicular non‐Abcb11‐mediated transport system is proposed to efflux these tetrahydroxyl bile salts. Furthermore, 6a‐OH‐TC is present in human amniotic fluid, neonatal urine and urine of cholestatic patients. Here we investigate human MRP2 as a possible alternative transporter for 6a‐OH‐TC.

Methods

Tauro‐Δ22‐3a, 6a, 7a, 12a ‐tetrahydroxy‐5 β‐cholan‐24‐oic acid was synthesized, reductively tritiated, and purified by TLC and HPLC (98% purity). Human MRP2 was expressed in Sf9 insect cells and the kinetic parameters (Km, Vmax, Hill coefficient [HC]) of ATP dependant transport of 3H‐6a‐OH‐TC determined in Sf9 plasma membrane vesicles.

Results

Saturation kinetics studies (6a‐OH‐TC 0.1‐1mM) yielded uptake that fit best to the Hill equation with Vmax = 509 pmol/mg/min; Km = 128 µM; Hill coefficient = 1.6.

Conclusion

6α‐OH‐TC is a substrate of MRP2 with evidence of positive cooperativity. (HD 58299)
Synergy between microtubule destabilizing agents and neurokinin 1 receptor antagonists identified by an siRNA synthetic lethal screen
Authors: Kitchens Carolyn A , McDonald Peter R , Pollack Ian F , Wipf Peter , Lazo John S
Published: June 2021

Coordinated microtubule polymerization and depolymerization is essential for numerous biological functions, including mitotic spindle formation, making microtubules a prime target for cancer therapy. Disruption of microtubules with microtubule destabilizing agents (MDAs) induces cell cycle arrest in mitosis and typically leads to the induction of apoptosis. To uncover proteins and signaling pathways involved in MDA cytotoxicity, we developed a short interfering RNA (siRNA) synthetic lethal screen, which identified gene products that sensitized human glioblastoma cells to sub‐lethal concentrations of two MDAs. The neurokinin 1 receptor (NK1R) was selected for further studies due to its expression in various cancer cells, involvement in regulating cell death pathways and the availability of various pharmacological inhibitors. The NK1R antagonist L‐733,060 phenocopied NK1R siRNA and produced a synergistic increase in the cytotoxicity of four different MDAs. This synergistic toxicity was seen in glioblastoma, bladder, cervical and breast cancer cells. Microtubule stabilizing agents (MSAs) and DNA interacting agents failed to induce synergistic toxicity when treated in combination with L‐733,060 suggesting an effect that is specific to MDAs. These results illustrate the potential for synthetic lethal siRNA screens to identify chemosensitivity nodes for MDAs that might be of clinical value.
CD‐NP: A novel aldosterone‐suppressing designer natriuretic peptide in a first‐in‐human study
Authors: Lee Candace Y.W. , Chen Horng H. , Lisy Ondrej , Lieu Hsiao D. , Burnett John C.
Published: June 2021

Background

CD‐NP is a novel chimeric natriuretic peptide (NP) consisting of the full‐length human C‐type natriuretic peptide (CNP), a NP receptor (NPR)‐B agonist, and the C‐terminus of Dendroaspis NP (DNP), a NPR‐A agonist. In the first‐in‐human clinical trial, CD‐NP activated plasma and urinary cGMP, the second messenger for the NPs, and increased Na+ excretion in normal subjects. Here we report for the first time plasma aldosterone (ALDO) and angiotensin II (ANG II) levels.

Methods

In the double‐blind, PLB‐controlled phase of this trial, adults received CD‐NP 17.5 ng/kg/min (n = 6) or PLB (n = 4) i.v. for 4 hours. Plasma ALDO and ANG II were quantified by RIA. Data were analyzed by 2‐tailed paired t‐test and signed‐rank (S‐R) test (SAS v9).

Results

In subjects who received CD‐NP, plasma ALDO (mean±SD) decreased from a baseline of 21.9±2.7 to 9.5±3.2 ng/dL (P<0.001 t‐test, P<0.05 S‐R test) at the end of infusion. Plasma ANG II tended to decrease (29.2±5.2 to 23.0±3.5 ng/L, P=0.086 t‐test, P=0.69 S‐R test). In the PLB group, ALDO (20.3±4.2 to 13.6±4.5 ng/dL) and ANG II (23.3±2.2 to 19.8±8.2 ng/L) did not change.

