bluuebunny/crossref_metadata_2025 huggingface dataset:
Trends in metabolic syndrome in the US adolescents ‐ NHANES 1999‐2004
Authors: Kafai Mohammad R , Ganji Vijay
Published: June 2021

We investigated the trends in indicators and prevalence of metabolic syndrome in the US adolescents (12–19 y) utilizing the data from NHANES 1999–2004 (n = 2481). Non‐Hispanic white (NHW) compared to non‐Hispanic black (NHB) adolescents had significantly lower mean HDL‐cholesterol (49 vs. 54 mg/dL), lower systolic blood pressure (109 vs. 111 mm Hg), and higher triglycerides (96 vs. 72 md/dL). The prevalences (%) of metabolic syndrome in NHW were 6.3, 4.9, and 5.7 for 1999–2000, 2001–2002, and 2003–2004, respectively; in NHB were 3.5, 3.1, and 1.3 for 1999–2000, 2001–2002, and 2003–2004, respectively; and in Mexican American/Hispanics were 7.2, 7.7, and 6.3 for 1999–2000, 2001–2002, and 2003–2004, respectively. The prevalence of metabolic syndrome was higher in boys compared to girls in 1999–2000 (6.5 vs. 3.8), 2001–2002 (7.4 vs. 2.4), and 2003–2004 (7.3 vs. 3.0). The odds ratio of having metabolic syndrome in adolescent girls is significantly lower compared to boys (OR: 0.3), lower in those who had high cardiovascular fitness level compared to those who did not have high fitness level (OR: 0.39), higher in those who had higher serum insulin than those who had lower serum insulin (OR: 7.0), and higher in those who had high serum c‐reactive protein (CRP) than those who had lower CRP (OR: 5.7). Prevalence of metabolic syndrome has shown a decline in NHB adolescents from 1999–2000 to 2003–2004.
Food insecurity is inversely associated with perceived diet quality and vegetable intake among impoverished women from rural Appalachian Ohio
Authors: Hazen Crystal L , Holben David H , Holcomb John P , Struble Celia J
Published: June 2021

The effect of produce delivery on food security (FS) and produce intake/behaviors of food stamp‐eligible, rural Appalachian OH women with at least 1 child <18 years were examined using a pre/post pilot study. Intervention group [n=30, 90.0% retention rate (RR)] received $60 of home‐delivered produce for 4weeks, and control group (n=31, 93.9% RR) received $60 food/fuel gift cards. Groups (35+/‐9 years, 4+/‐2 household members, $913+/‐728 monthly income) did not differ (t‐test, P>0.05) at baseline. After 4 weeks, both household (t‐test, p=0.028) and adult (t‐test, p‐0.020) FS decreased among produce group. Child FS and produce intake/behaviors (perceived benefit of, barriers to, and control of produce access; amount of produce eaten; readiness to change produce intake) did not change (p>0.05). At completion (n=61), adult FS was associated with both greater vegetable intake (r=0.253, p=0.049) and perceived diet quality (rho=0.276, p=0.032). The short duration of the study, small sample size, and lack of nutrition education may have contributed to both the decrease in FS and poor diet. Future studies should incorporate ongoing nutrition education during a study of a longer time period.

Grant Funding Source: Ohio University Research Challenge Award, Ohio University Diabetes Research Initiative
Effects of dietary chromium (III) picolinate on growth performance, respiration rate, plasma variables, and carcass traits of pigs fed high‐fat diets
Authors: Kim Beob G. , Lindemann Melin D. , Cromwell Gary L.
Published: June 2021

Effects of dietary chromium (III) picolinate (CrPic) on leanness and fat deposition of pigs have been inconsistent. We investigated the effects of supplemental CrPic on pigs fed high‐fat diets (HFD). Crossbred pigs (n = 32; 9.6 ± 1.6 kg BW) were allotted to 4 treatments with 4 blocks and 2 pigs/pen. Treatments included: 1) low‐fat diet (fat < 3.5%; LFD) with no CrPic, 2) HFD (fat > 30%) with no CrPic, 3) HFD with 1,000 ppb Cr as CrPic, and 4) HFD with 2,000 ppb Cr as CrPic. During d 0 to 56, pigs fed HFD gained faster, consumed less feed, and had lower feed:gain (P < 0.05). CrPic resulted in a quadratic increase (P < 0.05) in feed:gain of pigs fed the HFD. Pigs fed HFD had higher respiration rates than pigs fed LFD on d 41 (P < 0.01) and d 55 (P = 0.06) with no effect of CrPic. Plasma insulin on d 14 linearly decreased with CrPic (P = 0.05). Consumption of HFD resulted in greater carcass weight, perirenal fat, and backfat measures (P < 0.01) and in lower carcass fat‐free lean percentage (P < 0.01) compared with the LFD group. CrPic resulted in linear reductions of carcass weight (P = 0.08) and average carcass backfat (P < 0.05). The effects of CrPic on carcass fat measures were more pronounced in barrows than in gilts. These results indicate that CrPic attenuates some effects of HFD, mainly body fat accretion of pigs, and especially in barrows.
Induction of Snail family transcription factors on ultraviolet‐irradiated keratinocytes
Authors: Choi Changsun , Lee Bog‐Hieu
Published: June 2021

Zinc finger transcription factors of the Snail family play critical roles in embryonic development, in cancer progression and metastasis, and in normal physiologic processes. During development, expression of the Snail family members Slug and Snail is largely driven by growth factor binding to receptor tyrosine kinases and subsequent stimulation of downstream signaling cascades. Our initial studies showed that ultraviolet radiation (UVR) induced transient expression of Slug and Snail in cultured keratinocytes. Expression of Slug and Snail mRNA was stimulated by UVR in a dose‐dependent manner at physiological doses. Because growth factors have been shown to induce Snail family members through both MAPK and phosphatidylinositol 3‐kinase (PI3‐K) signaling pathways, we tested the contribution of these pathways to UVR induction of Slug and Snail in keratinocytes. In addition, because UVR is an environmental stressor, we also examined the role of the JNK stress response pathway in Snail family induction.

