bluuebunny/crossref_metadata_2025 huggingface dataset:
MnSOD protects against COX1‐mediated endothelial dysfunction in chronic heart failure
Authors: Miller Jordan D. , Peotta Veronica A. , Weiss Robert M. , Chu Yi , Brooks Robert M. , Heistad Donald D.
Published: June 2021

Endothelial function is impaired by oxidative stress in chronic heart failure (CHF). Mechanisms that protect against increases in oxidative stress in CHF are not unclear. We examined endothelial function in aorta in wild‐type mice (MnSOD+/+) and mice deficient in manganese superoxide dismutase (MnSOD+/−) 12 weeks after ligation of the left cornary artery (LCA). LCA ligation produced similar size infarctions in MnSOD+/+ and MnSOD+/− mice. Maximal relaxation in response to acetylcholine was 78 ± 3% (mean ± SE) and 66 ± 8% in sham‐operated MnSOD+/+ and MnSOD+/−mice, respectively. Induction of CHF increased MnSOD expression ~1.7 fold in MnSOD+/+ mice, but not in MnSOD+/− mice. Maximal relaxation to acetylcholine was impaired in MnSOD+/+ mice (68 ± 4%), with greater impairments in endothelial function in MnSOD+/− mice with CHF (46 ± 5%). Incubation of vessel rings with Tempol did not improve vasorelaxation in response to acetylcholine. However, incubation with nonspecific cyclooxygenase (COX) inhibitor indomethacin or the COX1‐specific inhibitor valeryl salicylate significantly improved relaxation to acetylcholine in CHF mice (maximum relaxation = 74 ± 5% and 91 ±1%, respectivley). Collectively, these data identify a novel mechanism whereby chronic increases in mitochondrial‐mediated oxidative stress impair vascular function via a COX1‐mediated endothelium‐derived contracting factor in CHF.
The Biologic Properties and Ultra‐Microstructure in the Senescence of Endothelial Cell Induced by AngiotensinII
Authors: Bai Xiaojuan , Shan Haiyan , Chen Xiangmei
Published: June 2021

Objective

The aim of this study was to investigate the biologic effects and morphology senescence of human umbilical vein endothelial cell (HUVEC) induced by angiotensinII and its possible action mechanism.

Methods

HUVEC were cultured in vitro and intervened by AngII. The cell living rate was measured by MTT assay; SA‐β‐gal stain and cell cycle analysis were used to identify cell aging status; the early stage apoptosis was detected by flow cytometry with Annexin V‐FITC/PI double staining, morphologic changes and percentage of apoptosis were assayed with acridine orange fluorescence staining and were observed under fluorescent microscope, transmission electron microscopy was used to evaluate the ultra‐microstructure of HUVEC.

Results

Induction of AngII promoted a growth arrest with phenotypic characteristics of cell senescence, such as enlarged cell shapes, increased senescence‐associated β‐galactosidase (SA‐β‐gal) staining and depressed cell proliferation. Apoptotic number and percentage was increased in senescent cell, which a small subset of the senescent cells showed aberrant morphology such as remarkable nuclear fragmentation or multiple micronuclei.

Conclusions

Our results suggest cell apoptosis is possibly an important factor for the process of pathologic and physiologic endothelial cells senescence and vascular aging.
NH4+ secretion in the avian colon. An actively regulated barrier to ammonium permeation of the colon mucosa
Authors: Laverty Gary , Holtug Klavs , Árnason Sighvatur S. , Skadhauge Erik
Published: June 2021

Experiments were done to characterize active, electrogenic transport of NH4+ ions across the colonic epithelium of the domestic fowl (Gallus gallus). Stripped colonic mucosal segments were mounted in Ussing chambers and voltage‐clamped to measure the short‐circuit currents (ISC). Bilateral addition of NH4+ caused a dose‐dependent outward current (negative ISC), with a Km of 34 + 8 mM and a maximal current response of 311 + 47 μA cm−2 (12 + 2 μEq cm−2h−1). A similar effect was seen with unilateral addition of NH4+ to the serosal (s) side, but not with mucosal (m) addition. Pre‐treatment with 10−4 M amiloride exposed a net outward (negative) ISC, and NH4+ addition (s) further increased this outward current (Km of 53 + 24 mM). Unidirectional NH4+ flux measurements revealed a net secretory flux (8.8 + 3.1 μmol cm−2h−1 s‐m, versus 2.6 + 1.4 μmol cm−2h−1 m‐s), which closely matched the change in ISC with NH4+ (s), showing that the transepithelial flux of NH4+ could account for the outward current response. Addition of 50 nM bafilomycin A to the mucosal solution completely eliminated s‐m NH4+ transport, implicating an apical V‐type H+‐ATPase in this transport process. Active NH4+ extrusion, combined with low permeability to NH3 in this tissue, allow for maintenance of steep ammonia gradients across the colonic epithelium and protection from ammonia toxicity. The hen colon may be a useful system for the study of NH4+ transport.
High blood glucose is not associated with oxidative stress or vascular dysfunction in birds
Authors: Sweazea Karen L. , Wolf Blair O. , Braun Eldon J. , Walker Benjimen R.
Published: June 2021

