bluuebunny/crossref_metadata_2025 huggingface dataset:
Effect of α‐ and γ‐Tocopherols on 4‐Hydroxyestradiol Induced Oxidative Stresses in MCF‐10A Breast Epithelial Cells
Authors: Sung Mi‐Kyung , Lee Eun‐Ju , Oh Seung‐Yeon
Published: June 2021

Both estrogen and oxidative stress are known risk factors for breast cancer development. Human breast epithelial cells(MCF‐10A) were incubated with 0.1uM 4‐ Hydroxyestradiol (4‐OHE2)with or without 30uM α‐, γ‐tocopherols(α‐T, γ‐T) for 24 to 96hrs. Intracellular ROS, glutathione and MnSOD expression as oxidative stress markers were measured. The level of DNA damage repair‐related protein including p53, PARP‐1, and BRCA1 were also measured. By oxidative stimuli, p53 is phophorylated and PARP‐1 was cleavaged, which leads to DNA repair. PARP‐1 is also essential in regulating BRCA1 in response to DNA damage. Results showed 4‐ OHE2 increased the intracellular ROS level with a down‐regulation of MnSOD protein. A significant negative correlation was found between ROS level and GSH/GSSG ratio. Cleavaged PARP‐1 was increased and BRCA1 and PARP‐1 depletion was observed with ROS accumulation. γ‐T reduced 4‐OHE2‐induced ROS, while increased GSH/GSSG ratio and MnSOD protein. γ‐T also up‐regulated phosphorylated p53 protein. α‐T is shown to up‐regulate BRCA 1 and PARP‐1 protein. In conclusion, 4‐OHE2 increases oxidative stresses which possibly down‐regulate protein related to DNA repair. α‐and γ‐T may prevent 4‐OHE2 induced oxidative stress and DNA damage and thereby retard breast tumor development.

This work was supported by the Korea Research Foundation Grant(KRF‐2005‐042‐C00194).
ALK1 signaling plays a pivotal role in regulation of genes involved in angiogenesis and vascular tone: implication on the pathogenetic mechanism for hereditary hemorrhagic telangiectasia 2 (HHT2)
Authors: Park Sung Ok , Lee Young Jae , Nguyen Ha‐Long , Han Chul , Choi Eun‐Jung , Oh S. Paul
Published: June 2021

ALK1 (Activin Receptor‐Like Kinase 1) belongs to the type I receptor family for TGF‐β family ligands. Heterozygous ALK1 mutations cause HHT2, a multisystemic vascular disorder, characterized by telangiectases and arteriovenous malformations (AVMs) in brain, lung, and visceral organs. Pathogenetic mechanisms underlying vascular malformations in HHT are poorly understood. To develop an animal model for HHT, we generated a Alk1 conditional knockout mouse line. Alk1 deletion in limited vascular endothelial cells by a novel endothelial Cre deleter line resulted in lethality by PN6 with massive hemorrhage in the lung. Numerous AVMs were detected in the cerebral and pulmonary vessels by corrosion casting and latex dye injections. The Alk1 mutants exhibited severe pulmonary vascular malformations mimicking all pathological features of HHT‐dilation of lumen, thinning of vascular walls, loss of capillaries, development of excessive tortuous vessels, and AVMs. Microarray analysis on PN3 and PN4 lungs of the control and mutant mice revealed that expression of genes involved in biosynthesis, metabolism or signaling of multiple pathways regulating angiogenesis and vascular tone was altered. These pathways include nitric oxide, angiotensin, endothelin, prostaglandins and sphingosine‐1‐phosphate pathways, providing important clues to understanding the pathogenesis of HHT.
Novel epigenetic mechanisms involved in enhanced inflammatory gene expression in vascular smooth muscle cells derived from diabetic db/db mice and relation to diabetic memory
Authors: Villeneuve Louisa M , Reddy Marpadga A , Lanting Linda L , Wang Mei , Meng Li , Natarajan Rama
Published: June 2021

Diabetes and associated inflammation are major risk factors for vascular complications despite glycemic control, thus implicating a “diabetic memory”. However, the molecular mechanisms are unclear. We hypothesized that dysregulation of histone H3 lysine trimethylation (H3K9me3), a key repressive epigenetic chromatin mark, may lead to diabetic memory. This was tested in vascular smooth muscle cells (VSMC) derived from insulin resistant type 2 diabetic db/db mice, which exhibit persistent pro‐atherogenic and inflammatory phenotype even in normoglycemic culture. H3K9me3 levels at promoters of key inflammatory genes (ChIP assays) and protein levels of the H3K9me3 methyltransferase Suv39h1 (W. Blot) were significantly decreased in db/db VSMC relative to non‐diabetic control db/+ cells. Furthermore, db/db VSMC were hypersensitive to inflammatory stimuli (TNFα) which induced dramatic reductions in promoter H3K9me3 and occupancies of Suv39h1. Suv39H1 gene silencing in non‐diabetic VSMC increased inflammatory genes, while conversely, Suv39H1 overexpression in db/db VSMC reversed this diabetic phenotype. These novel findings demonstrate protective roles for H3K9me3 and Suv39H1 in the pre‐activated diabetic cells. Aberrant epigenetic histone methylation may be a major mechanism for diabetic memory implicated in chronic inflammation and diabetic complications.

