bluuebunny/crossref_metadata_2025 huggingface dataset:
The Phosphatidylinositol 3‐Kinase/Akt Pathway Negatively Regulates Nod2‐Mediated NF‐kappaB Pathway
Authors: Zhao Ling , Lee Joo Y. , Hwang Daniel H.
Published: June 2021

Nucleotide‐binding oligomerization domain containing proteins (Nods) are intracellular pattern recognition receptors that recognize conserved moieties of bacterial peptidoglycan and activate downstream signaling pathways, including NF‐kappaB. Here, we show that Nod2 agonist MDP induces Akt phosphorylation in time and dose dependent manner. Pharmacological inhibitors of PI3K (LY294002 and wortmannin) and dominant negative form of p85 (the regulatory subunit of PI3K) or Akt enhance, while constitutive active form of p110 (the catalytic subunit of PI3K) or Akt inhibit, NF‐kappaB activation and the target gene IL‐8 induced by MDP. In addition, both LY 294002 and wortmannin enhance phosphorylation of NF‐kappaB p65 on Ser 529 and Ser 536 residues resulting in enhanced p65 transactivation activity. Furthermore, we show that the negative regulation of PI3K/Akt on MDP‐induced NF‐kappaB is in part mediated through inactivating glycogen synthase kinase (GSK)‐3Beta. Together, our results demonstrate that PI3K/Akt pathway is activated by MDP and negatively regulates NF‐kappaB pathway downstream of Nod2 activation, suggesting that PI3K/Akt pathway may involve in the resolution of inflammatory responses induced by Nod2 activation. This work was supported by grants DK064007, DK41868, and CA75613 from the National Institutes of Health.
MMP‐9 Mediated Loss of Airway Epithelial Integrity
Authors: Vermeer Paola Drapkin , Denker James , Moninger Thomas O. , Zabner Joseph
Published: June 2021

The family of zinc and calcium‐dependent matrix metalloproteases (MMP's) play an important role in remodeling of the airways in disease. Transcriptional regulation by pro‐inflammatory cytokines increases lymphocyte derived MMP‐9 levels in the airway lumen of asthmatics. Moreover, the levels of the MMP‐9 inhibitor, tissue inhibitor of metalloprotease (TIMP1), are decreased leading to increased protease activity. However, the mechanism by which MMP‐9 activity leads to asthma pathogenesis and remodeling remains unclear. Using a model of well‐differentiated human airway epithelia, we found that apical MMP‐9 significantly increases transepithelial conductance that is blocked by TIMP1. Moreover, apical MMP‐9 treatment decreased immunostaining of tight junction proteins suggesting disruption of barrier function. Consistent with this, viruses gained access to the epithelial basolateral surface after MMP‐9 treatment which increased infection efficiency. In addition, loss of epithelial integrity correlated with increased epithelial cell death. Importantly, in vitro translational data suggests that MMP‐9 cleavages the tight junction protein occludin. Thus we hypothesised that MMP‐9 exerts its effects on the epithelium by cleaving occludin and triggering anoikis. Taken together, these data suggest that a component of airway remodeling associated with asthma may be directly regulated by MMP‐9.
The oxidized phospholipid POVPC induces MMP3 expression and enhances migration of vascular smooth muscle cells
Authors: Cherepanova Olga A. , Pidkovka Nataliya A. , Leitinger Norbert , Owens Gary K.
Published: June 2021