Conclusion

CD‐NP suppressed plasma ALDO in normal subjects, consistent with NPR‐A activation by a novel NP with dual NPR‐A and NPR‐B activating properties. CD‐NP may be of therapeutic potential in disease states that are characterized by ALDO activation, such as in heart failure warranting further studies.

Sources of funding: Mayo Foundation and Nile Therapeutics, Inc.
A novel small molecule inhibitor of Id proteins (AGX‐51) blocks cell survival in vitro and diminishes angiogenesis and tumor growth in vivo
Authors: Chaudhary Jaideep , Garland William , Salvador Richard
Published: June 2021

The inhibitors of differentiation (Id) proteins (Id1, Id2, Id3, and Id4) are negative regulators of differentiation that act by sequestering basic helix loop helix (bHLH, e.g. E47) transcription factors. Ids are highly expressed during embryonic development but in adult tissues their expression is rare to absent. However, Id proteins are required for tumor angiogenesis and are also highly expressed in many cancer cells. As a proof of concept, decreased angiogenesis and tumor load as a result of blocking Id expression by antisense and siRNA molecules demonstrated that Id is a compelling anti‐cancer target. Based on a combination X‐ray crystal structure of Id1‐E47 interaction, in silico virtual screening and a high throughput electrophoretic mobility shift assay (EMSA), we discovered AGX‐51, a small molecule inhibitor of Id protein activity. In vivo and in vitro AGX‐51 disrupted Id1 and E47 interaction, blocked proliferation, induced apoptosis in cancer cell lines and significantly decreased neo‐vascularization in an in vivo matrigel plug assay. When used with Taxol, AGX‐51 also significantly decreased tumor growth by almost 50% compared to Taxol alone in nude mice implanted with MDA‐231 human breast cancer cells. In summary, a specific inhibitor of Id‐E47 interaction that has anti‐tumor activity is described for the first time. This research was supported by AngioGenex, Inc.
Role of Protein Kinase C and Phosphoinositide 3‐Kinase‐Akt in Substance P‐Induced Proinflammatory Pathways in Mouse Macrophages
Authors: Bhatia Madhav , Sun Jia
Published: June 2021

Neuropeptide modulation of immune cell function is an important mechanism of neuro‐immune inter‐system cross‐talk. Substance P (SP) is one such key neuropeptide involved. In this study, we investigated the yet unexplored cellular mechanisms of SP‐mediated inflammatory responses in macrophages using a mouse macrophage‐like cell line RAW 264.7 and isolated peritoneal macrophages. We found that the conventional PKCα and novel PKCδ and εwere selectively activated by SP via its primary neurokinin‐1 receptor (NK‐1R) on the cells. Activation of these PKC isoforms mediated the activation of downstream extracellular signal‐regulated kinase‐1/2 (ERK1/2) and the transcription factor NF‐kappaB (NF‐κB) which drove the transcription of inducible chemokines in macrophages. Additionally, phosphoinositide 3‐kinase (PI3K)‐Akt was also activated by SP/NK‐1R in macrophages. Inhibition of PI3K‐Akt pathway attenuated ERK1/2 and NF‐κB activation suggesting it also played a part in SP‐induced cellular inflammatory response. Kinetic analysis indicated that PKC isoforms induced early ERK1/2 activation while PI3K‐Akt contributed to the pathway at later time points. It was further demonstrated that PKC and PI3K‐Akt were activated independent of each other. Collectively, our results suggest that SP/NK‐1R activates two convergent proinflammatory signaling pathways, PKCs and PI3K‐Akt, resulting in ERK1/2, NF‐κB activation and chemokine production in mouse macrophages.
Endothelin‐1 (ET‐1) has Different Effects on Hypoxia‐inducible Factor 1 (HIF‐1) Expression in Aortic and Pulmonary Arterial Smooth Muscle Cells (PASMCs).
Authors: Shimoda Larissa A , Undem Clark , Sylvester J T , Pisarcik Sarah
Published: June 2021