Support: NIH, Myrtle Carr Fdn
TLR4‐signaling is required for NOD2 but not NOD1 activation in hepatocytes
Authors: Scott Melanie J , Liao Hong , Billiar Timothy R
Published: June 2021

NACHT‐LRR receptors (NLRs) are recently identified intracellular pattern recognition receptors. The NLRs NOD1 and NOD2 respond to microbial ligands, e.g. diaminopimelic acid (DAP) and muramyl dipeptide (MDP) respectively. Hepatocytes (HC) are microbial‐responsive cells that express and activate Toll‐like receptors (TLRs). We hypothesized that HC also express and activate NOD1 and NOD2 as part of the initiation and regulation of innate immunity. Protein expression of NOD1 and NOD2 was detected in whole cell lysates of primary isolated HC from C57BL/6 mice. Other HC were treated with LPS (100ng/mL), iE‐DAP (100ng/mL; NOD1 ligand), or MDP (10μg/mL; NOD2‐ligand) for up to 60min before collection of whole cell lysates or nuclear isolation. NOD ligands were endotoxin free as determined by sensitive colorimetric endotoxin assay. MAPK (ERK, JNK, and p38) were activated and NFκB translocated to the nucleus in HC treated with iE‐DAP (NOD1 ligand), to similar levels as with LPS‐stimulation. However there was minimal activation of NFκB with MDP (NOD2 ligand) unless cells were pretreated for 24h with LPS. Activation of NFκB after MDP stimulation (NOD2 ligand) was partially TLR4‐dependent but activation of NFκB with iE‐DAP was similar in isolated WT and TLR4−/− HC. Taken together these data show expression of NOD1/NOD2 in HC, and differential regulation of their activation by TLR4.

Work supported by NIH grant R01‐GM‐50441
Arachidonic acid increases MCF‐10A and MCF‐10CA1 motility dependent on PPAR‐gamma
Authors: Carter Jennifer C. , Church Frank C.
Published: June 2021

Recently, a high‐fat Western diet has been linked to an increase breast cancer risk. This high‐fat diet has an increased of omega‐6 (ω–6) poly‐unsaturated fatty acids (PUFAs) compared to omega‐3 (ω–3) PUFAs. We are investigating the role of one such ω‐6 PUFA, arachidonic acid (ArA), in breast cancer cell motility. ArA has been shown to be involved in increased cell motility and survival. Interestingly, ArA has also been shown to activate peroxisome‐proliferator activated receptor‐gamma (PPAR‐γ), a protein involved in adipocyte differentiation and plasminogen activator (PA) system regulation. We used two cell lines, MCF‐10CA1, an invasive carcinoma line and normal breast epithelial line, MCF‐10As, cells to study the effects of ArA. Chemotaxis to epidermal growth factor (EGF) was greatly increased in MCF‐10CA1 cells compared to the normal MCF‐10As. Arachidonic acid treatment increased chemotaxis in both cell lines about 2‐fold. Treatment with ArA increased uPA activity MCF‐10A cells, with a less dramatic increase in MCF‐10CA1 cells. To determine if ArA was acting on PPAR‐γ, we pretreated the cells with GW9662, a PPAR‐γ antagonist, and saw the ArA mediated increase in motility was abolished in both cell lines. This data suggests ArA increases cellular motility of normal and malignant cells through alterations in PA system components, through a PPAR‐γ dependent mechanism.
Computational Modelling Identifies Morphologic Predictors of Tumor Invasion
Authors: Bearer Elaine , Cristini Vittorio
Published: June 2021

We hypothesize that tumor morphology can be derived from first principles of physics: conservation of mass, energy and momentum. With a series of interdependent partial differential equations describing diffusion gradients of metabolites, cell division, adhesion and migration, we have designed a computational model that produces three‐dimensional architecture of tumor growth as a function of these variables. Through “constitutive” laws, these parameters are dictated in turn by a mutation array describing alterations of key cellular pathways in carcinogenesis. We calculated parameters from 36 human glioblastoma samples. Parameters affecting tumor growth in silica were compared statistically to behavior present in biopsy samples. Computer simulations demonstrate that morphological instability drives increased nutrient demand, introducing hypoxic diffusion gradients in the microenvironment. This results in nutrient gradients within the tumor that either cause necrosis or provoke hypoxic tumor cells to infiltrate adjacent tumor and/or brain. This invasive pattern is consistent with our experiments and with other in‐vitro and in‐vivo observations. We predict that increase in distance between capillaries at tumor margins is evidence of invading malignant cells and serves as a previously unrecognized histological marker of tumor‐brain margin. Supported by NINDS NS046810 and NIGMS GM47368.
The metastatic dichotomy between carcinomas and sarcomas is explained by differential lymphovasculogenic mimicry
Authors: Barsky Sanford H , Shott Robert , Jones Susie , Ostler Joe , Mahooti Sepi , Ye Yin
Published: June 2021

Human carcinomas are known to metastasize primarily through lymphatics whereas sarcomas metastasize hematogenously. However the reason for this metastatic dichotomy is not understood. With the newly emerging experimental evidence that lymphovascular invasion (LVI) and metastasis may be regulated by local lymphovasculogenesis, we wondered whether these phenomenon might differ in carcinomas v sarcomas and account for their metastatic disparity. We examined 20 human carcinomas and 20 sarcomas as well as derived cell lines to assess their comparative degrees of lymphangiogenesis and vasculogenesis. We conducted IHC studies with lymphatic (D2‐40), vascular (CD31), proliferation (Ki‐67) and myoepithelial / mesenchymal stem cell markers singly and in combination, designed to elucidate the nature of the lymphovasculogenesis in carcinomas v sarcomas. We also studied the expression profile of VEGF members: ‐A, ‐B, ‐C, ‐D in the tumors by real time PCR. The carcinomas exhibited a much greater density of lymphatics with LVI limited to only lymphatics whereas the sarcomas exhibited a much greater density of blood vessels with LVI limited to only blood vessels. However the lymphatics and blood vessels containing tumor emboli from carcinomas v sarcomas differed from each other and the resident vasculature in expression of myoepithelial v mesenchymal stem cell markers suggesting a different stem cell origin. The sarcomas paradoxically had 50 fold greater VEGF‐C whereas the carcinomas had 3 fold greater VEGF‐A. Our findings suggest that metastatic behavior of carcinomas v sarcomas is regulated by the type of LVI which, in turn, is governed not by stromal invasion nor VEGF‐mediated lymphovasculogenesis but rather by differential lymphovasculogenic mimicry.
Development of M cells within the follicle‐associated epithelium of Peyer's Patches depends on expression of TRANCE/RANKL by stromal cells
Authors: Freel Kathryn A , Taylor Rebekah T , Butler Betsy R , Kumar Nachiket , Williams Ifor R
Published: June 2021