Hyperglycemia is associated with the development of oxidative stress‐induced vascular dysfunction including reduced nitric oxide bioavailability. Plasma glucose levels in birds are 5–7 times that of mammals of similar body mass yet birds do not develop significant oxidative tissue damage. Therefore, we hypothesized that birds have evolved endogenous mechanisms to prevent hyperglycemia‐induced oxidative stress thereby maintaining normal vascular reactivity. Experiments were performed using adult mourning doves. Rats fed a high fat diet (HFD; 60% kcal from fat) for 6 weeks were used as positive controls for the development of oxidative stress and vascular dysfunction. Whole blood glucose levels were significantly greater in doves (341.2 ± 52.3 mg/dl) compared to HFD rats (97.4 ± 6.55 mg/dl). Consistent with our hypothesis, levels of the cell permeant reactive oxygen species fluorescent indicator, 5‐(and‐6)‐chloromethyl‐2′,7′‐dichlorodihydrofluorescein diacetate, acetyl ester, were significantly lower in isolated mesenteric arterioles from doves compared to HFD rats. In addition, despite having higher blood glucose levels, arterioles from doves were more sensitive to the endothelium‐dependent vasodilator acetylcholine, but less responsive to the vasoconstrictor endothelin‐1 than arteries from HFD rats. Thus, maintained vasodilation in avian arterioles in the presence of high glucose may be related to resistance to oxidative stress.
Effects of protein kinase C and NADPH oxidase on hypoxic vasoconstriction in lamprey dorsal aortas
Authors: McClain Robert L , Russell Michael J
Published: June 2021

Hypoxic vasoconstriction (HV) in aortas from cyclostomes, the most primitive vertebrates, has been suggested as the antecedent to hypoxic pulmonary vasoconstriction, and HV has been demonstrated in aortic segments from every vertebrate class except Mammalia. A protein kinase C (PKC) dependent role for NADPH oxidase has been shown in hypoxic pulmonary vasoconstriction in mammals. In the present study we investigated the role of PKC and NADPH oxidase pathways on HV in isolated lamprey (petromyzon marinus) dorsal aortas (LDA) using standard myography. Treatment of LDA with bisindoylmaleimide (BIM), a PKC inhibitor, reduced HV, whereas the PKC activator farnesyl thiotriazole (FTT) augmented HV, but had no effect on an alpha‐adrenergic contraction. However, the PKC inhibitor H7, and the PKC activator phorbol myristic acid (PMA) had no apparent effect on HV in LDA. In addition, the NADPH oxidase inhibitor diphenyleneiodonium (DPI) did not affect the magnitude of HV, but significantly impaired relaxation upon return to normoxia. These findings suggest that PKC, but not NADPH oxidase, may be an important component of the phylogenetically ancient lamprey HV. Supported by a grant from the MedCen Foundation of the Medical Center of Central Georgia.
Long‐term calorie restriction does not protect mice against a secondary infection with the parasitic nematode Heligmosomoides bakeri
Authors: Acosta Wendy , Kristan Deborah M
Published: June 2021

Benefits of long‐term calorie restriction (CR) include increased lifespan and enhanced immune function which should protect against pathogen infection. Most studies use antigen or mitogen stimulation and it is unknown if long‐term CR protects against an intact pathogen infection. The objective of our study was to examine if long‐term CR would change host susceptibility to a secondary infection with the nematode Heligmosomoides bakeri. Two dietary groups of C57BL/6 mice were used. One group was fed ad libitum (AL) while the CR group was fed 60% of AL. After 8 months, mice received a primary infection of 125 H. bakeri larvae. This first infection was cleared with an anti‐helminth drug and then a secondary infection of 300 larvae was given. Sixteen days post‐secondary infection, immune response, infection intensity, and worm reproduction were measured. CR mice had similar infection intensity as AL fed mice despite having increased CD4+ T‐cell numbers. Worm length and reproduction were similar for worms taken from all mice. Long‐term CR yielded the predicted increase in immune system function, but this response was not sufficient to reduce infection intensity or to affect worm growth and reproduction.
Revealing Anatomy Through Medical Imaging
Authors: Payer Andrew F.
Published: June 2021

Medical students have a great deal of difficulty in being able to comprehend the anatomy demonstrated in static or dynamic ultrasound imaging because the imaging found in most textbooks or intranet resources does not usually indicate the location of the ultrasound transducer on the body or the plane of image section. We purchased a portable ultrasound unit for use during the anatomy laboratory sessions. Students signed a waiver form agreeing that the imaging was not intended for diagnostic purposes. They were encouraged to use the unit on each other during the lab sessions to observe the anatomy they were studying on the cadaver. They could also save the images and download them to their laptop computers to share with other students in their groups. For the past three years, an ultrasonographer from a local radiology group practice volunteered time to attend laboratory sessions at the beginning of each week. She was able to demonstrate the clinical methods of using ultrasound, and answer questions about the technology. She also used female student volunteers to demonstrate suprapubic viewing of the uterus and ovaries. Attendees of the workshop will have hands on experience using the ultrasound equipment to demonstrate anatomical structures, discuss various ways to incorporate hands‐on ultrasonography in their respective courses, and learn about purchasing options for ultrasound equipment.
Incorporating Imaging into a Case‐Based First‐Year Anatomy Curriculum
Authors: Ruit Kenneth , Carr Patrick , Carlson Edward , Fogarty Ted , Vari Richard
Published: June 2021

One of the challenges of implementing a case‐based medical curriculum is creating a “continuity of learning” that minimizes compartmentalization of the learning that occurs in a variety of settings and promotes students’ conception of their learning as an “integrated whole”. For anatomists at the University of North Dakota, our role in meeting that challenge has occurred in two distinct steps: 1) integrating a meaningful Year 1 human gross anatomy experience into a case/problem‐based curriculum, and 2) integrating the teaching of clinical imaging into Year 1 in a way that faithfully represents global educational objectives for the learning of clinical imaging established for all four years of the medical curriculum. Four principal factors emerged as critical for successfully accomplishing this: efficient use of less available time, implementation of an objective‐driven approach to learning anatomy, selection of cases that create a clear context for learning of anatomy, and meaningful dissection laboratory experiences. Each of these factors has also proved critical for integrating clinical imaging into the Year 1 curriculum, including an investment in state‐of‐the‐art digital technology for navigating and interpreting clinical imaging of patients. As a result, students have a clear sense that their learning is “patient‐centered” and relevant to their future practice of medicine.
Responses to selenium supplementation in healthy Americans
Authors: Combs Gerald F , Watts Jennifer C , Johnson LuAnn K , Canfield Wesley K , Davis Cindy D , Milner John A
Published: June 2021