Numerous cellular responses to hypoxia are mediated by the transcription factor, HIF‐1. We recently demonstrated that ET‐1 amplifies HIF‐1 expression and activity in PASMCs. In this study, we examined whether ET‐1 also induces HIF‐1 expression in systemic vascular smooth muscle. Primary cultures of rat PASMCs or aortic smooth muscle cells (ASMCs) were treated with ET‐1 (10−8; 48hr). Protein levels of the oxygen‐sensitive α subunit of HIF‐1 (HIF‐1α) were markedly increased in nuclear extracts from PASMCs, but not ASMCs, treated with ET‐1 under non‐hypoxic conditions. Real‐time PCR revealed that ET‐1 increased Hif1a mRNA expression in PASMCs, but not ASMCs. Moreover, ET‐1 decreased mRNA and protein expression of prolyl hydroxylase 2 (PHD2), the protein responsible for targeting HIF‐1α for degradation, in PASMCs but not ASMCs. From these data, we conclude that ET‐1 induces HIF‐1α by upregulation of HIF‐1α synthesis and downregulation of PHD2‐mediated degradation in PASMCs. Furthermore, the induction of HIF‐1α by ET‐1 appears to be cell type specific, as it was not observed in ASMCs. Since ET‐1 levels increase during hypoxia, and we have shown previously that ET‐1 is required for upregulation of HIF‐1 in PASMCs during moderate hypoxia, our data suggest that the differential effect of ET‐1 on HIF‐1 may underlie the divergent responses to hypoxia observed in pulmonary and systemic vascular smooth muscle.
O‐GlcNAcylation inhibits NFκB activation in rat aortic smooth muscle cells through proteasome inhibition
Authors: Wang Kai , Xing Dongqi , Ho Shiuh‐Rong , Zhang Yun , Chen Yiu‐Fai , Oparil Suzanne , Paterson Andrew
Published: June 2021

O‐GlcNAcylation is a dynamic protein posttranslational modification that adds the monosaccharide N‐acetylglucosamine (GlcNAc) to specific serine or threonine residues on target proteins. We have found that O‐GlcNAc modification is an endogenous inhibitor of the proteasome, the primary cellular complex to degrade proteins. A crucial element of the vascular injury response is the activation of NFκB dependent inflammatory genes. NFκB interacts with IκBα in the cytosol and is activated by IκBα degradation, allowing NFκB to translocate into the nucleus to induce gene expression. In this study, we tested if O‐GlcNAc can inhibit NFκB mediated inflammation by blocking IκBα degradation through proteasome inhibition. Rat aortic smooth muscle cells (RASMCs) were treated with 5 mM glucosamine (GlcN) for 1, 2, 4, 6, 12 hrs. At 12 hrs of GlcN treatment, O‐GlcNAc was increased 2‐fold and IκBα level by 21% compared to vehicle control. GlcN pretreatment significantly inhibited TNF‐α induced degradation of IκBα. RASMCs were pretreated with 5 mM GlcN and then incubated with TNF‐α (10 ng/ml) for an additional 60 min, resulting in a 60% increase in IκBα levels after 12 hrs of GlcN treatment. A 32% decrease in proteasome activity was detected after 12 hrs GlcN treatment (Figure). This study indicated that the O‐GlcNAc might inhibit TNF‐α induced inflammatory response in RASMCs by blocking NFκB activation through proteasome inhibition.
Diesel Exhaust Particle (DEP)‐Induced Acute Loss of Respiratory Neutral Endopeptidase
Authors: Wong Simon S. , Sun Nina N , Miller Hugh B , Desmarais Thomas J , Witten Mark L , Burgess Jefferey L
Published: June 2021