Studies were performed using the 12F SCC human keratinocyte cell line, which was derived from a squamous cell carcinoma. Cells were allowed to reach confluence and serum‐starved for 48 hours. Two hours before UVR exposure, cells were pretreated with pharmacologic inhibitors (10 μM) of the MAPK pathway members MEK and p38, with an inhibitor of JNK activation, or with an inhibitor of the PI3‐K kinase pathway. Cells were exposed in PBS to 300 J/m2 UVR and harvested at 2 hours post‐exposure. Standard techniques were employed for RNA isolation, cDNA synthesis, and quantitative RT‐PCR. Levels of Slug and Snail mRNA were standardized to GAPDH levels and indexed to values in cells not exposed to UVR. Expression of Slug and Snail was significantly blocked by MAPK and PI‐3K pathways inhibitors. Inhibition of JNK activation had no effect on Slug induction.
Dietary plant sterols regulate genes involved in cholesterol metabolism in mouse liver but not intestine
Authors: Jesch Elliot D , Lee Ji‐Young , Carr Timothy P
Published: June 2021

Dietary plant sterols reduce intestinal cholesterol absorption, but their role in the modification of genes involved in cholesterol transport and metabolism is uncertain. Several intestinal genes and transcription factors are candidates for regulation by plant sterols. We tested the hypothesis that dietary plant sterols regulate gene expression of putative sterol transporters in the small intestine. Male C57BL/6 mice were fed modified versions of the AIN‐93M purified rodent diet with either stigmasterol or sitosterol at 3.94 g/kg diet to reflect the 2 g/day recommendation for humans, based on a 2000 kcal diet. Total RNA was extracted from the duodenum and liver of each mouse and NPC1L1, SRBI, SREBP‐2, HMG CoA Reductase, PPARδ, ABCA1, and ABCG5 mRNA was quantified by real time PCR. Fecal neutral sterol and bile acid output, gallbladder bile, plasma and liver lipids were also quantified. While plant sterols had no effect on plasma lipids or gene expression in the intestine, liver lipids and gene expression of SREBP‐2, HMG CoA Reductase, and ABCA1 in the liver of mice fed plant sterols were significantly reduced compared to controls. Furthermore, sitosterol—and, to a lesser extent, stigmasterol—significantly increase fecal neutral sterol excretion. These data indicate that not only do individual plant sterols increase neutral sterol excretion, but they also play a role in regulating the expression of cholesterol homeostasis genes in the liver. [Supported by USDA‐NRI Competitive Grant No. 2007‐35200‐18298]
Anaplastic Angiocentric Glioma: A Report of Two Cases
Authors: Donev Kliment , Scheithauer Bernd , Ketterling Rhett
Published: June 2021

Introduction: Angiocentric gliomas (ACG) are low grade, epileptogenic tumors with a good prognosis. Only one case of ACG with anaplastic features has been described. We report two examples.

Methods: Routinely processed tissue used for histology, immunochemistry and FISH analysis.

Results: Case1: 38‐man Two years chronic, worsening seizures. Resection frontal lobe primary (1995) and recurrence (2007).

Case 2: 79 woman. Memory deficit and expressive dysphasia. Enhancing temporal lobe mass.

Conclusions: The spectrum of ACG includes ependymal and astrocytic features. High grade transformation is uncommon and evidenced by histology, MIB‐1 elevation, strong p53 reactivity and a complex karyotype. The behavior of anaplstic ACG remains to be determined.
Coccidioides spp. presenting as hyphal form in a basilar artery mycotic aneurysm
Authors: Eschbacher Jennifer Mary , Ganatra Vipul , Gutierrez Felipe , Gonzalez Fernando , Coons Stephen William
Published: June 2021

Coccidioides ssp. is a parasitic dimorphic fungus predominantly found in the western and southwestern regions of the United States. The spherule form of this opportunistic organism is responsible for a host of tissue infections in humans, most commonly pneumonia. CNS involvement by coccidioides is an uncommon but well recognized complication, typically manifesting as basilar meningitis. The hyphal form, which existsts naturally in soil, is rarely diagnosed in human infections. Most case reports note infection with the hyphal form only in oxygenated tissues such as lung or osteomyelitic sinus tracts. There are only very rare reports of the hyphal form in the CNS and always in association with aerated surgical devices. We report a coccidioides meningitis and mycotic anyseursm of the basilar artery in a 24‐year‐old pregnant female with a history of HIV. Histologic examination of GMS‐stained sections showed numerous branching hyphae, arthrospores and occasional spherule forms, admixed with amultinucleated giant cells and acute inflammation. Microscopic granulomas were also seen in the subarachnoid space distributed in a military pattern. We believe this to be one of the first reports of a coccidioides ssp. infection with prevalent hyphae forms in the nonoxygenated CNS.
Regulatory T cells alleviate allergic airway hyper‐responsiveness by modulating angiogenesis of airway remodelling via Notch signaling pathway
Authors: Huang Miao‐Tzu , Kang Chi , Chou Yu‐Bin , Wang Chi‐Chiang , Chen Pei‐Jer , Chiang Bor‐Luen
Published: June 2021

Chronic inflammation and airway remodelling are two key features of asthma. Whilst CD4+CD25+ Treg cells have been shown to prevent allergen induced airway inflammation, the role of Treg cells in asthma with established airway remodelling has not been investigated. To address this issue, we induced airway remodelling in OVA‐sensitized mice by chronic aerosol OVA exposure, followed by adoptive transfers of OVA peptide‐specific Treg cells. Without further allergen challenges, residual leukocyte infiltrates in the lungs can be found regardless of the cell types transferred. Airway hyper‐responsiveness (AHR) and pulmonary vasculature were significantly reduced after Treg transfers (P<0.001), suggesting a relationship between pulmonary vasculature and AHR and an anti‐angiogenic effect of Treg cells in airway remodelling. A direct anti‐angiogenic function of Treg cells was validated by both in vitro and in vivo angiogenesis assays which showed a significant reduction of vessel formation at the presence of Treg cells (P<0.001). Notch signaling pathway is known to regulate both peripheral T cell differentiation and angiogenesis. We found that the anti‐angiogenic effect of Treg cells was abrogated by pre‐incubation with recombinant Notch‐Fc proteins. This study demonstrates a novel function of Treg cells in chronic airway inflammation where Treg inhibits angiogenic remodelling via Notch signaling pathway.