To determine dose‐response relationships of selenium (Se) intake and markers of Se status in healthy, non‐deficient subjects, we conducted a 12 mo. intervention using Se supplements (0, 50, 100 or 200 ug Se [as L‐selenomethionine]/d) in a group of 106 men and 155 women. At baseline, this cohort, none of whom took supplements with >50 ug Se/day, had plasma Se of 141.5±23.7 ng/ml, urinary Se of 58.2±21.5 ng Se/mg creatinine, and buccal cell Se of 10.2±6.5 ng Se/mg protein. Over the intervention, rates of drop‐out (7%) and compliance (98%) were very good, and each marker increased in a dose‐dependent manner. The 12 mo.‐increases in plasma and urinary Se in response to effective Se dose (corrected for body mass [kg0.75]) were similar: change in plasma Se (ng/ml)=18.2 × dose (ug/kg0.75) + 12.6 [r2=0.60]; change in urine Se (ng/mg creat.)=18.5 × dose (ug/kg0.75) + 17.4 [r2=0.44]. Men and women showed comparable plasma Se responses; however, the urinary Se response of women (20.9 ng/mg creat./ug Se/kg0.75) was nearly twice that of men (11.5 ng/ugSe/kg0.75) (P<0.001). Baseline plasma Se varied by glutathione peroxidase (GPX1) genotype: 198Leu/Leu, 135.7±19.0 ng/ml; 198Leu/Pro, 139.2±23.8 ng/ml; 198 Pro/Pro, 145.9±24.4 ng/ml (P<0.05). The 12‐mo. plasma Se response was not related to GPXi genotype; however, the urinary Se response of 198Leu/Leu individuals was only 63% of that of 198Pro/Pro individuals (P<0.006).
Selenium regulation of selenoproteome expression in rats
Authors: Sunde Roger A , Barnes Kimberly M , Raines Anna M , Evenson Jacqueline K
Published: June 2021

Glutathione peroxidase‐1 (Gpx1) mRNA levels as well as enzyme activity decrease dramatically in Se deficiency in rats, and both have been used to assess Se status and Se requirements. To determine the Se regulation of the complete selenoproteome, male weanling rats were supplemented with graded levels of Se (0 to 0.8 μg Se/g) for 28 d. There was no effect of Se deficiency on growth. Liver and kidney Se concentrations in Se‐deficient rats were 3 and 14% of Se‐adequate levels, respectively, and reached breakpoints at 0.1 μg Se/g. Se‐deficient plasma Gpx3 activity, and liver and kidney Gpx1 activities were 2, 2, and 6%, respectively, of Se‐adequate levels, with plateau breakpoints at 0.1 μg Se/g. Liver Gpx1, Sepw1, Selk, Selh, and Gpx3 mRNA levels were 11, 21, 28, 29, and 33%, respectively, of Se‐adequate plateau levels, with plateau breakpoints at 0.05–0.07 μg Se/g. In kidney, Gpx1, Sepw1, Selh, and Gpx3 were also highly regulated by Se status, with similar plateau breakpoints. In addition, 6 mRNAs in liver and 3 mRNAs in kidney were moderately down‐regulated by Se status. The majority of selenoprotein mRNAs in liver (12) and kidney (16) were not significantly down‐regulated by Se deficiency. The plateau breakpoints for Se‐regulated mRNAs are similar, and all are less than breakpoints for Gpx1 activity, suggesting a common mechanism for this regulation. (Supported by UW WIS04909 and by NIH T32‐DK07665)
Abnormal mitochondrial dynamics in Alzheimer disease
Authors: Zhu Xiongwei , Wang Xinglong , Su Bo , Perry George , Smith Mark A.
Published: June 2021

Mitochondrial dysfunction is a prominent and early feature of Alzheimer disease, although the cause is unclear. Nonetheless, emerging evidence suggest that mitochondrial function is dependent on the dynamic balance of fission and fusion events. While an impaired balance of mitochondria fission and fusion is being increasingly implicated in neurodegenerative diseases, few studies have examined this aspect in AD. To address this issue, in this study, we investigated mitochondria morphology and distribution in biopsy brains from normal subjects and those from AD patients. We found disease‐related changes in mitochondrial morphology and distribution as well as changes in expression levels and distribution of mitochondrial fission and fusion proteins. To understand the underlying mechanisms of these mitochondria alterations in AD, we overexpressed or knocked down functional DLP1 and other mitochondrial proteins in rat primary neurons. Interestingly, in situations where functional protein changes mimicking that in AD, we found similar changes in mitochondrial morphology and distribution to that observed in AD neurons. We further demonstrated that elevated oxidative stress and increased amyloid‐β production are likely the potential pathogenic factors that cause impaired balance of mitochondrial fission/fusion.
CAP37, an inflammatory mediator in Alzheimer's disease
Authors: Pereira H. Anne , Logan Sreemathi , Beverly Kandice , Lanis Jordi , Hinsley Heather , Fung Kar‐Ming , Adesina Adekunle
Published: June 2021