M cells are specialized epithelial cells found in the follicle‐associated epithelium (FAE) covering Peyer's patches (PP). M cells sample small particulate antigens from the intestinal lumen and transport these antigens to intraepithelial pockets found on the basal aspect of M cells. PP from mice lacking B cells (μMT knockout mice) have a marked reduction (over 150‐fold) in the overall number of intestinal M cells, but the specific receptor‐ligand interaction(s) central to promoting normal M cell differentiation within the FAE have not yet been identified. We now report that PP from mice deficient in the TNF superfamily member TRANCE/RANKL (TNFSF11) display a defect in M cell development that is even more pervasive than that of μMT mice. M cells identified by whole mount immunofluorescence microscopy using rhodamine‐conjugated Ulex europaeus lectin (UEA‐1) are essentially absent from PP in the proximal and central portions of the small intestine. Rare UEA‐1+ M cells are present in a few follicles from the most distal PP of TRANCE‐deficient mice, but these M cells don't have the normal radial symmetry of FAE M cells and are instead abnormally clustered near the apex of the dome. The M cell deficiency in the PP of TRANCE‐deficient mice is associated with impaired formation of PP germinal centers and decreased fecal IgA. TRANCE is selectively expressed by subepithelial stromal cells located in the dome area of PP. These findings indicate that local production of TRANCE by PP stromal cells is required for the normal induction and differentiation of M cells within the FAE.

Supported by the NIH (DK‐64730, DK‐64399).
Guanylate binding protein‐1 (GBP‐1) is upregulated in intestinal epithelia under inflammatory conditions, localizes to tight junctions and regulates epithelial barrier function
Authors: Schnoor Michael , Betanzos Abigail , Parkos Charles
Published: June 2021

GBP‐1 belongs to the family of large GTPases, is strongly induced in leukocytes and endothelial cells after IFN‐γ treatment and regulates endothelial cell proliferation under inflammatory conditions. Given the dependence of GBP‐1 expression on IFN‐γ, and the elevated level of IFN‐γ within intestinal mucosa of individuals with inflammatory bowel disease (IBD), we investigated whether GBP‐1 is expressed in colonic epithelial cells. Analyses of different epithelial cell lines revealed that GBP‐1 expression was low to absent in all cells tested under resting conditions, but was strongly induced in all tested epithelial cell lines after IFN‐γ treatment. Localization studies revealed that GBP‐1 is expressed at the plasma membrane and colocalized with the tight junction protein coxsackie‐ and adenovirus receptor (CAR) only in colonic epithelial cells. Immunofluorescence labeling of human colonic mucosa revealed upregulation of GBP‐1 expression in the epithelium of individuals with active IBD. Intriguingly, colocalization of GBP‐1 with CAR was also observed in IBD patients. Functional studies employing siRNA‐mediated knock down of GBP‐1 in colonic epithelia demonstrated delayed epithelial barrier formation and increased susceptibility to apoptosis. These data suggest that GBP‐1 expression in colonic epithelia may play a role in regulation of the epithelial barrier under inflammatory conditions.
Traffic Control: Cellular Dynamics in the Lymph Node
Authors: Bradley Linda M
Published: June 2021

Appropriate cellular trafficking with regulated entry, retention, and egress of mononuclear cells is a fundamental feature of lymph node biology. The cells of the immune system are constantly in motion after entering a lymph node from the blood. The movements of the principal residents, T cells, B cells, and dendritic cells (DCs) are controlled via adhesion receptors and chemokines that determine positioning within the tissue. The T cells move into the cortex while the B cells move into the follicles. In the absence of an immune response, the naive cells undergo regulated egress via the sinuses into efferent lymph. An immune response results in profound changes within the lymph node. The response is initiated by antigen‐presenting migrating DCs that enter the lymph node via the afferent lymph, and resident DCs. The DCs serially engage T cells in the cortex, ultimately culminating in T cell division and differentiation to effector cells. B cells that capture antigen migrate to the border of the cortex and follicles where they form conjugates with T cells. B cells then undergo the germinal center response culminating in the development of plasma cells. Both T and B cells are initially retained in lymph nodes, but they are ultimately released as effector cells. As the response resolves, homoestasis returns. The current understanding of these cellular dynamics and their molecular regulation will be presented.
Regulation of blood vessel patterning and branching
Authors: Eichmann Anne
Published: June 2021

The vertebrate vascular system is comprised of a highly branched network of arteries, capillaries and veins that penetrates all body tissues. To build a functional network during development, endothelial cells must navigate through tissue corridors and precisely project to their targets. Recent results showed that specialized endothelial cells, resembling axonal growth cones, are located at the tips of growing capillaries. These endothelial tip cells guide outgrowing capillaries in response to gradients of extracellular matrix‐bound vascular endothelial growth factor. We have identified several negative regulators of vessel branching, including the Notch ligand Delta‐like4 and the UNC5B receptor for the axon guidance cue Netrin‐1. Roles for these molecules in vessel morphogenesis during development and in pathological angiogenesis will be described.
Study of the impact of trans fatty acids from ruminants on blood lipids and other risk factors for cardiovascular disease
Authors: Motard‐Bélanger Annie , Charest Amélie , Grenier Geneviève , Paquin Paul , Chouinard Yvan , Lemieux Simone , Couture Patrick , Lamarche Benoit
Published: June 2021