The project was supported by grants from the Taiwan National Science Council and National Taiwan University Hospital.
Functional Imaging of Cannabinoid‐Mediated G‐Protein Activity in the 3D Reconstructed Mouse Brain
Authors: Nguyen Peter , Selley Dana , Sim‐Selley Laura
Published: June 2021

Receptor‐mediated G‐protein activity has been examined in select regions that mediate the effects of cannabinoids (CBs). However, the relationship between ligand type and region specific CB receptor function has not been systematically addressed using a novel 3D unbiased image analysis approach. We first mapped the anatomical profile of the relative efficacy of the CB agonists WIN 55,212‐2 (WIN), CP‐55,940 (CP), methanandamide (M‐AEA) using agonist‐stimulated [35S]GTPγS autoradiography, and data were normalized to %WIN stimulation. M‐AEA and CP were less efficacious than WIN in most regions, however both were equally or more efficacious than WIN in the thalamus and subset of cortex. We then used a voxel‐based approach, Statistical Parametric Mapping, to functionally map WIN‐ v.s. M‐AEA‐stimulated [35S]GTPγS binding in the 3D reconstructed mouse brain. WIN activity was significantly greater than M‐AEA in most regions, which is in agreement with WIN acting as a full agonist. However, WIN activity was not different from M‐AEA in most thalamic nuclei, in agreement with our relative efficacy maps. The ability to reconstruct a 3D dataset from autoradiographs enhances our ability to examine ligand dependent regulation of CB receptor function. Using a whole‐brain approach allows us to comprehensively localize the spatial extent and regional differences in CB‐mediated G‐protein activity.

Supported by DA14277
Escalating cocaine intake during extended self‐administration enhances cocaine‐primed reinstatement of operant behavior in rhesus monkeys
Authors: Henry Porche' Kirkland , Nichols Carol , Howell Leonard L.
Published: June 2021

The present study assessed the effects of extended cocaine exposure on the magnitude and persistence of cocaine‐primed reinstatement as a model of relapse. Rhesus monkeys (n=6) with an extensive cocaine self‐administration history under a second‐order schedule were allowed extended self‐administration sessions under a fixed‐ratio 20 schedule. Subsequently, saline was substituted for cocaine under the second‐order schedule and subjects underwent extinction. Once extinction criteria were met (<20% of cocaine‐maintained responding), noncontingent cocaine (0.1 mg/kg, iv) was administered prior to an extinction session and peak response rates were measured as an index of reinstatement magnitude. Reinstatement experiments were repeatedly conducted until response rates returned to extinction levels. The number of sessions to this criterion was taken as an index of resistance to extinction. Peak response rates following extended cocaine access were greater for each reinstatement trial compared to the initial limited drug access. In contrast, sessions to reach extinction criteria decreased between cocaine access conditions. These results suggest that extended cocaine experience enhanced cocaine‐induced reinstatement, but not resistance to extinction. USPHS Grant DA016589, DA00517, and RR00165
Hyperemic response to different intensities isometric dorsiflexion contractions and brief arterial cuff occlusion
Authors: Sanchez Otto Alexander , Louie Elizabeth A , Damon Bruce M
Published: June 2021

Introduction: Reactive hyperemia can induce vascular injuries. The purpose of this study was to compare the changes in blood volume in response to isometric contractions with arterial cuff occlusion of short duration. Methods: apparently healthy subjects. One each testing day, the subjects performed a maximal voluntary contraction (MVC), 50% MVC (50MVC) or 50% MVC with arterial cuff occlusion (50MVCcuff). MRI data acquisition and analysis: MRI data were obtained on a 3Tusing a dual gradient recalled MRI. Sscans were acquired with TR/TE= 1000/6, 46 ms, slice thickness=7.5 mm, FOV= 18×18, matrix size=128×128, NEX=1. The TE=6 ms and TE=46 ms signal intensities (SI6 and SI46, respectively) plotted as a function of time. The difference between pre and post contraction peak SI (ΔSI) SI6 and SI46 reflects changes in blood volume and oxygenation, respectively (1). Statistical analysis: A general linear model repeated measures analysis was performed to test for differences in ΔSIavg, between 50MVC, 50MVCcuff and MVC. Results: ΔSIavg, for TE6 showed a trend towards a main effect by condition and ΔSIavg, for TE46 demonstrated a main effect for condition, p = 0.04. Conclusion: This procedure could be used in subjects with muscular weakness and to reduce the potential risk of arterial cuff occlusion of longer durations.
Acute retinal ischemia inhibits endothelium‐dependent nitric oxide‐mediated dilation of retinal arterioles: role of endothelin‐1 and superoxide
Authors: Hein Travis W. , Yuan Zhaoxu , Xu Wenjuan , Ren Yi , Roberts Elizabeth , Newman Joseph M. , Kuo Lih
Published: June 2021