Neutral endopeptidase (NEP) is a key cell surface peptidase in the maintenance of airway homeostasis involving the development of pulmonary disorders. However, little information is available about the effect of respiratory particles on airway NEP. Cross‐shift spirometry and sputum NEP were measured on twelve normal subjects following acute diesel exhaust exposure. A significant increase in soluble NEP in sputum was observed with 31% average net increases when compared with pre‐exposure. Pearson's correlation analyses indicated that changes in sputum NEP activity were significantly associated with diesel exhaust particle (DEP) exposure. DEP exposure also induced a significant decline in forced expiratory volume in one second (FEV1, 4.03 ± 0.29 vs. 3.78 ± 0.29; p = 0.005 pre‐ vs. post exposure) and forced vital capacity (FVC, 5.16 ± 0.36 vs. 4.86 ± 0.39, p = 0.017), which were significantly associated with sputum NEP activity, not with DEP exposure. The changes in NEP activity may be an accurate endpoint for DEP exposure, be mechanistically linked to lung function, and indicate a greater susceptibility to the risk of pulmonary diseases.
Effect of aging on arm and leg muscle sympathetic nerve activity in humans
Authors: Monahan Kevin D. , Sauder Charity L. , Ray Chester A.
Published: June 2021

Aging is associated with heterogeneous changes in sympathetic nervous system outflow to various organs. Previous studies have consistently reported pronounced increases in leg muscle sympathetic activity (MSNA) at rest with age. We tested the hypothesis that aging is associated with similar increases in arm and leg MSNA at rest. Moreover, we tested the hypothesis that baroreflex unloading elicits similar increases in arm and leg MSNA in older adults. To address these hypotheses we simultaneously measured arm and leg MSNA using microneurography in young (N=7; 23±1 years old; mean±SE) and older adults (n=5: 63±2) at rest and in response to lower body negative pressure (LBNP of ‐10, ‐20, and ‐40 mmHg) (older subjects only). MSNA at rest increased similarly with age in the arm (8±2 vs. 34±6 bursts/min for young and older, respectively; P<0.05) and leg (9±2 vs. 35±6 bursts/min; P<0.05). Moreover, LBNP elicited similar increases in leg (Δ4±2, Δ8±4, and Δ16±6 bursts/min, change from baseline for ‐10, ‐20, and ‐40 mmHg LBNP, respectively; main effect P<0.05) as well as arm MSNA (Δ4±2, Δ2±2, and Δ15±6; main effect P<0.05) in older adults. Collectively, these data indicate that aging is associated with similar increases in arm and leg MSNA at rest. Moreover, parallel increases in leg and arm MSNA occur during baroreflex unloading in older humans.

Support: NIH AG24420, HL77670, M01 RR10732
Effects of oral contraceptives on sympathetic baroreflex sensitivity during Valsalva's maneuver
Authors: Lawrence Johnathan E , Klein Jenna C , Carter Jason R
Published: June 2021

A recent study reported increased cardiovagal baroreflex sensitivity (BRS) in women taking oral contraceptives (OC) compared to naturally eumenorrheic women (EW) during low hormone phases of the menstrual cycle (OC placebo vs. early follicular), but sympathetic BRS was not reported. We hypothesized an increased sympathetic BRS in OC compared to EW. Muscle sympathetic nerve activity (MSNA), arterial pressure (systolic: SAP, diastolic: DAP), and heart rate were recorded during three Valsalva maneuvers in 23 healthy females (12 EW, 11 OC) during the low hormone phase of the menstrual cycle. Resting MSNA, arterial pressure, and heart rate were not different between groups. The phase II SAP decrease was exaggerated in the OC users (OC: 32 ± 4 mmHG vs. EW: 20 ± 2 mmHG; p < 0.05), but phase II MSNA and sympathetic BRS (%Total MSNA/ΔDAP) did not differ between groups. The rise in SAP (OC: 35 ± 5 mmHG vs. EW: 18 ± 2 mmHG; p < 0.05) and DAP (OC: 18 ± 3 mmHG vs. EW: 10 ± 1 mmHG; p < 0.05) during phase IV were also augmented in the OC group, but the cardiovagal BRS (slope of R‐R interval and SAP) was not different between OC and EW. In conclusion, our results indicate cardiovagal and sympathetic BRS in women taking oral contraceptives are similar to naturally menstruating women during the low hormone phase of the menstrual cycle. However, oral contraceptives appear to exaggerate arterial pressure changes during Valsalva's maneuver. Supported by NIH (HL‐088689).
Hypoxia Inducible Factor‐1 Improves the Actions of Positive Inotropic Agents in Stunned Cardiac Myocytes
Authors: Tan Tao , Luciano Jason A , Scholz Peter M , Weiss Harvey R
Published: June 2021