While the intake of trans fatty acids from industrially hydrogenated oils (iTFA) is known to have deleterious effects on cardiovascular health, the effects of TFA from ruminants (rTFA) are unknown. The purpose of this study was to compare the effects of rTFA and iTFA on LDL‐C and other cardiovascular disease risk factors in human. In a double‐blind, randomized controlled crossover study, 38 healthy men were fed each of 4 experimental isoenergetic diets lasting 4 weeks each. The 4 diets were 1‐ high in rTFA (10.2 g/2500 kcal), 2‐ moderately high in rTFA (4.2 g/2500 kcal), 3‐ high in iTFA (10.2 g/2500 kcal) and 4‐ the control diet low in TFA from any sources (2.2 g/2500 kcal). LDL‐C levels were higher after the high rTFA diet than after the control (p=0.03) and the moderate rTFA diets (p=0.002). LDL‐C levels were also higher after the iTFA diet compared to the moderate rTFA diet (p=0.02). HDL‐C were lower after the high rTFA diet than after the moderate rTFA diet (p=0.02). All risk factors were comparable between the control and the moderate rTFA diets. While a very high dietary intake of TFA from ruminants may have a deleterious impact on cholesterol homeostasis, consumption of rTFA corresponding to the upper limit of current human consumption has neutral effects on plasma lipids and other cardiovascular risk factors. Financial support: Dairy Farmers of Canada, Novalait, Canada Research Chair in Nutrition and Cardiovascular Health.
High intakes of fructose are associated with alterations in liver fatty acid composition in rats
Authors: Riediger Natalie Diane , Chen Qilin , Abdullah Mohammad , Kroeker Amy , Lanoo Christy‐Anne , Othman Rgia , Xu Zuyuan , Zou Jitao , Azordegan Nazila , Moghadasian Mohammed H
Published: June 2021

Background: Diets high in fructose may result in abnormalities in glucose and lipid metabolism.

Purpose: To investigate the effects of a high‐fructose diet on liver fatty acid composition.

Methods: Male Sprague‐Dawley rats were divided into two groups: a control group (n=5) receiving standard chow diet, and treatment group (n=7) receiving a diet containing 10% w/w lard and 60% w/w fructose for 20 weeks. Fatty acid profile of various lipid fractions in the liver of these animals was characterized.

Results: Total liver lipids including cholesterol and triacyglycerol (TAG) were significantly higher in the treated group as compared to controls. The livers from treated animals had significantly lower levels of C16:0 and higher levels of C18:1 in the TAG fraction, and lower levels of C18:1c11, C18:2c9, C20:1c11, C20:1c13 along with higher levels of C16:1 and C18:1c9 in the free fatty acid fraction. The levels of C16:0, C18:2c9, C20:1c11, C20:1c13, C20:2c11 and C24:0 were significantly increased in the phosholipid fraction of the treated animals.

Conclusions: High intakes of fructose change liver lipid composition and fatty acid profile in rats. TAG and phospholipid fractions seem to be affected more. These changes may lead to alterations in hepatocyte functions including lipid metabolism and may modify cardiovascular risk.
An impaired gut immune response to a high‐fat diet is associated with visceral adipose inflammation and peripheral insulin resistance in leptin‐deficient mice
Authors: Gieng Sin Hoa , Eshraghi Aria , Hawley Elizabeth , Sanger Patrick , Menshenina Julia , Shigenaga Mark K
Published: June 2021

The role of leptin as an immune modifier in the gut following standard chow versus high‐fat, no‐fiber (HF) diet feeding was examined in Ob/Ob and wild‐type (WT) mice. Ileum and mesenteric adipose gene expression, cecal digesta bacterial composition, and fasting levels of plasma glucose, insulin, and cholesterol were determined. Inflammatory markers (Saa3, Mcp1, and F4/80) in the ileum increased in response to HF diet in WT but not in Ob/Ob mice. In contrast, these markers in mesenteric adipose increased in Ob/Ob versus WT mice but were not affected by HF diet. Concurrently, total bacteria and Bacteroides decreased and Enterococcus and Lactobacillus increased in response to HF diet in both WT and Ob/Ob mice. However, Enterobacteriaceae increased in response to HF diet in Ob/Ob but not WT mice. Insulin resistance, as measured by HOMA‐IR, was higher in Ob/Ob mice in response to HF diet at week 8 and was further, but similarly, elevated in both Ob/Ob diet groups at week 14, a metabolic alteration that we postulate is associated with impaired gut immunity and shifts in gut bacteria. In conclusion, leptin deficiency is associated with an impaired gut immune response to a HF diet and is associated with alterations in gut bacterial composition, an accelerated increase in peripheral insulin resistance, and increased plasma cholesterol. Establishing a mechanistic link between gut immunity and insulin resistance will require experimental manipulation of gut bacteria, a current focus of our ongoing studies. Funded by NIH P60MD00222.
Bone, diet and physical activity in a group of Puerto Rican obese adults.
Authors: Palacios Cristina , Soltero Sandra , Rios Ruth , Beltran Jose Juan
Published: June 2021