Increases in endothelin‐1 (ET‐1) and oxidative stress are associated with retinal vascular disease. Herein, we assessed whether acute retinal ischemia increases local ET‐1 and superoxide (O2−), and alters nitric oxide (NO)‐mediated dilation of retinal arterioles. Retinal ischemia was established by elevating intraocular pressure (IOP) to 90 mmHg for 30 or 90 min via infusion of saline into the anterior chamber of the porcine eye. Following ischemia, retinal arterioles from control and ischemic eyes were isolated and pressurized without flow for in vitro study. Dilation to endothelium‐dependent NO‐mediated agonist bradykinin but not endothelium‐independent NO donor nitroprusside was significantly reduced in arterioles following only 90‐min ischemia. Dihydroethidium staining and ET‐1 ELISA revealed increase of O2− inarterioles and ET‐1 in the vitreous after ischemia. Pre‐ischemia intravitreal treatment with O2− scavenger TEMPOL or ET‐converting enzyme inhibitor phosphoramidon prevented elevation of O2− and preserved dilation to bradykinin. Phosphoramidon but not TEMPOL reduced vitreous ET‐1. Our data show that acute retinal ischemia inhibits endothelium‐dependent NO‐mediated dilation of retinal arterioles. The impairment is mediated by elevation of local ET‐l and subsequent NO inactivation via increased vascular O2−. Support: Retina Research Foundation and Scott & White Research Foundation.
Role of nitric oxide and prostanoids in the adrenergic contraction of goat cerebral arteries after ischemia‐reperfusion
Authors: Garcia‐Villalon Angel Luis , Salcedo Adely , Fernandez Nuria , Monge Luis , Sanchez Ana , Narvaez Raul , Dieguez Godofredo
Published: June 2021

To analyze the role of nitric oxide and prostanoids in the adrenergic cerebrovascular response after ischemia‐reperfusion (I‐R), left middle cerebral artery (MCA) was occluded for 120 min followed by 60 min of reperfusion in anesthetized goats. After this, the animals were sacrificed, the brain was removed and 3 mm‐long arterial rings were taken from branches of the left (I‐R) and right (control) MCA, and prepared for isometric tension recording in organ baths. Noradrenaline (NA) (10−8 – 10−4 M, in the presence of beta‐adrenoceptor blockade) produced concentration‐dependent contractions, and the maximal contraction but not the sensitivity was higher in I‐R than in control arteries. The inhibitor of nitric oxide synthesis L‐NAME (10−4 M) increased the contraction to NA similarly in control and I‐R arteries, while the cyclooxygenase inhibitor meclofenamate (10−5 M) augmented the maximal response to NA in control but not in I‐R arteries. The relaxation induced by ADP (10−8–10−5 M) in the arteries precontracted with the thromboxane A2 analogue U46619 was similar in control and I‐R arteries. Therefore, ischemia‐reperfusion may increase the adrenergic cerebrovascular reactivity due, at least in part, to reduced formation of prostacyclin; the modulatory role of NO in this reactivity may be preserved after I‐R. (Supported, in part, by FMMA and MEyC)
Nitric oxide buffers effects of angiotensin II on arterial baroreflex control of heart rate in conscious lambs
Authors: Wehlage Stephanie J , Smith Francine G
Published: June 2021

Previously, we showed that both nitric oxide (NO) and angiotensin II (ANG II) modulate the arterial baroreflex control of heart rate soon after birth. To test the hypothesis that NO buffers the effects of ANG II through activation of AT1 receptors early in life, three experiments were carried out at intervals of 48 h and in random order in two age groups of conscious, chronically instrumented lambs: ∼one week (N=9) and ∼six weeks (N=11): The arterial baroreflex was assessed before and after administration of the selective AT1 receptor antagonist, ZD 7155, and vehicle (experiment one), ZD 7155 and L‐NAME (experiment two), or vehicle alone (experiment three). There were no effects on any of the parameters governing the arterial baroreflex at six weeks after ZD 7155 with or without L‐NAME or vehicle. In contrast, after ZD 7155 at one week there was a decrease in the heart rate range, an increase in minimum heart rate, and an increase in maximum gain. After L‐NAME, in the presence of ZD 7155, these changes were exacerbated at one‐ but not six weeks. These findings support the postulate that there is an age‐dependent interaction between ANG II and NO in modulating the arterial baroreflex control of heart rate.
Neural pathways of cardiovascular depressor reflex during gastric distension and its modulation by electroacupuncture
Authors: Hsiao An‐Fu , Tjen‐A‐Looi Stephanie , Zhou Wei , Li Peng , Cabatbat Ranier , Longhurst John C
Published: June 2021

Mild mechanical gastric distension (GD) frequently evokes a decrease in blood pressure and bradycardia. We hypothesized that the GD‐induced cardiovascular depressor reflex is transmitted through both sympathetic and parasympathetic afferent pathways. We also hypothesized that it can be modulated by electroacupuncture (EA) and is mediated by caudal ventral lateral medulla (cVLM) and nucleus ambiguus (NA). Sprague‐Dawley rats were anesthetized, ventilated, and their mean arterial blood pressures (MAP) and heart rates (HR) monitored. Repeated balloon inflation of the stomach every 10 minutes to 15–20 mmHg gastric wall tension induced consistent falls in MAP and HR. This inhibitory response was reversed by chemical denervation of celiac ganglia and intra‐abdominal vagal nerves with 0.1mL of 1% lidocaine, suggesting that the decreased MAP was mediated by both the withdrawal of sympathetic tone and increased parasympathetic tone. Similarly, the reduced HR was reversed after vagotomy and celiac ganglionectomy. Bilateral stimulation with EA at cardiovascular acupoints P5‐6 for 30 min significantly decreased the depressor response. This modulation lasted for 50 min. Unilateral microinjection of atropine into cVLM immediately after termination of EA reversed the inhibition by EA, whereas microinjection of normal saline into cVLM did not reverse it. Microinjection of gabazine into the NA reversed the inhibition by EA. These data suggest that GD activates both sympathetic and vagal afferents to evoke reflex cardiovascular sympatho‐ and parasympatho‐inhibition through the cVLM and NA respectively. EA attenuates the cardiovascular depressor reflex through a cholinergic mechanism in the cVLM and a gabanergic mechanism in the NA.
Angiotensin II‐induced ERK1/2 activation is mediated by PKC delta and intracellular calcium in cardiac fibroblasts
Authors: Shamhart Patricia , Olson Erik , Naugle Jennifer , Doane Kathleen , Meszaros J. Gary
Published: June 2021