Oxidized phospholipids (oxPLs) including 1‐palmitoyl‐2‐(5‐oxovaleroyl)‐sn‐glycero‐3‐phosphoholine (POVPC) which are active components of minimally modified low density lipoprotein, are primary stimuli of multiple proatherogenic events. Previously we showed that oxPLs including POVPC induced profound phenotypic switching of vascular smooth muscle cells (VSMCs) including suppression of multiple SMC differentiation marker genes as well as activation of multiple inflammatory genes (Pidkovka et al, 2007, Circ Res). Little is known about the effects of oxPLs on genes that play a key functional role in the synthesis and degradation of extracellular matrix proteins that are critical in plaque formation and/or stability. Real time RT‐PCR, Western blot and Zymography analyses showed the increase of MMP3/stromelysin‐1 mRNA and protein level in cultured VSMCs after treatment with POVPC. Furthermore, POVPC‐induced elevation of MMP3 mRNA level was demonstrated in vivo after application of the pluronic gel containing POVPC to carotid arteries of rats. In addition, we found that POVPC enhances the VSMCs migration and invasion in Boyden chambers. The results provide new insights into the possible contribution of oxPLs to the atherogenesis through matrix degradation via MMP release and VSMCs migration leading to plaque destabilization and rupture. Supported by NIH grants R01 HL 38854, R37HL57353 and P01 HL19242.
Neuronal migration and layer formation in the developing cerebral cortex
Authors: Nakajima Kazunori
Published: June 2021

Mammalian cerebral cortex has a six‐layered structure, where the neurons are aligned depending on their birth‐date. Since the migrating neurons invariably stop migrating just beneath the marginal zone (MZ), the cortical plate (CP) is formed in an inside‐out manner. These well‐organized developmental events are orchestrated by Reelin, which is secreted by Cajal‐Retzius neurons in the MZ. Using reaggregation culture system of dissociated migrating neurons, we found that the cells acquired a birth‐date‐dependent segregation property (or the fate to acquire this property) in a Reelin‐independent manner before their somas reach beneath the MZ. Ectopic overexpression of Reelin on the way of migration revealed that Reelin affected the cell aggregation patterns of the migrating neurons in vivo. These results suggest that the appropriate layer formation in the cortex might be accomplished through the interplay between the cell‐intrinsic mechanism such as segregation properties and the extracellular signals including Reelin.
Molecular regulation of endocardial heterogeneity during semilunar valve development and disease
Authors: Baldwin H Scott , Misfeldt Drew , Zhou Bin
Published: June 2021

A critical event in cardiac morphogenesis is differentiation of heterogenous endocardial subpopulations with distinctive developmental capabilities. However, little is known about the factors that regulate endocardial differentiation. We have utilized NFATc‐Cre mediated lineage tracing in vivo to identify a subpopulation of valvular endocardial cells within the outflow tract that is resistant to transformation and is required for semilunar valve formation. Bacterial artificial chromosome (BAC) transgenesis containing endogenous regulatory elements of the NFATc1 locus was used to study the earliest events of endocardial differentiation. These studies have defined the endocardium as a unique subpopulation of endothelium during early mesodermal differentiation. Further analysis of in vitro endocardium differentiation utilizing ES cells derived from transgenic animals confirms that in vitro differentiation mirrors the temporal and spatial organization seen in vivo. Furthermore, manipulation of tissue specific culture conditions suggests that endocardial differentiation is not dependent on vascular or hematopoietic differentiation but is more closely associated and regulated by myocardial differentiation in a Wnt dependent process. These studies will be discussed in the context of developing a renewable resource of endocardial cells as mediators of tissue regeneration and repair.
Functional and Molecular Adaptation of Lymphatic Vessels
Authors: Wilson Emily , Laine Glen , Quick Christopher , Hardy Joanne , Ngo Bruce , Zawieja Dave , Gashev Anatoliy , Stewart Randolph
Published: June 2021

We developed a bovine model of mesenteric venous hypertension (MVH)by surgically occluding mesenteric veins for 3 days to investigate the functional and molecular adaptations of the mesenteric lymphatic vessels. The MVH model was characterized by an increase in mesenteric vein pressure from 10 to 30 mm Hg, a decrease in lymph protein concentration from 6 to 5 g/dL and an increase in intestinal water content from 72 to 78%. After 3‐days of MVH, isolated mesenteric lymphatic vessels demonstrated decreased contraction frequency, decreased ejection fraction and decreased stroke volume at each transmural pressure tested. Microarray analysis revealed that 117 genes were upregulated and 79 genes were down regulated in MVH lymphatics. A large number of the regulated genes are muscle contractile proteins, consistent with the changes in function. We further characterized changes in the mRNA of actin isoforms expressed in lymphatic muscle in response to venous hypertension. The smooth muscle actins (alpha and gamma) and skeletal actin were increased and cardiac actin decreased in lymphatics from the MHV model. Actin protein levels were analyzed by western blot analysis and revealed similar directional changes in response to MHV. These data suggest that lymphatic vessels undergo functional and molecular adaptation in response to changes in lymphdynamics in a time frame that may be important in resolution of edema. This work was supported by a LSI grant from Texas A&M University and CDC grant 623086.
Cellular responses to genetic change
Authors: Fire Andrew
Published: June 2021