CAP37 is an inflammatory mediator that plays a key role in innate immunity. Recently, we showed that CAP37 is present in the vasculature of patients with Alzheimer's disease (AD) and that it is a potent regulator of microglial functions. The objective of this study was to extend these observations to determine the role of CAP37 as an inflammatory mediator in AD. Tissue was obtained from temporal, parietal, midbrain, and hippocampus from 10 AD patients, patients diagnosed with Pick's disease, Diffuse Lewy Body Dementia with AD‐aging, vascular dementia with no AD pathology, and sex‐ and age‐matched controls. Immunohistochemistry showed that in addition to the previously observed endothelial expression of CAP37 in AD patients, CAP37 was expressed in hippocampal neurons including dentate gyrus as well as pyramidal neurons of regions CA1–4 and the subiculum. Tissues obtained from patients with other neurodegenerative diseases and age‐ and sex‐matched controls lacked staining for CAP37. In vitro studies demonstrated that CAP37 is expressed in a dose‐ and time‐dependent fashion in HCN‐1A neurons in response to beta‐amyloid peptide 1–40 and tumor necrosis factor‐alpha but not interleukin 1‐beta. These findings lead us to hypothesize that CAP37 induced in neurons in AD patients activates microglia resulting in the exacerbation of the inflammatory cascade leading to neuronal death. Support: Alzheimer's Association.
Phosphorylation of beta‐amyloid precursor protein (APP) cytoplasmic tail facilitates amyloidogenic processing during apoptosis
Authors: Gottardi‐Littell Numa R , Sodhi Chhinder P , Perez Ruth G
Published: June 2021

Secretion and progressive cerebral accumulation of β‐amyloid peptides (Aβ) derived by endoproteolytic (‘amyloidogenic’) processing of β‐amyloid precursor protein (APP) represent collectively an early and necessary event in the pathogenesis of Alzheimer's disease. We previously demonstrated that secretion of the neurotoxic species Aβ42 increases during staurosporine‐induced apoptosis in undifferentiated PC12 cells, in an endocytosis‐dependent manner. In the present study, we tested whether phosphorylation of the APP cytoplasmic‐tail is contributory to this apoptosis‐related increased Aβ‐secretory response. We demonstrate that cytoplasmic‐tail phosphorylation specifically at amino‐acid residue T668 (APP‐695 numbering) increases during staurosporine‐induced apoptosis, in parallel with activation of the mitogen‐activated, proline‐directed serine/threonine protein kinase ERK1. We demonstrate additionally that specific ERK inhibition during staurosporine induction, with serum‐free conditions, results in down‐regulation of APP phosphorylation at T668, together with attenuation of the increased Aβ‐secretory response. These results are consistent with APP cytoplasmic‐tail phosphorylation at T668 during apoptosis as contributory to increased Aβ42 secretion originating from the endocytotic pathway, likely with cell‐line restriction. This work was supported by NIH grant AG15066 awarded to NR Gottardi‐Littell.
Activation of oncogenic k‐ras in glioneuronal precursor cells results in simultaneous expansion of the subventricular zone and infiltrating glioma
Authors: Abel Ty William , Clark Cara , Bierie Brian , Chytil Anna , Aakre Mary , Gorska Agnes , Moses Hal
Published: June 2021

A subset of neoplastic cells within human high‐grade gliomas has been shown to possess stem cell‐like properties. These cells may sustain glioma growth, and their unique properties may confer resistance to current glioma treatments. Whether glioma stem cells derive from indigenous neural stem cells or from tumor cells that have re‐acquired stem cell‐like properties is unknown. To address this, we activated oncogenic K‐ras in mouse glioneuronal precursor cells using GFAP‐Cre. GFAP‐Cre/K‐rasG12D mice showed a marked expansion of the subventricular zone/rostral migratory stream (SVZ/RMS), a stem cell population in the mature rodent brain. Each mouse (n =7) also developed bilateral, intermediate grade, infiltrating glioma. Tumors were characterized by hyperchromatic, angular cells, satellitosis of neurons, scattered mitotic figures, elevated Ki67 labeling, and GFAP expression. Tumors were consistently located in the amygdalohippocampal region and nearby cortex, often in association with the lateral ventricle. In some cases, foci of infiltrating tumor were contiguous with the expanded SVZ/RMS. Tumor cells expressed markers associated with neural progenitor cells, including Olig2 and PDGFR‐α. These data strongly suggest that the infiltrating tumor cells are derived from neural stem cells transformed by activation of oncogenic K‐ras and provide an animal model for investigating the role of neural stem cells in gliomagenesis. This work was supported by a Vanderbilt Physician Scientist Development award (TWA).
Novel origins of medulloblastoma from Olig2+ progenitors of the rhombic lip
Authors: Ligon Keith Lloyd , Schuller Ulrich , Heine Vivi , Dillon Allison , Mao Junaho , Kho Alvin , Han Young Goo , Sun Tao , Ma Qiufu , Alvarez‐Buylla Arturo , McMahon Andrew , Rowitch David
Published: June 2021

The most common malignant pediatric brain tumor, medulloblastoma (MB), arises from so far undefined progenitor cells within the cerebellum. Here, we identify a novel population of progenitor cells within the rhombic lip defined by expression of the transcription factor Olig2. Conditional activation of Sonic hedgehog signaling in these progenitors produces MB globally similar to those originating from unipotent Math1+ granule neuron precursors. All tumors exhibit multilineage differentiation and proliferating Olig2+ cells, similar to human medulloblastoma. These studies suggest that MB may originate from diverse populations of progenitor cells, in addition to those in the EGL, but have a convergent stem‐cell phenotype even when derived from non‐stem cell populations.
Transcriptional regulation in the MAPT H1/H2 Haplotype Block
Authors: Rhodes C. Harker , Haroutunian V , Katsel P , Tsongalis G J , Lefferts J A , Sibille E , Lathrop M J , Fiering S N
Published: June 2021

The protective effect of the MAPT H2 haplotype against neurodegenerative diseases suggested that a genome wide screen comparing normal control individuals with and without an H2‐containing chromosome might provide insights into the molecular biology of those diseases.