We tested hypothesis that upregulation of hypoxia inducible factor‐1 (HIF‐1) would improve the actions of positive inotropic agents in cardiac myocytes after simulated ischemia‐reperfusion. HIF‐1α was upregulated with deferoxamine (150 mg/kg/day for 2 days). Rabbit cardiac myocytes were subjected to simulated ischemia (15 min, 95% N2‐5% CO2) and reperfusion (reoxygenation) and compared to control myocytes. Cell contraction and calcium transients were measured at baseline and after forskolin (10−7, 10−6M) or ouabain (10−5, 10−4M). Under control conditions, high dose forskolin and ouabain increased percent shortening by +20% and +18%. Deferoxamine‐treated control myocytes responded similarly. In stunned myocytes, forskolin (+8%) and ouabain (+9%) did not significant increase shortening. Deferoxamine restored the effects of forskolin (+26%) and ouabain (+28%) in stunning. The results for maximum shortening and relaxation rates were similar. The increased calcium transients caused by forskolin and ouabain were depressed in stunned myocytes, but were maintained after HIF‐1 upregulation. These results suggested that simulated ischemia‐reperfusion impaired the functional and calcium transient effects of positive inotropic agents. Upregulation of HIF‐1 protected cardiac myocyte function after ischemia‐reperfusion by maintaining calcium release.
NBCe1 (SLC4A4) functional dimer assembly
Authors: Chang MinHwang , Holmes Heather L. , Ranatunga Wasantha K. , Chen Anping , Romero Michael F.
Published: June 2021

Mutations in the electrogenic Na+/HCO3− cotransporter (NBCe1) result in proximal renal tubular acidosis, glaucoma and cataracts in patients and are only recessive. Parents and siblings of these affected individuals are asymptomatic though their tissues should make some of the mutant NBCe1 protein. Biochemical studies with AE1 and NBCe1 indicate that both, and probably all, Slc4 members form dimers. However, the physiologic implications of dimerization have not yet been fully explored. For our studies we chose two severe NBCe1 mutations (<10% WT function individually): S427L (naturally occurring) and E91R (for N‐terminal structure studies). When we co‐expressed S427L and E91R, we measured ~55% wild‐type function which can only occur if the S427L‐E91R heterodimer is the functional unit. To further investigate this functional dimerization, we have engineered concatemeric hkNBCe1 constructs. When the hkNBCe1 tandem‐dimer (WT‐WT) cRNA was expressed in Xenopus oocytes, the electrophysiology data demonstrate the WT‐WT homodimer functions as a WT hkNBCe1. The electrophysiology data also showed that the WT‐S427L or WT‐E91R heterodimers functioned very similarly to WT hkNBCe1. Intriguingly, we observed ~50% WT function in both the S427L‐E91R and E91R‐S427L heterodimers constructs. These data illuminate why the "NBCe1 phenotype" only occurs recessively. An eYFP fragment (1‐158, YFPN) was fused to the N‐ or C‐terminus of hkNBCe1 and another eYFP fragment (159‐238, YFPC) was fused to N‐ or C‐terminus of hkNBCe1. The electrophysiology data demonstrate the eYFP‐hkNBCe1 fusion proteins have WT hkNBCe1 like function. The hkNBCe1 dimerization can also be visualized by biomolecular fluorescence complementation (BiFC) method. We plan to further use these molecular tools to probe the nature of the hkNBCe1 dimer interactions. Supported by EY017732.
SOD1‐deficiency causes salt‐sensitivity and aggravates hypertension in hydronephrosis
Authors: Carlström Mattias , Brown Russell D. , Sällström Johan , Larsson Erik , Zilmer Mihkel , Zabihi Sheller , Eriksson Ulf J. , Persson Erik G.
Published: June 2021

We investigated the role of oxidative stress for salt‐sensitivity and for hypertension in hydronephrosis (HN).