Activated cardiac fibroblasts (CFs) proliferate, differentiate, and migrate to regulate cardiac remodeling. ANG II activates extracellular signal‐regulated kinase (ERK) 1/2 to induce proliferation, but the signaling pathways leading to ERK 1/2 activation have not been elucidated in CFs. The goal of the current study was to identify the intracellular mediators of ANG II‐induced ERK 1/2 activation in adult rat CFs. We determined that ANG II does not transactivate the epidermal growth factor receptor (EGFR) in adult CFs, since pretreatment with AG 1478 did not inhibit [3H]‐thymidine incorporation or ERK 1/2 activation. We found that ANG II‐induced ERK 1/2 phosphorylation is inhibited by simultaneous chelation of cytosolic Ca2+ and downregulation of PKC by phorbol ester or by the specific PKC inhibitor rottlerin as well as PKC siRNA, but not by inhibition of each agent alone. We next investigated the role of specific PKC isoforms in migration of the activated CFs using in vitro wound healing assays. Through pharmacological blockade we determined that inhibition of PKC slows migration of CFs and inhibition of PKC also decreases the rate migration but to a lesser extent than blockade of PKC. These data reveal that PKC is a key mediator in both ANG II‐induced proliferation with concurrent Ca2+chelation and migration of CFs.
Nitric oxide (NO) released from a new NO donor has lower activity in vascular smooth muscle cells from renal hypertensive rat
Authors: Lunardi Claure N. , Rodrigues Gerson J. , da Silva Roberto S. , Bendhack Lusiane M.
Published: June 2021

Increased production of superoxide anion (O2−) reduces the biovailability of NO released by the new NO donor cis‐[Ru(bpy)2(py)NO](PF6)2 (RUNOCL) in rat aortic smooth muscle cells (SMC). NO is a messenger molecule that plays an important role in the control of vascular tone due to the activation of soluble guanylyl cyclase (sGC)‐ cGMP‐ protein kinase G pathway that causes the decrease of cytosolic Ca2+concentration ([Ca2+]c). This study aimed to investigate the effect of the NO released from RUNOCL on the [Ca2+]c in SMC from normotensive (2K) and renal hypertensive rats (2K‐1C). [Ca2+]c, [NO]c and [O2−]c were measured with fluorescent probes FLUO‐3AM (510 nm), DAF‐2DA (545 nm) and DHE (595 nm), respectively, excited by 488 nm using a laser scanning confocal microscopy. [NO]c released from RUNOCL (10 μmol/L) was greater in 2K cells (15.9±0.6%) than in 2K‐1C cells (10.9±1.1%, n=3, P<0.001). On the other hand, in 2K‐1C cells the basal [O2−] was 2.6 times greater than in 2K cells (P<0.05). RUNOCL decreased [Ca2+]c from 100% to 60.0±10%, n=3) in 2K cells and from 100% to 77.2 ± 2%, n=3 (P<0.001) in 2K‐1C cells. These results indicate that the NO donor RUNOCL has greater effect in reducing [Ca2+]c in 2K than in 2K‐1C cells. This impaired effect of the NO donor is related to the lower [NO]c available in 2K‐1C cells. It could be due to an increased production of O2− that reacts with NO reducing its bioavailability. Supported by FAPESP and CNPq.
ERK, p38 and JNK MAPK are differently distributed and activated in the anoxic‐reogygenated embryonic heart
Authors: Gardier Stephany , Pedretti Sarah , Sarre Alexandre , Raddatz Eric
Published: June 2021

Involvement of Mitogen Activated Protein Kinases (MAPK) ERK, p38 and JNK in the response of the developing heart to anoxia‐reoxygenation is not known. In an embryonic heart model we sought to determine the activation of MAPKs in atria (A), ventricle (V) and outflow tract (OT) during anoxia‐reoxygenation and after a severe oxidative stress.

Content of MAPKs and their profile of activation during 30 min of anoxia and 60 min of reoxygenation were assessed by western immunoblotting and kinase assay in A, V and OT of hearts isolated from 4‐day‐old chick embryos. MAPKs phosphorylation was also determined after exposure to H2O2 (1mM, 1h).

Basal total and phosphorylated forms of ERK, p38 and JNK were the highest in V. Phosphorylation of ERK decreased twofold in A during anoxia, that of p38 increased twofold in A and V and returned to basal level at the end of reoxygenation. In OT, phosphorylation of ERK and p38 was not detectable. JNK activity peaked fivefold only in V after 30 min of reoxygenation. Exogenous H2O2 increased ERK and p38 phosphorylation more markedly in A and OT, with no effect on JNK.

Thus, in the embryonic heart ERK, p38 and JNK are differently distributed and modulated by anoxia‐reoxygenation and oxyradicals. Such findings provide a first step in understanding modulation of the MAPK cascades in the fetal heart subjected to intrauterine hypoxia.