RNA from the anterior cingulate and amygdala from 18 individuals with no neurologic disease was examined by U133 expression array analysis. Four of the subjects were heterozygous for the MAPT H1/H2 haplotype, one subject was an H2 homozygote, and the remaining 13 were H1 homozygotes. Comparison of H1 homozygotes and H2 carriers revealed 5 transcripts which were expressed at significantly higher levels in H2‐carriers. This result was confirmed by a quantitative PCR assay of one of the transcripts and by U133 array analysis of RNA from multiple brain areas in an independent cohort. All five transcripts map to the 1 Mb inversion containing the MAPT locus. None of the transcripts comes from a named gene or contains a known miRNA.

The coordinated up‐regulation of these transcripts suggests a global difference in transcriptional regulation in the H1 and H2 chromosomes. This hypothesis is in contrast to, but is not mutually exclusive with, the usual explanation that the protective effect of the H2 haplotype is due to specific functional polymorphisms.
The distinct pattern of tau degeneration in dementia pugilistica
Authors: McKee Ann Carolyn , Stern Robert A , Gavett Brandon E , Cantu Robert C
Published: June 2021

We examined the brains of 3 demented male ex‐boxers, including one middleweight world champion, age 72–79 years, using immunocytochemistry for phosphorylated tau (AT8, PHF‐1, CP‐13) and Aβ. We found dense tau immunoreactive neurofibrillary tangles (NFTs), glial tangles, neuropil threads and spindles, often in clusters, in the superficial layers of cerebral cortex (I‐III). Cortical tau pathology was most prominent in the sulcal depths of the frontal and temporal lobes, especially the medial temporal lobe, often in a vascocentric pattern. Tau positive glia and spindle‐shaped neurites were also found in the subcortical white matter and corpus callosum. The olfactory bulb, thalamus, hypothalamus, nucleus basalis, striatum, globus pallidus, substantia nigra, raphe, periventricular gray, locus ceruleus, oculomotor nucleus, red nucleus, pontine base, tegmentum, reticular nuclei, inferior olives, and dentate nucleus also showed dense NFTs and glial tangles. Spindle‐shaped neurites and tau positive glia were pronounced in the midline white matter tracts of the brainstem. Aβ neuritic plaques were found in 2 cases. The pattern of tau pathology in dementia pugilistica is distinct from AD; its distribution in superficial cortex, deep nuclei, brainstem and white matter tracts may reflect its origin from traumatic neuronal and axonal disruption. Supported by NIA P30 AG13846, and by the Dept of Veteran's Affairs.
Involvement of α‐Synuclein in Neurodegeneration in Multiple Sclerosis?
Authors: Lu Jian‐Qiang , Fan Yan , Mitha Alim P , Yong Wee V
Published: June 2021

Multiple sclerosis (MS) is associated with neurodegenerative features including widespread brain atrophy, neuronal/axonal loss in the deep gray matter nuclei, as well as demyelination in the cortex and in normal appearing white matter. The cause of neurodegeneration in MS is unclear. We aimed to test the hypothesis that the neurodegeneration in MS may be contributed by the mechanisms underlying classic neurodegenerative diseases such as α‐synucleinopathies. Seventy‐nine MS lesions from 9 patients were examined, compared with the corresponding regions of 6 neuropathologically normal controls. α‐Synuclein immunoreactivity was found in the cytoplasm of neurons, activated microglia, and oligodendrocytes within and surrounding the MS lesions. The immunoreactive cells were identified in the active [15/15 within the brainstem; 5/8 within the cerebral hemispheres (CH)] and chronic active [11/12 within the brainstem; 6/15 within CH] lesions, but not in the inactive lesions or controls. This α‐synuclein immunoreactivity was preferentially expressed in lesions of the brainstem (p < 0.05, vs. CH). Analysis of double‐immunofluorescence labeling shows co‐localization of α‐synuclein with neuronal (NeuN), microglial (Iba1) and oligodendroglial (Nogo‐A) markers, but not with astrocytes (GFAP). We suggest that α‐synuclein regulated by inflammatory signals may be involved in the pathology of MS.
Regulation of valvular interstitial cell phenotype by matrix mechanics involves alpha‐smooth muscle actin
Authors: Yip Cindy Ying Yin , Chen Jan‐Hung , Simmons Craig A
Published: June 2021

Introduction: Remodeling of the extracellular matrix (ECM) is a hallmark of valvular calcification. Remodeling may alter the mechanical properties of the ECM and consequently regulate the fate of valve interstitial cells (VICs) to a pathologic phenotype. Objectives: To determine if matrix stiffness regulates calcification by VICs in vitro, and to investigate the role of alpha‐smooth muscle actin (SMA) in mechanically‐regulated cell responses. Methods: Primary porcine aortic VICs were cultured on topographically identical stiff and compliant Type I collagen matrices. We compared the morphology, proliferation, and differentiation of VICs by (immuno)staining, RT‐PCR, and biochemical analyses. Results: Compared with VICs grown on stiff substrates, VICs grown on compliant substrates proliferated more rapidly and formed more bone‐like nodules (P<0.05) that displayed alkaline phosphatase activity, calcium accumulation, bone gene expression, and viable cells. VICs on stiff substrates expressed abundant filamentous SMA in stress fibres, while those on compliant substrates did not. Disruption of SMA stress fibres with Swinholide A had no effect on VICs on compliant matrices, but further reduced the number of bone‐like nodules on stiff matrices. Conclusion: This study demonstrates for the first time that mechanical cues from the matrix, mediated in part by SMA, determine VIC phenotype.
DDR1: a novel regulator of intimal calcification
Authors: Ahmad Pamela Janet , Speer Mei Y , Giachelli Cecilia M. , Bendeck Michelle P.
Published: June 2021