This talk will describe the use of C. elegans and other genetic systems as models to understand the responses of biological systems to genetic change.
The Cullin‐ROC familyof E3 Ubiquitin Ligases
Authors: Xiong Yue
Published: June 2021

The concentrations of many cellular proteins change rapidly as the result of alterations in cellular physiological conditions. They are regulated, to a large extent, by the ubiquitin pathway that involves E1 activating, E2conjugating and E3 ligating enzymes. Three issues central to our understanding of E3 ligases are the molecular nature of E3, the mechanisms controlling the assembly and activity of E3 ligases, and how individual protein substrates are recruited to specific E3 ligases. Currently, two major families of E3 ligases, the HECT and the RING families, have been described. Cullins are a family of evolutionarily‐conserved proteins that assemble the largest number of distinct RING‐type E3 ligase complexes with estimated number of E3 compelxes to be in the range of 300 – 500. I will discuss our recent progresses on the catalytic mechanism, substrate recruitment and regulation of the cullin‐ROC1 family E3 ligases.
Healthy School Lunch Behavior and the Invisible Hand
Authors: Wansink Brian , Just David , Payne Collin
Published: June 2021

Restricting the use of prepaid lunch accounts use may improve food selection without eliminating free choice.

In a school lunch cafeteria, 166 students were allowed to purchase their lunch in one of three ways: 1) cash only, 2) cash and an unrestricted debit card, or 3) cash and a restricted debit card that could be used to purchase items marked as healthy. Those given restricted debit cards ate significantly fewer calories than those given the unrestricted card (p=0.045), and they consumed healthy foods (78.6%, p=0.000).

Because this payment procedure still allowed students to use their own cash to purchase less healthy options, it did not unduly restrict dining options or reduce cafeteria revenue.
Canine dietary protein influences glomerular filtration rate in adult dogs
Authors: Jewell Dennis E.
Published: June 2021

Seventy normal adult dogs were randomly assigned to five dietary protein levels with and without amino acid fortification. This study was run to determine the effect of different protein and amino acid concentration on renal function as measured by glomerular filtration rate (GFR) and body composition in a six month study. The experimental protocol was reviewed and approved by the Institutional Animal Care and Use Committee. Dogs had daily socialization through interacting with other dogs and daily walks with people. Glomerular filtration rate was measured by iohexol clearance while body composition was determined via dual energy x‐ray absorptiometry (DXA). GFR and DXA were measured before and after six months on treatment. Foods were formulated to contain 15, 19, 22, 25, or 30% protein. All treatments had numeric increases in body lean but change in lean was not influence by diet. Dietary protein had a significant quadratic and linear effect (P<0.05) on GFR. Increased GFR was attained through either increasing above or reducing below 21% protein. These data show that renal function as measured by GFR may be increased with increasing protein and through restricting excess protein.
In vitro evaluation of methanogenesis in the dog
Authors: Middelbos Ingmar S. , Bauer Laura L. , Fahey George C.
Published: June 2021