Supported by the Swiss National Science Foundation n°3100A0‐105901
Increased myogenic precursor cell number in human skeletal muscle with 12 weeks of training at low intensity
Authors: Mackey Abigail Louise , Holm Lars , Reitelseder Søren , Pedersen Troels Gravers , Kadi Fawzi , Døssing Simon , Kjaer Michael
Published: June 2021

Satellite cells increase in number with heavy resistance training, but the response to very light muscle loading is unknown. We recruited 12 young healthy male (mean age 25 ± SD 3 yrs) volunteers to train one leg with a heavy load (H) and the other leg with a light load (L), for 12 weeks. We hypothesized that, compared to L training, H training would induce a greater response of the satellite cell pool. Satellite cells were visualised by immunohistochemistry on muscle biopsies collected from the m. vastus lateralis of both legs before and after the training period. Compared to the pre training values, there was a significant increase in the number of NCAM+ cells (per myonuclear number) post training in both groups (H: from 4.16 ± 1.01 to 5.32 ± 1.22; L: from 4.01 ± 0.78 to 4.72 ± 1.72; mean ± SD, p < 0.05). The finding that 12 weeks of training skeletal muscle with very low loads can induce an increase in the number of satellite cells reveals a new aspect of myogenic precursor cell activation. This knowledge could be relevant in considering training regimens where heavy resistance training is not possible.

Funded by Lundbeck Foundation and the Danish National Research Foundation.
Estrogen receptor (ER) α and β and Aromatase knock‐out (ArKO) effects on muscle development in male mice
Authors: ferreira Andries Johan , Lubahn Dennis B , Brown Marybeth
Published: June 2021

ERs have been identified in male muscle but whether estrogen (E2) exerts its effects through ERα or ERβ is unclear. Direct E2 effects on developing muscle were also determined in ArKO. All comparisons were made to corresponding wild type (WT) controls. Muscle mass, particularly in fast‐fibered gastrocnemius (Gast) and Tibialis Anterior (TA) (not soleus or plantaris) was diminished in ERα but not ERβ. Most absolute mass measures, and 3 of 4 muscles normalized to body weight were significantly lower in ArKO mice compared with WT. Normalized muscle mass/body weight was still lower in ERAα Gast and TA but not in the ERβ group. There was no difference between WT and KO mice in peak force, force/area, force/velocity, time to peak tension or ½ relaxation time. Total protein and contractile protein myosin in Gast and TA were similar in WT and KO, further indication that contractile function was not affected by KO status. Results suggest ERαKO and ArKO may directly regulate muscle growth without affecting contractile function. The presence or absence of ERβ does not alter skeletal muscle function in male mice. Estrogen may be required for complete muscle size during development.

Support: Spinal Injuries Board, Research Board
Are areas other than the pre‐Botzinger complex capable of maintaining rhythmic breathing and normal blood gases?
Authors: Krause K L , Forster H.V. , Davis S. , Kiner T. , Bonis J.M. , Pan L.G. , Quian B.
Published: June 2021

Abrupt neurotoxic destruction of >70% of the pre‐Botzinger complex (PBC) in awake goats caused hypoxemia and hypercapnia, ultimately resulting in respiratory and cardiac failure (J. Appl Physiol 97:1629–36, 2004). We hypothesized that gradual, incremental destruction of the PBC will enable another rhythmogenic area to compensate for the loss of this nucleus. Microtubules were bilaterally implanted into the PBC of 6 adult goats. After recovery and baseline parameters were established, increasing volumes (0.5, 1, 5, and 10 uL) of ibotenic acid (50 mM) were injected into the PBC, with one week between injections. Minutes after each injection, breathing frequency (f) increased and eupneic airflow and respiratory muscle pattern were disrupted. Full recovery in each goat occurred within days; blood gases and pH were unchanged from baseline (p>0.05). One week post the 10 uL injection, all eupneic breathing variables, and breath‐to‐breath variation, were unchanged from baseline, except for a significant decrease in f (p = 0.03). Histological analysis showed >85% reduction in total PBC neurons and >90% reduction in neurokinin I receptor neurons. Thus, incremental, near total elimination of the PBC area allows the respiratory system to completely compensate for the loss of this area, resulting in a respiratory rhythm appropriate to sustain homeostasis. (Supported by the Department of Veterans Affairs and NIH HL25739)
Determination of Energy Requirements for P‐glycoprotein Function in Eastern Oysters, Crassostrea virginica
Authors: Reynolds Kristen Nicole , Sokolova Inna
Published: June 2021

Cadmium (Cd) is an important pollutant in aquatic habitats, and Cd exposure results in increased energy demand in oysters Crassostrea virginica. However, mechanisms of this increase are not well understood. To determine whether activity of ATP‐dependent multixenobiotic resistance pumps (P‐glycoproteins, or P‐gp) contributes to higher metabolic rate in Cd‐exposed oysters, we measured oxygen consumption and P‐gp substrate (calcein‐AM) uptake in the presence and absence of specific P‐gp inhibitors (verapamil, cyclosporin A and JS‐2190) in isolated gills from control and Cd‐exposed (50 μg L−1, 30–70 days) oysters. Lowest effective concentrations of inhibitors (66.7 μM verapamil, 13.3 μM cyclosporin A and 16.7 μM JS‐2190) were selected in dose‐response experiments. In control oysters, addition of P‐gp inhibitors had no effect on calcein‐AM uptake indicating low P‐gP activity, whereas in Cd‐exposed oysters inhibition of P‐gp‐mediated efflux led to a significant increase of calcein accumulation by 50–100%. Respiration of isolated gills was decreased by 4–10% by P‐gp inhibitors; however, this decrease was statistically not significant indicating that contribution of P‐gp activity to energy balance was negligible in control and Cd‐exposed oysters. Supported by United Negro College Fund, Undergraduate Summer Research Fellowship of the American Physiological Society and NSF CAREER (IBN‐0347238).
Evolution of an RNP assembly system: A minimal SMN‐complex facilitates formation of U snRNPs in Drosophila melanogaster
Authors: Kroiss Matthias , Schultz Jörg , Wiesner Julia , Chari Ashwin , Sickmann Albert , Fischer Utz
Published: June 2021