Intimal calcification is a serious complication of advanced atherosclerotic disease. Low density lipoprotein receptor‐deficient mice (Ldlr−/−) fed a high fat diet develop complicated atherosclerotic lesions. Here we report that Ldlr−/−mice develop foci of intimal calcification in the aortic arch, which are positive for Alizarin Red S (AR) and Von Kossa (VK), two histological stains for mineralized tissues. Cells within these foci are surrounded by a type II collagen‐rich matrix and express a chondrocyte‐specific transcription factor, Sox‐9. Atherosclerotic lesions are rich in collagens, which have been shown to promote smooth muscle cell‐mediated calcification. Thus, we hypothesized a role for the discoidin domain receptor 1 (DDR1), a collagen receptor, in intimal calcification. Male mice with a combined deficiency in LDLR and DDR1 (Ldlr−/−;Ddr1−/−) and controls (Ldlr−/−;Ddr1+/+) were fed a high fat diet for 12 weeks. DDR1‐deficiency attenuated the incidence of intimal calcification, measured by positive staining for AR and VK. Aortic arch calcium extraction confirmed a reduction in the extent of calcification in the Ldlr−/−;Ddr1−/− mice compared with control mice (45 ± 22 vs 123 ± 52 μmol/g dry weight). This study demonstrates regions of calcification within the intimal lesions of Ldlr−/− mice fed an atherogenic diet and provides novel evidence of a role for DDR1 in this process.
Cell and tissue dynamics during gastrulation
Authors: Rongish Brenda J , Zamir Evan A , Cui Cheng , Little Charles D
Published: June 2021

During avian gastrulation epiblastic/presumptive mesodermal cells ingress through the primitive streak, subsequently moving away to re‐epithelialize and form tissue sheets. Our time‐lapse studies demonstrate the extent of cell‐autonomous motility compared to passive tissue motion during gastrulation. Particle image velocimetry allowed computation of tissue displacement fields, using the motion of ECM fibrils as in situ markers for tissue displacements — while simultaneously tracking individual cells in the same embryo. This approach allows calculation of active cell‐autonomous motility versus passive convective tissue motion. The data demonstrate that passive tissue motion contributes significantly to apparent mesodermal cell trajectories. Notably: 1) ingression of epiblastic cells, into and through the streak, is accompanied by corresponding passive ingression of ECM fibers; and 2) cell‐autonomous motility decreases as “new” mesodermal cells move away from the streak and are incorporated into ECM rich tissue sheets. These data greatly clarify the biophysical mechanisms that drive mesodermal cell movements in warm‐blooded embryos. The work has profound implications for understanding cell guidance mechanisms, presumptive morphogen gradients and tissue morphogenesis during gastrulation. Supported by the G. Harold and Leila Y. Mathers Charitable Foundation (CDL, BJR).
Peer teaching in medical gross anatomy
Authors: Suarez‐Quian Carlos Andres , Weissinger Peggy
Published: June 2021

Georgetown developed a competency based curriculum consisting of knowledge‐, skill‐, and values‐related competencies. These included (1) knowledge of biomedical science and ability to, integrate and apply this knowledge to patient care, (2) an understanding of the scope and limits of one's knowledge and skills, (3) ability to communicate and collaborate with colleagues and (4) the ability to learn independently. The first year gross anatomy course was redesigned to support these curricular‐level objectives by assigning students to participate in peer teaching. Students were placed into groups of five and assigned to prepare in advance the day's prelab talks. Over the life of the course, each student was responsible for presenting 5 talks. Prosected cadavers and extensive web and library based material were available for students to preview prior to their talks, but only limited, faculty guidance. At the end of the course, students were encouraged to self‐assess and to assess their peers. The surveys consisted of three questions, two using a Likert scale (Knowledge, Preparation and Organization), and one open‐ended question for self reflection and anonymous feedback to peers. Student performance improved markedly on the laboratory component of the mid‐term, and we are now awaiting results from the final exams. We suggest that to truly learn something, it helps to teach it.
GABAergic circuits in the hippocampus
Authors: Klausberger Thomas
Published: June 2021

In the hippocampal CA1 area a relative homogenous group of pyramidal cells is accompanied by 21 different classes of GABAergic interneurons. Different interneurons make synapses with distinct compartments of pyramidal cells, but why is such a large diversity of distinct interneurons required?

We recorded from identified interneurons in vivo and showed that interneurons targeting different domains of pyramidal cells exhibited distinct firing patterns. The spike timing of parvalbumin expressing basket and axo‐axonic cells indicates roles in synchronizing the sub‐threshold oscillations and output of pyramidal cells, respectively. The spike timing of bistratified and O‐LM cells, which innervate dendrites of pyramidal cells in the stratum radiatum and lacunosum‐moleculare, respectively, suggests their role in phasing EPSPs and de‐inactivating voltage‐gated ion channels of pyramidal cells; bistratified cells also synchronise oblique pyramidal cells dendrites to field gamma oscillations. We discovered GABAergic neurons projecting to subiculum, retrosplenial cortex and medial septum, possibly supporting synchronisation across brain areas. Another class of interneuron expresses neuronal nitric oxide synthase and provides slowly‐rising GABAergic inhibition in pyramidal cells.

Our results indicate that different GABAergic interneuron release GABA at distinct times to different domains of pyramidal cells explaining the need of diverse classes of interneurons.
Mechanism of Ubiquitin Chain Formation by the human Anaphase‐Promoting Complex
Authors: Rape Michael
Published: June 2021