Diet, physical activity and bone mineral content (BMC) and density (BMD) were measured in a group of Puerto Rican obese adults, 21–50 years old. A total of 29 subjects, 23 females and 6 males, participated in this study. Dietary intake was estimated from 3 24‐h diet records and daily physical activity level was measured by the Framingham Physical Activity Index during the same 3 days. BMD was measured by Dual‐Energy X‐Ray Absorptiometry (Hologic). BMI was 40.2±5.9 kg/m2, and 39.9±6.7 kg/m2, in females and males, respectively. Calcium intake was 528±146 mg/d and 624±197 mg/d, magnesium intake was 184.6±65 mg/d and 219±75 mg/d, vitamin K intake was 30±20 mcg/d and 17±6 mcg/d, and physical activity index score was 29±2.6 and 31±3, in females and males, respectively. Total BMC was 2325±389 g and 2856±577 g and BMD was 1.164±0.121 g/cm2 and 1.270±0.139 g/cm2, in females and males, respectively. BMI was significantly correlated to BMC (r=0.45, p<0.05). In this group of young obese adults, dietary intake was not related to BMC or BMD. Calcium, magnesium and vitamin K intakes were much lower than recommended intakes and most subjects had a sedentary lifestyle. A larger study is needed to determine the major determinants of bone in Puerto Ricans.

This investigation was supported by the RCMI Clinical Research Infrastructure Initiative (RCRII) Award, 1P20 ‐ RR11126, from NCRR, NIH.
Blood glucose response to a novel starch
Authors: Lee Sun‐Ok , Li Li , Hendrich Suzanne
Published: June 2021

We hypothesized that a new starch, which is resistant to human starch digestion, would cause less blood glucose response than a control starch of the same glucose content. The study used a crossover design of 15 healthy adult male subjects; 7–8 subjects/treatment/day, at each of two days spaced one week apart. Subjects consumed 200 mL beverage stock (containing the non‐caloric sweetener, sucralose, lemon flavoring, and yellow food color) mixed with 50 g of the control or resistant starch in a single meal at breakfast. All subjects completed a detailed questionnaire on gastrointestinal distress symptoms over the 24h after drinking the beverage. Subjects fed the resistant starch showed significantly lower blood glucose level compared with those fed the control starch at 15, 45 , 60 , 75 and 90 min after starch ingestion. The incremental AUCs (Area Under the Curve) for glucose were significantly less in subjects who consumed a new resistant starch beverage (4047 + 361 mg·(−15∼240)min/dL) than those who consumed control beverage (5247 + 404 mg·(−15∼240)min/dL). Minimal adverse side effects were seen among participants, and no difference between treatments in gastrointestinal distress. This effect of digestion resistant starch may help people to prevent diabetes and obesity. Funded by Grain Processing Corporation, and supported by the Center for Designing Foods to Improve Nutrition, Iowa State University.
EMAP II Overexpression Induces Endothelial Apoptosis and Emphysema in Murine Lungs
Authors: Clauss Matthias , Rajashekhar Gangaraju , Sigua Ninotchka , Kamocki Krzysztof , Ahlbrecht Katrin , Yildirim Ali Oender , Fehrenbach Heinz , Voswinckel Robert , Petrache Irina
Published: June 2021

Objective: Endothelial Monocyte‐Activating Polypeptide II (EMAP II) is a monocyte attracting, anti‐angiogenic and endothelial cells apoptosis inducing pro‐inflammatory cytokine. Based on our previous work linking endothelial apoptosis to emphysema we tested the hypothesis that inducible overexpression of EMAP II leads to apoptosis and emphysema in murine lungs. Methods: A transgenic murine model of tetracycline‐inducible lung specific EMAP II expression was employed. EMAP II overexpression was analyzed by Western immunoblotting of bronchoalveolar lavage fluid and immunohistochemistry of lung tissue. Endothelial apoptosis was determined in lung sections treated with active caspase‐3‐ and VE‐cadherin‐specific fluorescent‐labeled antibodies using confocal microscopy. Lung sections were stained with H&E for morphometric analysis of airspace enlargement. Results: Lung‐specific EMAP II overexpression resulted in a significant increase in caspase‐3 positive cells in lung parenchyma. A marked decrease in alveolar surface density was observed in transgenic mice after 3 months of induction compared to controls (39.4 + 2.4 vs 46.2 + 0.6 mm2/mm3, p=0.001). Conclusion: Transgenic EMAP II overexpression leads to increased endothelial apoptosis and emphysema‐like morphologic changes in murine lungs. These results suggest that EMAP II may contribute to the pathophysiology of emphysema development.
Role of beta3 Integrin in Pathological Angiogenesis
Authors: Feng Weiyi , Mahabeleshwar Ganapati H , Phillips David R , Byzova Tatiana V
Published: June 2021

Background: Angiogenesis depends on the coordinated action of numerous cell types. A key adhesion molecule expressed by most of these cells and having a regulatory function in angiogenesis is αvβ3 integrin. However, the exact role of this integrin in vascular pathologies remains unclear.

Method: Using a knock‐in mouse (diYF) characterized by impaired β3 integrin phosphorylation and signaling, the role of β3 integrin in angiogenesis was investigated.

Results: Analysis of angiogenesis in wounds and in implanted tumors revealed that the process of neovascularization is impaired in DiYF mice. The key regulatory function in angiogenesis appears to be dependent on the β3 integrin function in recruitment of bone marrow‐derived cells (BMDC) to the sites of neovascularization. BMDC did not directly incorporate into the tumor neovasculature, but were situated in the close proximity of blood vessels and might affect angiogenesis by secretion of key effectors. αvβ3 integrin controls BMDC recruitment and retention at the sites of angiogenesis in vivo. Impaired α3 integrin signaling in CXCR4+BMDC, but not in endothelium, diminishes adhesion and transmigration of CXCR4+cells through endothelial monolayer and their recruitment in angiogenic tissues.