Gut microbiota are becoming increasingly recognized as a factor affecting host phenotype. Microbial energy harvest in the intestinal tract by fermentation has been implicated as contributing to obesity, and methanogenic Archaea are believed to increase efficiency of energy harvest by eliminating hydrogen to form methane. Methanogenesis and methanogens have been described in many species, but not the dog. We evaluated methanogenesis of dog intestinal microbiota using an in vitro fermentation system. Selected substrates were subjected to 12‐h fermentation in gas‐tight tubes after inoculation with fecal slurry derived from three canine donors. Short‐chain fatty acids and gas production (methane, hydrogen) were measured. Short‐chain fatty acid production was higher (P < 0.05) for pectin and β‐fructan substrates compared with beet pulp and yeast cell wall. Significant methane production was detected for all substrates (305 – 6135 μg/g substrate) except wood cellulose. Beet pulp generated more (P < 0.05) methane than any other substrate and was moderately fermentable. These data suggest that the dog intestinal tract harbors methanogenic Archaea that have yet to be identified.
Hierarchical contributions of IgM and complement C1q in protection against murine atherosclerosis
Authors: Lewis Myles Jonathan , Malik Talat H. , Ehrenstein Michael R. , Boyle Joseph J. , Botto Marina , Haskard Dorian O.
Published: June 2021

Natural IgM antibodies and complement C1q have been implicated in atherogenesis through their ability to bind tissue debris such as denatured low‐density lipoprotein (LDL) and apoptotic cells. To investigate their relative contributions, mice deficient in serum IgM (sIgM), C1q or both C1q and sIgM were crossed with LDL receptor deficient mice (Ldlr−/−) and studied at 22 weeks. sIgM.Ldlr−/− mice showed increased lesion size in comparison to Ldlr−/− mice, both en face (~7‐fold, p<0.0001) and at the aortic root (~5‐fold, p<0.0001). Similar findings were noted on a high fat diet with a 98% increase en face (p=0.0009) and a 23% increase in aortic root lesion area (p=0.006) in sIgM.Ldlr−/− mice, and increased smooth muscle cell infiltration (p=0.007). En face and aortic root lesions were substantially larger in sIgM.Ldlr−/− than C1q.Ldlr−/− mice on either diet (all p<0.005), and the effects of combined C1q and serum IgM deficiency were no different to those of sIgM deficiency alone. Thus although IgM antibodies and C1q are both significantly atheroprotective, IgM makes a more prominent contribution. The major mechanism of protection by IgM therefore appears to be independent of classical pathway activation.

Funded by the Wellcome Trust and British Heart Foundation
Stress‐activated TRPV4 channels mediate integrin to integrin mechanical signaling that is required for angiogenesis
Authors: Thodeti Charles K , Ingber Donald E.
Published: June 2021

Physical forces applied to extracellular matrix (ECM) influence the direction of capillary endothelial cell outgrowth during angiogenesis, but little is known about how capillary cells sense and transduce mechanical signals responsible for these spatially oriented responses. Here we show that capillary cells reorient perpendicular to the direction of the applied force when cyclic mechanical strain is exerted on their integrin‐ECM adhesions, and that this results from activation of a mechanical signaling pathway that induces calcium influx through stress‐activated TRPV4 ion channels that, in turn, stimulates phosphatidyl inositol‐3‐kinase, thereby inducing activation and binding of additional β1 integrin receptors that promotes Rho‐dependent cytoskeletal remodeling. Moreover, knockdown of TRPV4 channels using specific siRNA inhibits angiogenesis in vitro, whereas control siRNAs have no effect on capillary formation. Thus, physical forces may guide angiogenesis through an “integrin to integrin” signaling mechanism that is mediated by stress‐induced activation of mechanically‐gated TRPV4 ion channels on the cell surface. (Supported by NIH and AHA)
NRF2 Protects Mice From Cigarette Smoke‐Induced Emphysema
Authors: Biswal Shyam , Rangasamy Tirumalai , Sussan Thomas E , Yates Melinda , Sporn Michael , Kensler Thomas , Tuder Rubin
Published: June 2021