The ubiquitin ligase Anaphase‐Promoting Complex (APC/C) is essential for proliferation in all eukaryotes. It orchestrates progression through mitosis by catalyzing the disassembly of spindle checkpoint complexes and by promoting the sequential degradation of mitotic regulators. The APC/C mediates its functions by decorating substrates with ubiquitin chains of unknown topology. Previously, we had found that the number of substrate binding events required for ubiquitin chain formation, referred to as the processivity of ubiquitination, can determine the timing of substrate degradation. However, the mechanism that enables the human APC/C to assemble ubiquitin chains within a single substrate binding event has remained elusive. Similar to many ubiquitin ligases, the APC/C nucleates ubiquitin chain formation by transferring ubiquitin to a lysine in its substrate, but elongates chains by modifying lysine residues in attached ubiquitin molecules. Here, we have determined the topology of the ubiquitin chains that are assembled by the human APC/C and its specific E2 UbcH10. This allowed us to identify a patch of charged amino acids on the surface of ubiquitin, the TEK‐box, which is required for the APC/C‐dependent elongation of ubiquitin chains. Strikingly, sequences highly related to the TEK‐box in ubiquitin are found in APC/C‐substrates, where they facilitate the modification of substrate lysine residues, and thus, ubiquitin chain nucleation. We propose a mechanism of ubiquitin chain formation by the human APC/C, in which recognition of TEK‐boxes first orients lysine residues within substrates and later in ubiquitin relative to the active site of the E2 to allow the rapid formation of specific ubiquitin chains.
A replicase sliding clamp bound to DNA and a novel chemical compound that inhibits DNA replication
Authors: O'Donnell Mike , Georgescu Roxana E , Kuriyan John , Kong Xiang‐Peng
Published: June 2021

Cellular replicases utilize a sliding clamp that holds the replicase to DNA for high processivity. We have solved the structure of the E. coli β clamp in complex with a peptide of the Pol III replicase and compare it to the structures of β–δ and Pol IV‐β. In overview, all proteins that bind the clamp utilize a common protein binding pocket, although they also bind unique positions on β. Using this information we have devised a high‐throughput assay to screen chemical libraries for small organic chemicals that disrupt interaction between β and Pol III. We have obtained several compounds, some of which inhibit E. coli cell growth. The clamp binding pocket is conserved and some chemical compounds also inhibit gram positive clamp‐DNA polymerase interactions.

We have also solved the structure of β clamp bound to primed DNA. The duplex is highly tilted within the ring. This may facilitate switching of DNA among different polymerases bound to one clamp. Surprisingly, template ssDNA fits into the protein binding pocket of β. In fact, a polymerase peptide competes the ssDNA off of β, implying that ssDNA binding to β serves a “placeholder” function that locks the clamp to the primed site after clamp loading. This interaction is severed upon association of Pol III to β, and this frees the clamp for rapid sliding on dsDNA during fast processive replication.

(Funded by the NIH).
Rapamycin blocks leucine‐induced protein synthesis by suppressing mTORC1 activation in skeletal muscle of neonatal pigs
Authors: Suryawan Agus , Jeyapalan Asumthia S. , Orellana Renan A. , Nguyen Hanh V. , Wilson Fiona A. , Davis Teresa A.
Published: June 2021

Skeletal muscle in the neonate grows at a rapid rate due in part to an enhanced sensitivity to the postprandial rise in amino acids, particularly leucine (Leu). To elucidate the molecular mechanism by which Leu stimulates protein synthesis in neonatal muscle, overnight fasted 7‐day‐old piglets were treated with rapamycin (an inhibitor of mammalian target of rapamycin complex 1/mTORC1) for 1 h and then infused with Leu for 1 h. Fractional rates of protein synthesis and activation of signaling components lead to mRNA translation were determined. Rapamycin completely blocked Leu‐induced muscle protein synthesis. Rapamycin markedly reduced raptor‐mTOR association, an indicator of mTORC1 activation. Consequently, phosphorylation of mTOR, S6K1, and 4EBP1, and eIF4E·eIF4G complex abundance were decreased and eIF4E·4EBP1 complex abundance was increased. Rapamycin had no effect on the association of mTOR with rictor, a crucial component for mTORC2 activation. Neither Leu nor rapamycin affected the phosphorylation of AMPK, PKB, or TCS2, signaling components that reside upstream of mTOR. eEF2 phosphorylation was not affected by Leu or rapamycin, although current dogma indicates that eEF2 phosphorylation is mTOR‐dependent. Taken together, these in vivo data suggest that Leu stimulates muscle protein synthesis by enhancing mTORC1 activation and its downstream effectors. NIH AR44474, USDA 58‐6250‐6‐001
The level of nitrogen supply modulates the expression of arginine metabolizing enzymes in Caco‐2/TC7 intestinal cells.
Authors: Ventura Gabrielle , Moinard Christophe , Carrière Véronique , Chambaz Jean , Cynober Luc , De Bandt Jean‐Pascal
Published: June 2021

Portal arginine (Arg) plays a key role in liver ureagenesis. Intestine ability to control Arg availability from the diet may depend on a modulation of arginase, glutaminase, ornithine aminotransferase (OAT), argininosuccinate lyase (ASL) and synthetase (ASS) expression, according to protein supply. The aim of this work was to study the influence of amino acid (AA) supply on the intestinal expression of these enzymes.

Caco‐2/TC7 cells were exposed for 3 days to different media containing AA at 0 (0), 1 (1X), 2 (2X) or 4 (4X) fold plasma post‐prandial concentrations. Enzymes mRNA were quantified using real time PCR. 18S rRNA was used as an internal control. Results (mean ± SEM) were analyzed by linear regression.

There was an inverse relationship between AA supply and glutaminase (p= 0,028), OAT (p= 0,035), ASS (p= 0,028) and arginase (p= 0,058) expressions.