Conclusion: β3 integrin controls angiogenesis and angiogenesis‐dependent processes of tumor growth and wound healing by regulating BMDC recruitment to neovasculature.
Ang II‐Inducible PDGF‐D Transcription Requires Ser/Thr Residues In The Second Zinc Finger Region of Sp1: Novel Phosphospecific Antibodies Demonstrate that Sp1 is Phosphorylated by SMC Injury
Authors: Khachigian Levon M , Berndt Michael C , Tan Nicole Y
Published: June 2021

Sp1, the first identified and cloned transcription factor, regulates gene expression via multiple mechanisms including direct protein‐DNA interactions, protein‐protein interactions, chromatin remodelling and maintenance of methylation‐free CpG islands. Sp1 is itself regulated at different levels, for example, by glycosylation, acetylation and phosphorylation by kinases such as the atypical protein kinase C‐zeta. Although Sp1 controls the basal and inducible regulation of many genes, the post‐translational processes regulating its function and their relevance to pathology are not well understood. Here we have used a variety of approaches to identify 3 amino acids (Thr668, Ser670 and Thr681) in the zinc finger (ZNF) domain of Sp1 that are modified by PKC‐zeta, and have generated novel anti‐peptide antibodies recognising the PKC‐zeta‐phosphorylated form of Sp1. Angiotensin II (Ang II), which activates PKC‐zeta phosphorylation (at Thr410) via the Ang II Type I receptor, stimulates Sp1 phosphorylation and increases Sp1 binding to the PDGF‐D promoter. All 3 residues in Sp1 (Thr668, Ser670 and Thr681) are required for Sp1‐dependent PDGF‐D activation in response to Ang II. Immunohistochemical analysis revealed that phosphorylated Sp1 is expressed in SMCs of human atherosclerotic plaques, and is dynamically expressed together with PDGF‐D in SMCs of the injured rat carotid artery wall. This study provides new insights into the regulatory mechanisms controlling the PKC‐zeta‐phospho‐Sp1 axis and Ang II‐inducible gene expression.
Cis‐regulatory control of the nodal gene, initiator of the sea urchin oral ectoderm gene network
Authors: Nam Jongmin , Su Yi‐Hsien , Lee Pei Yun , Robertson Anthony , Coffman James , Davidson Eric
Published: June 2021

Expression of the nodal gene initiates the gene regulatory network which establishes the transcriptional specification of the oral ectoderm in the sea urchin embryo. The cis‐regulatory system of the nodal gene transduces anisotropically distributed cytoplasmic cues that distinguish the future oral and aboral domains of the early embryo. Here we establish the genomic basis for the initiation and maintenance of nodal gene expression in the oral ectoderm. A 5′ cis‐regulatory module functions both to initiate expression of the nodal gene and to maintain its expression by means of feedback input from the Nodal signal transduction system. These functions are mediated respectively by target sites for bZIP transcription factors, and by SMAD target sites. At least one SMAD site is also needed for the initiation of expression. An intron module also contains SMAD sites which respond to Nodal feedback, and in addition acts to repress vegetal expression. These observations explain the main features of nodal expression in the oral ectoderm: since the activity of bZIP factors is redox sensitive, and the initial polarization of oral vs aboral fate is manifested in a redox differential, the bZIP sites account for the activation of nodal on the oral side; and since the immediate early signal transduction response factors for Nodal are SMAD factors, the SMAD sites account for the feedback maintenance of nodal gene expression.
FGF signaling is a major regulator of coronary tubulogenesis in the murine embryo
Authors: Zheng Wei , Christensen Lance P , Schatteman Gina C , Murakami Masahiro , Simons Michael , Tomanek Robert J
Published: June 2021

Our previous work has documented that FGF‐2 is a potent stimulator of both coronary endothelial cell proliferation and tube formation in quail embryos. The current study addressed the role of FGF signaling and Sca‐1+ bone marrow cells (BMC) in coronary tubulogenesis. Embryonic day 6 mouse hearts were explanted onto collagen gels and incubated for 7 days. Tubulogenesis (aggregate tube length), migration distance and cell number were quantified. Exogenous FGF‐2 enhanced tube formation 3‐fold, while adenovirus FGFR‐1DN, which virtually abolishes all FGF signaling, decreased tubulogenesis by about 50%, and markedly inhibited cell proliferation. Soluble adenovirus FGFR‐3 IIIb (or soluble R3IIIb‐Fc), which can bind and inhibit FGF‐1 and FGF‐9, did not significantly inhibit tubulogenesis, but reduced the number of endothelial cells. Addition of sca‐1+ BMC caused only a small (20%) increase in tubulogenesis. The data support the hypothesis that cell proliferation and tube formation are regulated by different FGF ligands.
Role of Non‐coding RNAs in Heterochromatin Formation
Authors: Moazed Danesh
Published: June 2021

Assembly of heterochromatin at the fission yeast centromeres requires components of the RNAi machinery as well as histone binding proteins and modifying enzymes. We have identified several multiprotein complexes that link the RNAi pathway to heterochromatin assembly, and have analyzed their activities in vitro and in vivo. Our results show that RNAi complexes associate with nascent noncoding transcripts in centromeric repeat regions. This association requires repeat‐associated small interfering RNAs (siRNAs) as well as histone H3 lysine 9 (H3K9) methylation and creates a positive feedback loop that generates more siRNA and maintains H3K9 methylation. In addition to transcriptional repression, the mechanism of silencing involves the co‐transcriptional degradation of RNAs that are transcribed in heterochromatic domains by two distinct pathways. One pathway involves RNAi and the second pathway involves polyadenylation‐coupled recruitment of the exosome, a multisubunit RNA degradation machine. The coupling of histone H3K9 methylation to a chromosome‐associated siRNA positive feedback loop may provide a mechanism for the epigenetic inheritance of silent chromatin domains.
Bioinformatics for Undergraduate Biochemistry Courses at Kalamazoo College
Authors: Furge Laura Lowe
Published: June 2021