HN was induced in SOD1‐transgenic (SOD1‐tg), SOD1‐deficient (SOD1‐ko) and wild‐type mice. Telemetric blood pressure measurements were performed during normal and high sodium conditions. Excretion of F2‐IsoP and histology were investigated and the effects of Tempol on blood pressure and TGF were studied.

HN wild‐type mice developed salt‐sensitive hypertension (114±1 to 120±2 mmHg) which was augmented in SOD1‐ko mice (125±3 to 135±4 mmHg), but abolished in SOD1‐tr mice (109±3 to 108±3 mmHg). SOD1‐ko controls displayed salt‐sensitive blood pressure (108±1 to 115±2 mmHg), which was not found in wild‐type or SOD1‐tg mice. HN wild‐type and SOD1‐ko mice exhibited increased urine flows, increased urinary excretion of F2‐IsoP, and impaired urine concentration ability. An increased excretion of F2‐IsoP was also found in SOD1‐ko controls. The renal histopathological changes found in HN wild‐types were augmented in SOD1‐ko and diminished in SOD‐tg mice. Tempol attenuated blood pressure and normalized the abnormal TGF in HN.

Oxidative stress due to SOD1‐deficiency is associated with salt‐sensitivity and plays an important role for the development of hypertension in HN. Increased superoxide formation can enhance the TGF response and thereby be involved in the development of hypertension.
Protection of podocytes from hyperhomocysteinemia‐induced injury in the kidneys of mice lacking gp91phox gene
Authors: Zhang Chun , Hu Jun‐Jun , Xia Min , Brimson Christopher , Li Ning‐Jun , Li Pin‐Lan
Published: June 2021

NADPH oxidase (NOX) has been reported to contribute to glomerulosclerosis induced by hyperhomocysteinemia (hHcys). In the present study, we used a mouse model lacking gp91phox (gp91−/−), a membrane subunit of NOX, to test whether the pathogenic role of NOX is associated with podocytes injury induced by hHcys. gp91−/− and wild‐type mice (gp91+/+) were fed on folate‐free (FF) or normal chow for 6 weeks. HPLC analysis showed that both strains of mice had higher plasma homocysteine levels on FF diet compared to the mice on normal chow (9.86 ± 1.06 vs. 4.98 ± 0.52 μM in gp91−/− mice and 10.53± 1.27 vs. 5.05 ± 0.68 µM in gp91+/+ mice). ESR analysis showed that hHcys failed to markedly increase NOX‐dependent superoxide production in kidneys from gp91−/− mice compared with gp91+/+ mice. Glomerular sclerosis index was markedly lower in gp91−/− than in gp91+/+ mice on FF diet. RT‐PCR showed that hHcys significantly decreased nephrin mRNA expression in gp91+/+ mice but not in gp91−/− mice. Correspondingly, desmin mRNA expression was less elevated in gp91−/− mice on FF diet. Immunohistochemistry demonstrated decreased nephrin and increased desmin staining in gp91+/+ mice on FF diet, rather than in gp91−/− mice. Our results suggest that knock‐out of gp91phox leads to a substantial reduction of local oxidative stress in glomeruli, which protects podocytes from hHcys‐induced injury (supported by NIH grants DK54927 and HL075316).
Endogenous nitric oxide minimizes vasoconstriction during low frequency renal sympathetic nerve stimulation
Authors: Kretzer Julie , Collett Jason , O'Brien Michelle , Osborn Jeffrey
Published: June 2021