In vertebrates, assembly of spliceosomal UsnRNPs is mediated by the SMN‐complex, a macromolecular entity composed of the proteins SMN and Gemins 2–8. Here we have studied the evolution of this machinery using complete genome assemblies of multiple model organisms. The SMN‐complex has gained complexity in evolution by a block‐wise addition of Gemins onto an ancestral core complex composed of SMN and Gemin2. In contrast to this overall evolutionary trend to more complexity in metazoans, orthologs of most Gemins are missing in dipterans. In accordance with these bioinformatic data a novel biochemical purification strategy elucidated that the dipteran Drosophila melanogaster contains a SMN‐complex of remarkable simplicity. Surprisingly, this minimal complex not only mediates the assembly reaction in a manner very similar to its vertebrate counterpart, but also prevents mis‐assembly onto non‐target RNAs. Our data suggest that only a minority of Gemins is required for the assembly reaction per se, whereas others may serve additional functions in the context of U snRNP biogenesis. The evolution of the SMN‐complex is an interesting example of how the simplification of a biochemical process contributes to genome compaction.
Interactions between the G quadruplex forming Microtubule‐Associated Protein 1B RNA and the Fragile X protein Family
Authors: Menon Lakshmi , Mihailescu Mihaela Rita
Published: June 2021

Fragile X Mental Retardation Syndrome, the most common cause of inherited mental retardation, is caused by the absence of the fragile X mental retardation protein (FMRP). The Arginine‐Glycine‐Glycine (RGG) Box domain of FMRP has been shown to bind to target messenger RNA (mRNA) sequences with high affinity to RNA targets that have been proposed to contain G quadruplex structures. One such potential mRNA target of the FMRP is the MAP1B RNA,which encodes for the Microtubule‐Associated Protein 1B (MAP 1B) protein, and is expressed at high levels in actively extended neurons. FMRP has been shown to bind in vivo to MAP1B RNA and to control the translation efficiency of the MAP1B protein in neurons during neonatal development. It has been proposed that the FMRP interaction with this RNA is G quadruplex dependent. In this study, we present biophysical evidence for the formation of an intramolecular parallel G quadruplex structure in MAP1B RNA and a thermodynamic analysis of the binding between this RNA and the FMRP RGG box. To determine if the recognition of the MAP1B RNA structure is unique to FMRP, we also studied the binding of the FMRP autosomal paralogs, the FXR1 and FXR2 proteins, to this RNA.
Predictable Transitive RNA Interference Induced by mRNA hairpins in C. elegans
Authors: O'Bier Jennifer , Cox Jessica , Doty Matthew , Schwartz Caitlin , Rawn J. David , Dwyer Timothy M.
Published: June 2021

RNA interference (RNAi), is one of the newest and yet most prevalent areas of research today. While the protein machinery necessary to cause this phenomenon has been well characterized, it is not always possible to predict the silencing of one specific gene. At times, when a target gene is silenced, it can lead to the silencing of other RNAs, known as off‐target effects. We are trying to understand this process, termed transitive RNAi, and trying to determine if there are scientific rules governing it. Through the use of an algorithm, we have mapped gene networks that may cause these off‐target effects. One of these networks is now being examined in the nematode C. elegans. Two plasmid constructs have been created. The first (37hp) targets four genes within the network for silencing through a common hairpin sequence found in each of the genes. The second (37a) construct targets just one these genes through sequence homology in an area outside of the hairpin. The two constructs, as well as a control construct, have been introduced into bacteria, induced to express the dsRNA and fed to the C. elgans to induce RNAi. C. elegans fed the bacteria containing the two constructs show slight phenotypic variations. These phenotypic variations lead us to believe that the pattern of silenced genes is similar between the two groups and that the silencing of the one gene with the second construct might have led to the silencing of the other three genes in a manner that we could then begin to predict. The effect of 37a and 37hp constructs against genes within the network are being analyzed, using Real‐time PCR. Preliminary results suggest that the constructs can silence Y37.e3.13 but do not affect Ankyrin. Characterization of the other genes will also be discussed.
Structural Insight into the Kinetics and ΔCp of interactions between TEM1 β‐lactamase and BLIP
Authors: Wang Jihong , Zhang Zhen , Palzkill Timothy , Chow Dar‐Chone
Published: June 2021

Our previous report of the thermodynamics of binding between TEM‐1 β‐lactamase and BLIP mutants indicated that Y51A and W150A mutants have distinctive TEM‐1‐binding thermodynamics (Wang J., et al. JBC. 2007; 282:17676–84). The binding heat capacity changes for Y51A/TEM‐1 and W150A/TEM‐1 interactions are −0.8 kcal/mol/K and −0.3 kcal/mol/K, respectively, while the W150A TEM‐1 interaction is ∼1000 fold weaker than the wild type. Our kinetic measurements using stopped‐flow and enzymatic activity‐based methods show that these complexes have similar association constants but that W150A has an at least 100 fold faster dissociation constant. To understand the structural basis of the altered thermodynamic and kinetic properties, we determined structures of the complexes of the Y51A/TEM‐1, W150A/TEM‐1 complexes and the W112A BLIP mutant using x‐ray crystallography. The weak‐binding complex of W150A/TEM‐1 exhibits significant structural rearrangements around the mutated residue W150A and more water molecules trapped in the interface compared to the wild type complex. Our results show that W150 is important for structural complementary between BLIP and TEM‐1 in that its mutation introduces structural mismatches that disrupt many direct binding forces resulting in more trapped interfacial water molecules, reduced binding heat capacity changes and enthalpy driving forces, and a faster dissociation rate.
The ubiquitin‐interacting motifs of 26S proteasome subunit S5a induces the cellular vacuolization and ER stress in A549 lung cancer cells
Authors: Elangovan Muthukumar , Yoo Yung Joon
Published: June 2021