In 2004 Kalamazoo College received a gift from the Howard Hughes Medical Institute to support development of bioinformatics education in the Biochemistry and Molecular Biology Concentration – a joint program between the Chemistry and Biology Departments. Bioinformatics modules were introduced over a period of four years into 10 courses in Biology, Chemistry, and Computer Science. Bioinformatics modules introduced in Biochemistry and Advanced Biochemistry are presented here. Modules described include use of the following tools, databases, and search engines: BLAST, KEGG, OMIM, BRENDA, Protein Data Bank, ExPASy, Protein Explorer, Swiss Deep View, and VAST. In addition, a new module using tools for investigating intrinsic protein disorder is described. Student assessment of the experiences is given. (This work was supported by an HHMI grant to Kalamazoo College).
InvestiGators research honors society
Authors: Borum Peggy R.
Published: June 2021

The InvestiGators (IG) research honors society at the University of Florida allows outstanding undergraduate students to be professional basic and clinical research scientists. The usual paradigm of the undergraduate student working closely with a graduate student is altered to three interdisciplinary teams working directly with the professor. The Gator Team provides a detailed metabolic assessment for each pediatric HIV patient in a weekly clinic and addresses different hypotheses concerning HIV associated metabolic syndrome. The KetoGator Team has a primary role in a General Clinical Research Center project evaluating the clinical aspects of altered ratios of dietary macronutrient intake on changes in metabolism and electrical properties of the brain. The Carnitine Team uses clinical samples and a piglet model to evaluate the role of carnitine in normal metabolism and in the metabolism of patients seen in clinic by the other two teams. Members learn basic, clinical and translational science through direct experience. In addition to experimental design, careful implementation of experiments, data analyses, and reporting of results, IG members must learn how to work together in an interdisciplinary team of peers as well as more senior scientists. They learn at an early career stage what the professional world is really like and they leave significant footprints in translational science and clinical care.
Frontotemporal dementia associated with a Valosin‐Containing Protein mutation: report of three families
Authors: Spina Salvatore , Van Laar Amber D. , Murrell Jill R. , de Courten‐Myers Gabrielle , Hamilton Ronald L. , Farlow Martin R. , Quinlan John , DeKosky Steven T. , Ghetti Bernardino
Published: June 2021

Objective: The phenotype of inclusion body myopathy with Paget disease of the bone and frontotemporal dementia (FTD) (IBMPFD), associated with Valosin‐Containing Protein (VCP) mutations is described in 3 families. Methods: Probands were identified on the basis of a pathological diagnosis of frontotemporal lobar degeneration with neuronal intranuclear ubiquitin/TDP‐43‐positive inclusions (FTLD‐U type IV). VCP sequencing was carried out. Clinical data on affected family members were reviewed.

Results: Family A: 4 subjects had a myopathy (M): 2 showed also Parkinsonism (P) and FTD. Proband's brain had FTLD‐U type IV and brainstem‐type Lewy body pathology. Muscle displayed neurogenic changes and VCP(+)‐rimmed vacuoles. A VCP R191Q mutation was found in one allele. Family B: 8 subjects developed IBMPFD: FTD was seen in 4, M in 3, P in 2 and primary progressive aphasia (PPA) in one. Proband's brain showed FTLD‐U type IV, hippocampal sclerosis (HS) as well as Lewy neurites in the substantia nigra. A novel VCP T262A mutation was found in one allele. Family C: 2 subjects developed IBMPFD. The proband had FTD while a brother presented M and dementia. Proband's brain had FTLD‐U type IV and HS. A VCP R159C mutation was found in one allele.

Conclusions: We identified three new families with IBMPFD associated with VCP mutations. A novel T262A mutation was found. One individual had PPA: a novel finding in IBMPFD. Supported by AG10133
Signal transduction in melanoma transendothelial migration using a tissue‐engineered human blood vessel
Authors: Auger François A. , Fiola Marie‐Christine , Tremblay Pierre‐Luc , Lacroix Dan , Basoni Caroline
Published: June 2021

Transendothelial migration involves major modifications in cell‐cell contact, consequent morphological changes and cytoskeletal reorganization. In this context, GTPases are key modulators in signal transduction leading to cellular modifications. Rap1, a member of the GTPase Ras Family, is implicated in the stability of endothelial adherent junctions. Rap1 is also implicated in integrin‐mediated adhesion of leukocytes to the endothelium, and participate in cadherin‐modulated adhesion. Those mechanisms are poorly understood, so further study is needed to elucidate the steps inherent to this process. We used a tissue‐engineered human blood vessel to analyze the role of Rap1 in the modulation of endothelial junctions. In endothelial cells monocultures, we observed that melanomas increased activated Rap1 and activated Extracellular Signal‐Regulated Kinase (Erk). We further analyzed the distribution of Zonula Occludens‐1 (ZO‐1) in endothelial tight junctions, and we observed a translocation to the nucleus when endothelial cells are stimulated with forskolin, a potent cAMP activator. We have to correlate those activations both with Rap1 signalling pathway and endothelial junctions remodelling. In the future, we will transpose those results using our 3D tissue‐engineered human blood vessels.