Low frequency renal nerve stimulation (LFRNS) decreases medullary (MBF) and cortical (CBF) blood flows. The impact of endogenous nitric oxide (NO) on LFRNS mediated changes in MBF and CBF was determined in anesthetized Sprague Dawley (SD) rats. Animals (n=13) were anesthetized and Laser Doppler flow probes were positioned on or below the kidney surface to measure CBF and MBF. After control, LFRNS was conducted at 0.5 and 1.5 Hz for 15 minutes each. LFRNS (0.5 Hz) decreased MBF by ‐1.89 ± 0.39% and CBF by ‐2.12 ± 0.77% of control. At 1.5 Hz, LFRNS decreased MBF and CBF by ‐8.70+2.31% and ‐10.84 ± 3.7% of control, respectively. Following a 30 minute recovery period from LFRNS, L‐arginine (L‐ARG; 300 ug/kg/hr, i.v.) was infused for 30 minutes and LFRNS was repeated. L‐ARG infusion did not change control MBF or CBF but abolished renal vasoconstriction elicited by 0.5 Hz LFRNS. Furthermore, 1.5 Hz LFRNS mediated vasoconstriction was attenuated in both medullary and cortical regions (‐3.75 ± 1.78% and ‐6.87 ± 2.67%, respectively; p<0.05 vs. control constrictor responses). Thus, endogenous NO significantly decreases renal vascular constriction during LFRNS in renal medullary and cortical vessels. NO may provide protection against both cortical and medullary vasoconstriction during small and subtle changes in renal sympathetic outflow. (Supported by an APS Predoctoral Fellowship to J. Kretzer and UK Biology Ribble Foundation).
Anti‐inflammatory treatment improves renal autoregulation in DOCA‐salt hypertensive rats
Authors: Guan Zhengrong , Cook Anthony K , Inscho Edward W
Published: June 2021

The role of inflammation in afferent arteriole (AA) function in DOCA‐salt hypertension was studied using the blood‐perfused juxtamedullary nephron technique. DOCA rats receiving the anti‐inflammatory agent, pentosan polysulfate (PPS) for 3 weeks developed hypertension similarly to DOCA alone (181±5 vs. 180±7mmHg) and basal AA diameters were also similar (15.2±0.9 vs. 16.2±0.4μm, n=9‐11). AA autoregulation was normal in sham rats (uninephrectomy). Step (15mmHg) increases in perfusion pressure from 65‐170mmHg caused pressure‐dependent AA constriction to 68±3% of control at 170mmHg. Pressure‐mediated AA responses were markedly attenuated in DOCA rats. Diameter remained between 90 and 101% of control over the range of PP studied, indicating a loss of autoregulation. PPS partially restored autoregulation in DOCA rats. Diameters were 110±2 and 76±3% of control at 65 and 170mmHg, respectively. AA responses to a P2X1 receptor agonist, ?,γ ‐methylene ATP (0.01 to 100μM) were assessed in separate groups. PPS improved the attenuated AA constriction to ?,γ ‐methylene ATP in DOCA rats. The maximal vasoconstriction was 81±6% of control compared to 70±2% in sham and 90±1% in DOCA (p<0.05). In conclusion, inflammatory processes contribute to AA dysfunction in DOCA‐salt hypertension by impairing autoregulation and AA reactivity to P2X1 receptor activation.
Arterial segmentation of renal parenchyma with single and multiple renal arteries. A comparative study on corrosion casts
Authors: Zahoi Delia Elena
Published: June 2021

Out of 200 human renal corrosion casts, 158 presented a single renal artery and 42 ‐ multiple renal arteries. Of the latter, 38 casts had 2 renal arteries while 4 casts had 3 renal arteries.

The casts involved the injection of plastic (based on nitrocellulose) in the vascular and ductal elements and the corrosion of the renal parenchyma with hydrochloric acid.

For the casts with a single renal artery, the study describes 37 morphological types of segmentation. The number of the segments varied between 4 and 6 as follows: 6 casts (3.8%) showed 4 renal segments; 90 casts (57%) showed 5 renal segments; 50 casts (31.64%) showed 6 renal segments; 9 casts (5.7%) showed 7 renal segments; 3 casts (1.9%) showed 8 renal segments.

The casts with multiple renal arteries showed 5 to 8 segments of 24 morphological types. Most of the studied casts (24 cases / 57.14%) showed 5 renal segments; 14 casts (33.33%) showed 6 renal segments; 3 casts (7.14%) showed 7 renal segments; only 1 case (2.38%) showed 8 renal segments.

The analysis of the casts revealed that in the case of multiple renal arteries, the smallest number of segments was higher than in that of single renal artery casts. Both single and multiple renal artery casts showed 5 segments frequently and in almost the same percentage, which concurs with the Anatomical Terminology. In multiple renal arteries casts, the number of segments (6‐8) was higher than in that of single renal artery casts.