Rationale: NRF2, a redox‐sensitive transcription factor, protects the lungs against cigarette smoke (CS)‐induced oxidative stress and emphysema by up‐regulating antioxidant and cytoprotective pathways. We hypothesize that activation of NRF2 by a small molecule activator will attenuate CS‐induced oxidative stress and emphysema. Methods: Wild‐type (WT) and NRF2−/− mice were fed a diet containing 90mg/kg CDDO‐Im, and exposed to CS for 5.5 months. Lungs were examined for emphysema, oxidative stress, inflammation, and NRF2‐pathway expression. Results: Chronic CS‐exposure induced oxidative stress and pulmonary emphysema in both WT and NRF2−/− mice with dramatically increased oxidative stress and alveolar destruction in NRF2−/− mice. WT mice treated with CDDO‐Im had significant reductions in both oxidative stress and alveolar destruction with increased transcriptional induction of multiple NRF2‐regulated antioxidant genes. Furthermore, NRF2−/− mice had no significant reduction in alveolar destruction following treatment with CDDO‐Im. Conclusion: This data suggests that activation of the NRF2‐pathway in emphysema may represent a novel therapy for oxidative stress related lung disease, such as COPD.
Vascular tube formation on matrix metalloproteinase‐1 damaged collagen
Authors: VARANI JAMES J , PERONE PATRICIA , WARNER ROSCOE L , DAME MICHAEL K , KANG SEWON , FISHER GARY J , VOORHEES JOHN J
Published: June 2021

Endothelial cells maintained on a three‐dimensional lattice of intact, polymerized type I collagen formed a monolayer of cells with a cobblestone morphology. When the collagen was exposed to organ culture fluid from human skin (previously characterized and shown to contain a high level of active matrix metalloproteinase‐1 [MMP‐1]), degradation of the collagen occurred. The major degradation products were the ¾ and ¼ sized fragments known to result from the action of MMP‐1 on type I collagen. When endothelial cells were maintained on the partially fragmented collagen, the cells organized into a network of vascular tubes. Pretreatment of the organ culture fluid with either tissue inhibitor of metalloproteinase‐1 (TIMP‐1) or neutralizing antibody to MMP‐1 prevented degradation of the collagen lattice and concomitantly inhibited endothelial cell organization into the vascular network. Purified (activated) MMP‐1 duplicated the effects of skin organ culture fluid but other enzymes including MMP‐2 (gelatinase A), MMP‐9 (gelatinase B), elastase or trypsin failed to produce measurable fragments from intact collagen and failed to promote vascular tube formation. Based on the findings described here, we suggest that damage to the connective tissue is a major promoter of new vessel formation. This study was supported in part by NIH grant GM77724 from the USPHS.
E‐cadherin re‐expression in invasive breast cancer cells as a putative marker for tumor cell dormancy modeled by infiltration into hepatocyte spheroids
Authors: Shepard Christopher Reed , Wells Alan
Published: June 2021

Epithelial‐cadherin downregulation enables cancer cells to escape from the primary mass; however, E‐cadherin has been found to be expressed on metastatic foci, bringing into question the role of this molecule in tumor progression. We previously defined a novel role for the cellular adhesion molecule E‐cadherin, in which we reported the protein's re‐emergence due to loss of promoter methylation allowing potential carcinoma‐parenchymal interactions in ectopic sites. We now report that E‐cadherin re‐expression is concurrent with an upregulation of cytokeratin‐18 expression, consistent with our previously reported hypothesis of a mesenchymal‐epithelial reverting transition (MErT). E‐cadherin allows for EGFR‐dependent Erk‐MAPK activation that is localized to the plasma membrane and not the nucleus, further suggesting a cessation of the EMT‐related phenotype. This system can be reconstituted in 3D‐spheroid culture, in which the invasive breast cancer cells infiltrate and migrate to the center of the hepatocyte spheroid, and re‐express E‐cadherin that is well‐localized to the plasma membrane. We postulate that E‐cadherin re‐expression in ectopic sites may be a molecular marker for tumor cell dormancy.
Insights into the Anti‐inflammatory Action of the Soluble Epoxide Hydrolase Inhibitor through Systems Biology based in silico Modeling Approach.
Authors: Vali Shireen , Hegedus Christine , Bhattacharjee Chumki , Robinson  , Schmelzer Kara , Inceoglu Bora , Hammock Bruce D
Published: June 2021