This study shows for the first time a direct effect of AA supply on transcriptional regulation of arginine metabolizing enzymes. Such a regulation would enable in situations of low protein supply to increase Arg catabolism and to decrease hepatic AA oxidation thus promoting nitrogen sparing.
Exploring the role of REDD1 in mTOR activation
Authors: McGhee Nora K. , Jefferson Leonard S. , Kimball Scot R.
Published: June 2021

REDD1 is a protein of unknown function that has previously been shown to be important for repression of mTOR activity in response to DNA damage, hypoxia, glucose deprivation, and glucocorticoid treatment. We hypothesized that REDD1 expression would increase during fasting and rapidly fall after refeeding, thereby mediating mTOR signaling to mRNA translation via phosphorylation of its substrates S6K1 and 4E‐BP1. To test these hypotheses in vivo male Sprague‐Dawley rats were fasted overnight and were allowed to re‐feed for 45 min. In gastrocnemius muscle REDD1 protein and mRNA increase with fasting and fall with re‐feeding. To further examine the mechanism involved in regulation of REDD1 expression, Rat2 fibroblasts were starved of serum and leucine for 2 h then leucine or insulin was added back for 30 min. In these experiments the amount of REDD1 protein increases with starvation and decreases upon insulin, but not leucine, add‐back. The siRNA knock‐down of REDD1 in Rat2 cells enhances S6K1 and 4E‐BP1 phosphorylation that occur with insulin add‐back. These results are consistent with a role for REDD1 in inhibition of mTOR activity under low insulin conditions. Supported by grants DK13499 and DK15658
Changes in skeletal muscle protein synthesis in trained adults during recovery from endurance exercise
Authors: Vislocky Lisa , Gaine P. Courtney , Pikosky Matthew , Bolster Douglas , Ferrando Arny , Wolfe Robert , Rodriguez Nancy
Published: June 2021

Endurance exercise stimulates mixed muscle protein synthesis (MPS) for several hours post‐exercise. However, the time course of this response is not well characterized.

PURPOSE: The purpose of this study was to characterize changes in MPS in trained persons following an endurance run.

METHODS: Following an overnight fast, participants (n=14; men=9, women=5) underwent muscle biopsies at 30, 190, and 210 min after an endurance exercise bout (75 min, 70% VO2peak or 45 min, 65% VO2peak). MPS was determined using a primed, continuous infusion of [2H5]‐phenylalanine and muscle biopsies at 30 and 190 min (EARLY) and 190 and 210 min (LATE).

RESULTS: LATE MPS values were significantly higher than EARLY values (0.237 ± 0.036 % / hr vs 0.067 ± 0.006 % / hr for LATE and EARLY, respectively; P < 0.001), with the eta squared statistic (0.048) indicating a large effect size of time. A strong positive correlation (r=0.571, P < 0.001) existed between time and skeletal muscle MPS values.

CONCLUSION: These data document an increase in MPS three hours after completing an endurance exercise bout in the fasted state. Given that the source of amino acids to support the noted increase in MPS during the fasted state is endogenous proteins, studies should be conducted to evaluate the benefit of protein supplementation and feeding on post‐exercise on protein utilization during the recovery period.
Preferential uptake of xanthophylls by human retinal pigment epithelial cells is mediated by scavenger receptor B1
Authors: Harrison Earl Howard , During Alexandrine
Published: June 2021

The purpose of this study was to investigate mechanisms by which carotenoids are taken up by human retinal pigment epithelial cells (ARPE‐19). When ARPE‐19 cells were fully differentiated (7–9 weeks), the xanthophylls lutein (LUT) and zeaxanthin (ZEA) were taken up by cells to an extent about two‐fold higher than beta‐carotene (b‐C) (P < 0.05). At 9 weeks of differentiation, the extents of uptake were 1.6% for b‐C, 2.5% for LUT and 3.2% for ZEA under identical incubation conditions. Differentiated ARPE‐19 cells did not exhibit any detectable beta‐carotene oxygenase 1 enzyme activity or convert exogenous b‐C into vitamin A. In experiments using specific antibodies against the lipid transporters CD36 and SR‐B1 cellular uptake of b‐C and ZEA were significantly decreased (40–60%) by treatment with anti‐SR‐B1, but not with anti‐CD36. Transfection of siRNA for SR‐B1 led to marked inhibition of SR‐B1 protein expression (∼90% knock‐down), which resulted in a decrease of cellular uptake of b‐C and ZEA by 51% and 87%, respectively. Thus, RPE cells preferentially take up xanthophylls versus the carotene b‐C by a process that appears to be entirely SR‐B1‐dependent for zeaxanthin and partly so for beta‐carotene. This mechanism may explain, in part, the preferential accumulation of xanthophylls in the macula of the retina. Supported by NIH Grant R01 HL049879.
Specificity of epithelial cell adhesion by Group A Streptococcus pilus proteins in the M1 serotype SF370 strain
Authors: Abbot Emily Louise , Smith Wendy D. , Pointon Jonathan A. , Chiriboga Carlos , Wilson Janet A. , Hirst Barry H. , Kehoe Michael A.
Published: June 2021

Group A Streptococcus (GAS) express long polymeric pili which are encoded in the hypervarible FCT‐region. In the serotype M1 strain SF370 these pili are composed of a major subunit (Spy128) and two ancillary proteins (Spy130 and Spy125). Deletion mutants of each of the individual subunit genes failed to adhere to human tonsil epithelium or human keratinocytes (HaCATs), suggesting that pili are essential in promoting the initial stages of GAS infection. However, which of the subunits is functioning as the primary adhesin in this pilus‐dependent interaction remains undetermined. Each of the three pilus subunits were expressed as a recombinant protein and their ability to interact with various epithelial models was examined by immunofluorescence using subunit specific antisera. Both r130 and r125 proteins adhered to the surface of tonsil epithelium and HaCAT cells, however r128 failed to bind to either model. The ability of subunit‐specific antisera to reduce parent strain (WT) bacterial adherence to human tonsil or HaCAT cells was examined. Incubation of WT GAS with anti‐Spy125 sera inhibited the ability of GAS to adhere to either cell model. In contrast, anti‐Spy 128 and anti‐Spy 130 sera had no affect on bacterial binding. These results suggest that although both Spy130 and Spy125 proteins have the ability to bind host cells, pilus adhesion specificity resides in the Spy125 subunit.

Sponsored by MRC.