The subunit S5a is a key component for the recruitment of ubiquitinated substrates to the 26S proteasome. We previously found that the ectopic expression of ubiquitin‐interacting motifs of S5a (S5a‐UIMs) accumulated the ubiquitinated proteins in vivo and induced the A549 lung cancer cell death but not non‐cancer BEAS‐2B cell death. In this study, we further found that S5a‐UIMs induced cell cycle arrest in A549 cells and stabilized the p53, p21, and cyclins, but could not stabilize the unstable GFP, suggesting that it can be used as a selective upstream inhibitor of the proteasome pathway. In addition, S5a‐UIMs induced the cellular vacuolization in A549 cells, which may result from the perturbation of endocytosis. Ectopic expression of S5a‐UIMs also induced the ER stress in A549 cells, which was monitored by the increased expression of PDI, GRP78, GRP94, and caspase12. Taken together, our data suggest that S5a‐UIMs can induce the cellular vacuolization and ER stress, which may result in the A549 cell death.
Association of ADAM19 Polymorphism with the level of IgE in Asthmatic Patients
Authors: Kwon Ji‐Hee , Song Hyun‐mi , Chung Hun Soo , Park Byung‐Lae , Rhim Tai youn , Shin Hyung Doo , Park Choon‐Sik
Published: June 2021

ADAM19 is encoded on chromosome 5q32‐q33, which has been reported to be linked with the development of asthma. Aim of study is to investigate the association of polymorphisms on ADAM19 with the risk and the phenotypes of asthma. We found 22 SNPs on ADAM19 by direct sequencing. Among these polymorphisms, 5 SNPs were selected for genotyping in Korean considering the location of SNPs, MAF and LD. In the association analysis, we could not found any association between the polymorphisms and the risk of asthma. However,+56656T>C,+95719C>T and +97188T>C showed significant association with the totalIgE level in serum of asthmatics, especially in that of atopic asthmatics (P=0.001–0.048). Asthmatics having rare allele on those SNPs showed significantly higher levels of total IgE than those having common alleles. Asthmatics having haplotype 1, which is composed by the combination of major alleles on the SNPs, showed significantly lower levels of total IgE than those having other haplotypes (p=0.002). Overall, ADAM19 may be associated with IgE level in asthma, genotypes and haplotypes of ADAM19 could be used for the new genetic markers for the hyper IgE syndrome. This work was supported by a grant of the Korea health 21 R&D project.
Characterization of mitochondrial outer membrane proteins involved in mitochondrial protein import in Trypanosoma brucei
Authors: Sharma Shvetank , Singha Ujjal K , Chaudhuri Minu
Published: June 2021

Mitochondrial protein translocation machinery in parasites like Trypanosoma brucei is poorly characterized. Homologs for components of the translocase of mitochondrial outer membrane (TOM) in T. brucei are not known. Major component of the TOM complex in eukaryotes is Tom40. TOM complex also has two tetratricopeptide repeat containing proteins, Tom70 and Tom20 (receptors for mitochondrial precursor proteins). We identified two β‐barrel proteins from T. brucei gene database. Analysis of the subcellular fractions of T. brucei using antibodies developed against peptides from these proteins revealed that these are localized on the mitochondrial membrane. RNA interference studies showed that both these proteins are essential for cell growth in the procyclic T. brucei. We also identified three TPR containing proteins named TPR5, TPR7, TPR9 showed some homology to Tom70 of various species. The effect of expression of RNAi on cell growth and morphology was monitored for each of the proteins individually. Results showed that RNAi for TPR5 and TPR7 had little effect on cell growth. However, RNAi for TPR9 caused more than 70% inhibition of cell growth within 4 days in culture. In addition TPR9 knock‐down also changed the cell morphology. Further analysis of the functions of all the candidate proteins is under investigation. Grant Support: 3SO6GM08037‐30S1 and 1SC1GM081146‐01.
Overexpression of Sp1 transcriptoin factor in tumor cells reduced cell proliferation
Authors: Hung Jan‐Jong , Wu Chien‐Hsing
Published: June 2021

The transcription factor Sp1 is expressed in different cells and thereby regulates the expression of genes involved in many cellular processes including cell cycle regulation, apoptosis, and differentiation. Previous studies have revealed that the expression of Sp1 is maintained in several types of cancer such as gastric cancer. However, the recent studies and our preliminary data revealed that accumulation of Sp1 led to tumor cells death. In our data, we found that more expression of Sp1 in the most cancer tissues was observed compared to the normal tissues, especially lung cancer. In this study, we chose non‐small cell lung cancer (NSCLC): (A549) to character the role of Sp1 during tumorigenesis. At first, adeno‐constructs including adeno‐GFP and adeno‐GFP‐Sp1 have succeeded to be made to infect the HeLa cells completely. We found that Sp1 regulated cell proliferation negatively in A549 cell by counting the cell number and the MTS assay. In the nude mice system, HeLa and A549 cells infected with adeno‐GFP and adeno‐GFP‐Sp1 viruses to overexpress the Sp1, to be injected into the nude mice, the tumors in the mice injected with adeno‐GFP injected cells were larger than those injected with adeno‐GFP‐Sp1 injected cells. Both of sub‐G1 and cell cycle arrest were observed in adeno‐GFP‐Sp1 injected cells by FACS analysis. Furthermore, we also used the Nocodazole and thymidine to synchronize cells in Mitosis and G1 phase respectively to study the role of overexpressed Sp1 in cell cycle. We found that overexpression of Sp1 could repress the cell cycle progression, and stayed in G1/S phase. Recently we also used the Time‐lapse to observe the cell growth in living cells, and found the adeno‐GFP‐Sp1 infected cells could be inhibited in growth and led to death at last, but not occurred in adeno‐GFP infected cells. In the future, we will study which signal transduction pathway was involved in the death caused by overexpression of Sp1.