Financial support is provided by the Canadian Institutes of Health Research.
Dynamics of the polymerase manager protein UmuD: DNA damage tolerance in E. coli
Authors: Koleva Bilyana N. , Ordazzo Michael J. , Fung Amy , Sefcikova Jana , Wales Thomas E. , Engen John R. , Beuning Penny J.
Published: June 2021

In E. coli, the function of specialized Y family DNA polymerases is regulated by UmuD, a small manager protein, and its cleaved form, UmuD′. The different forms of the UmuD proteins play dramatically different roles in the cellular response to DNA damage: UmuD facilitates a primitive DNA damage checkpoint while UmuD′ enables DNA damage‐dependent mutagenesis. The β processivity clamp, which tethers DNA polymerases to the DNA, confers high processivity on normal DNA polymerases. UmuD is known to bind the β clamp and this may modulate the activity of β. The objective of this study is to understand the conformational dynamics of UmuD to determine its effect on DNA damage tolerance. We constructed a library of single cysteine derivatives of UmuD to facilitate labeling for fluorescence resonance energy transfer (FRET) and electron paramagnetic resonance (EPR) studies. All single cysteine derivatives of UmuD were shown to be active in UV‐induced mutagenesis, and will be labeled. Hydrogen‐deuterium exchange mass spectrometry experiments show that UmuD is flexible, while UmuD′ is slightly less so.

This work is supported by a Dreyfus Foundation New Faculty Award and the Northeastern University Office of the Provost.
Mechanistic studies of eukaryotic topoisomerase II: a cysteine footprinting and FRET approach
Authors: Collins Tammy , Guan Ziqiang , Sun Alfred , Durst Jennifer , Smiley Derike , Hammes Gordon , Hsieh Tao‐shih
Published: June 2021

Type II DNA topoisomerases are essential and ubiquitous enzymes that perform important functions in chromosome condensation and segregation, and in regulating intracellular DNA supercoiling. Topoisomerase II (topo II) carries out these DNA transactions by passing one segment of DNA through the other using a reversible, enzyme‐bridged double‐stranded break. The extensive conformational changes that both DNA and topo II undergo during the reaction cycle have been probed with fluorescence resonance energy transfer and cysteine footprinting, respectively. Using fluorescent oligonucleotides, we found that the DNA gate transition has comparable rate constants for both the opening and closing reaction, with an apparent equilibrium constant near unity. To complement the results obtained with fluorescent DNA, cysteine footprinting was used to observe conformational changes in topo II itself. We found contrasting changes in cysteine solvent accessibility in the presence of various drugs and/or cofactors, depending on the domain in which the cysteine is located. Our results provide direct evidence linking opposing movements in the topo II domains; as the ATPase domain closes, the domain directly beneath the ATPase domain opens. This work was supported by NIH grants and a grant from the Department of Defense Breast Cancer Research Program.
Research of bacteria at waters of river Morava Binqës
Authors: Kurteshi Kemajl , Vehapi Idriz , Ibrahimi Haqif , Salihu Dilaver , Sahiti Naim , Ismaili Muharem
Published: June 2021

During the spring 2006, we research the bacteria at upstream of river Morava Binqës. The samples taken in four stations. Qualitativ evaluation done according to presenc of saprophyt bacteria and coliform bacteria . With saprophyt bacteria eavaluate the pollution of waters by organic material and with coliform bacteria evaluate the of waters of river Morava Binqës by fecalic. According the Tampling, Sladecek and Kohl the waters of the river Morava Binqës belongs to second level of bonity
Genome‐wide analysis of RNA helicase A translation control
Authors: Hernandez Julia Marcela , Qian Shuiming , Yilmaz Alper , Boris‐Lawrie Kathleen
Published: June 2021

RNA helicase A (RHA) operates a novel translation control axis that is necessary for expression of several retroviruses and junD. Specificity is provided by selective interaction at the 5′ terminal post‐transcriptional control element (PCE) located in the 5′ UTR of RHA target mRNAs. This interaction represents an “RNA switch” that neutralizes the complicated structure of the 5′ UTR to derepress protein synthesis. Here, we executed a genome‐wide analysis to identify the collection of cellular mRNAs dependent on RHA for efficient translation. Identification of mRNAs that coimmunprecipitate with RHA and/or require RHA for polyribosome association was performed by microarray and functional analysis. Overlapping data sets identified five candidate genes, which included junD. Complementary bioinformatic analysis identified conserved sequence and/or structural features in cellular PCEs. In addition to the RHA binding site, these features may include binding sites for cofactors crucial for PCE activity under the conditions tested. This collection of genes is postulated to comprise a post‐transcriptional regulon that is coordinately regulated by RHA. Our work provides new insight into a mechanism that likely contributes to the role of RHA in human diseases such as cancer, Lupus, and pathogenic retrovirus infection.
AT‐1 is an ER membrane acetylCoA transporter and is required for the lysine acetylation of nascent membrane proteins in the ER/ERGIC system
Authors: Jonas Mary Cabell , Puglielli Luigi
Published: June 2021

Folding of newly synthesized membrane proteins in the endoplasmic reticulum (ER) is enhanced by enzymes that modify, either temporally or definitively, the protein. Our group has shown that the lipid second messenger ceramide regulates the molecular stability of the β‐site APP cleaving enzyme 1 (BACE1) by affecting a transient form of lysine acetylation in the ER/Golgi system (Biochem J 2007; EMBO J 2006; J Biol Chem 2003). Specifically, nascent BACE1 is initially acetylated in the lumen of the ER and then deacetylated by a Golgi‐based deacetylase. The apparent limiting factor in the above events is the translocation of acetylCoA, the donor of the acetyl group in the reaction of acetylation, from the cytoplasm to the lumen of the ER. Here we report the functional identification of AT‐1, a putative acetylCoA membrane transporter that is up‐regulated as result of ER‐induced stress. Biochemical fractionation studies indicated that AT‐1 is enriched in the ER membrane and overlaps with the endogenous acetylCoA membrane transport activity. In addition, ER fractions from AT‐1 expressing cells display increased acetylCoA membrane transport activity, which is inhibited by CoA suggesting an antiporter mechanism. Finally, overexpression of AT‐1 resulted in increased acetylation and steady‐state levels of BACE1. In conclusion, our results indicate that AT‐1 is an ER‐resident acetylCoA membrane transporter.