The arachidonic acid (AA) cascade is involved in inflammation, pain, and multiple pathological states. Products of AA metabolism include the inflammatory leukotrienes and prostaglandins as well as the anti‐inflammatory epoxyeicosatrienoic acids (EETs). We used a dynamic in silico platform comprising of AA, tumor necrosis factor alpha and toll‐like receptor signaling blocks. The goal of this study was to validate the in silico platform with experimental data and use it to probe into the mode of action of the anti‐inflammatory affects of the soluble epoxide hydrolase inhibitor (sEHI). Previous in vivo results demonstrated that use of sEHI attenuated the effects of LPS induced inflammation without affecting prostacyclin to thromboxane ratio and concurrent use of a cyclooxygenase (COX) inhibitor demonstrated synergistic effects. In silico analysis of sEHI demonstrated identical results. Both in vivo and in silico analyses indicate that increase in EETs may mediate the inhibition on the COX enzymes. The in silico results also gave insight that the EETs mediate reduction in expression of COX2 and inflammatory cytokines through inhibition at the level of nuclear factor kappa B transcription. Thus, this study shows that in silico analysis of AA metabolism is consistent with in vivo results and that predictive analysis is particularly useful and highly practical for streamlining hypothesis generation and testing.
Oxylipin Profiling Methods for Cyclooxygenase, Lipoxygenase and Epoxygenase derived Oxylipins using LC/MS/MS
Authors: Yang Jun , Schmelzer Kara , Georgi Katrin , Hammock Bruce D
Published: June 2021

Cyclooxygenase, lipoxygenase and epoxygenase derived oxylipins, especially eicosanoids play important roles in many physiological processes. A LC/MS/MS method is developed to monitor these metabolites. In 21 minute, 40 oxylipins were able to be quantified corresponding to eight surrogates. The limits of quantification were between 0.1–32 pg. As parallel a targeted method for the analysis of selected indicator eicosanoids is being developed targeted to small sample size and high throughput for support of epidemiological studies. This work was supported in part by NIEHS SBRP Grant P42 ES004699, NIEHS Grant R37 ES02710 and NIEHS Grant P30 ES05707.
Ether epoxyeicosatrienoic acid (EET) analogs lower blood pressure when administered in‐vivo to the spontaneously hypertensive rats
Authors: Elmarakby Ahmed A. , Falck John R. , Imig John D.
Published: June 2021

Epoxyeicosatrienoic acids (EETs) play an important role in the regulation of vascular tone. EETs rapidly degrade in‐vivo by the soluble epoxide hydrolase (sEH) enzyme. Using the pharmacophoric moiety of EET, ether EET analogs were designed with improved solubility and resistance to auto‐oxidation and metabolism by sEH. We tested the blood pressure lowering effects of these analogs in an established model of hypertension, the spontaneously hypertensive rats (SHR) using telemetry. Initially, 11,12–ether EET‐8‐ZE failed to lower blood pressure in SHR. Esterification of the carboxylic group of 11,12–ether EET‐8‐ZE prevented blood pressure increase in SHR when injected at 2 mg/day for 12 days (MAP Δ change at day 8 of injection was −0.3±2 for treated and 12± 1 mmHg for control SHR). Amidation of the carboxylic group with aspartic acid produced another EET analog with a blood pressure lowering effect when injected at 3 mg/day in SHR for 5 days (MAP Δ change at day 2 of injection was −8± 1.3 for treated and 1±1.3 mmHg in control SHR). Amidation of the carboxylic group with lysine amino acid produced another analog with a minimal blood pressure lowering effect. These data suggest that esterification of the carboxylic group of 11,12–ether EET‐8‐ZE produced the most effective ether EET analog in lowering blood pressure in SHR and provide the first evidence to support the use of EET analogs in treatment of cardiovascular diseases.
Functional Roles for C5a Receptors in Sepsis
Authors: Rittirsch Daniel , Sarma J. Vidya , Day Danielle E. , Nadeau Brian A. , Zetoune Firas S. , Chen Anthony J. , Huber‐Lang Markus S. , Flierl Michael A. , Ward Peter A.
Published: June 2021

The complement anaphylatoxin, C5a, is excessively generated during sepsis and plays adverse roles in survival. The function of the two C5a receptors (C5aR and C5L2) remains controversial; especially C5L2, originally termed a “default receptor”. The role of each receptor was investigated in the setting of cecal ligation and puncture‐induced sepsis in mice by antibody/antagonist‐induced blockade of C5a receptors or by using knockout mice (C5aR−/−, C5L2−/−). Cytokine/chemokine levels in plasma were determined by quantitative proteomic analyses. The content of high mobility group box 1 protein (HMGB1) in plasma and supernatant fluids was assessed by ELISA and Western blotting. Using a “mid‐grade” level of sepsis (30% survival), blockade or absence of either C5aR or C5L2 improved survival and attenuated the buildup of proinflammatory mediators in plasma. Release of HMGB1 in vivo or secretion from phagocytes in vitro required C5L2 and signaling via mitogen‐activated protein kinase (MAPK) pathways, but was independent of C5aR. When “high‐grade” sepsis was employed (100% lethality), the only protective condition was the combined blockade of C5L2 and C5aR. These data indicate that C5aR and C5L2 contribute synergistically to harmful consequences during sepsis and that C5L2 is required for the release of HMGB1. Contrary to earlier speculation, C5L2 is a functional receptor rather than merely a “default” receptor.
CRP Induces Superoxide Anion Release from Rat Macrophages In vivo Via Upregulation of NADPH oxidase
Authors: Devaraj Sridevi , Singh Uma , dasu Mohan r
Published: June 2021

C‐reactive protein (CRP), the prototypic marker of inflammation is a cardiovascular risk marker. Recent in vitro studies support a role for CRP in atherogenesis. CRP induces oxidative stress in vitro. However, there is a paucity of data examining effects of CRP on oxidative stress in vivo. Thus, we tested the effects of CRP in vivo administration on superoxide anion in a rat sterile air pouch model. Sterile air pouches were made on Male hooded Wistar rats (150 gm, n=5/group) by injecting 20ml sterile air. Human CRP, isolated from pleural fluid and purified of endotoxin/azide, (25ug/ml /intrapouch) or Human serum albumin (HA,25ug/mL) were injected. After 12 hours, blood and pouch exudates were used to assess superoxide production by DHE fluorescence and cytochrome C method. CRP treatment significantly increased DHE labeling and superoxide production in pouch macrophages (p<0.01). Also, CRP resulted in increased p47phox activity. Pretreatment with NADPH oxidase inhibitor, apocyanin significantly abrogated CRP‐induced superoxide anion release and DHE fluorescence (77% and 66% decrease, p<0.001). Pretreatment with ERK inhibitor, but not p38MAPK or JNK significantly decreased CRP induced superoxide in vivo (p<0.05). Thus, CRP induces superoxide production in vivo via upregulation of p47phox activity and ERK phosphorylation. This in vivo demonstration lends support to its role in atherothrombosis.
Endothelial cells induce expression of Notch3 in vascular support cells
Authors: Liu Hua , Lilly Brenda
Published: June 2021

The communication of endothelial cells and vascular support cells (pericytes and smooth muscle cells) is crucial for the assembly and subsequent function of blood vessels. To assess the significance of this heterotypic interaction on gene expression, we screened for factors that were modulated by coculturing of these different cell types. We identified Notch3 as one gene that is strongly induced in fibroblasts, pericytes, and smooth muscle cells upon coculture with endothelial cells. Notch3 belongs to an evolutionarily conserved family of transmembrane receptors that are known to govern cell fate decisions and differentiation in diverse cell types. In particular, Notch3, the causative gene of the neurovascular disorder CADSASIL, has been linked to smooth muscle differentiation. To investigate the mechanism that governs Notch3 induction by endothelial cells, we employed chemical inhibitors to select signaling pathways. Interestingly, Notch3 upregulation was completely abolished by the Notch signaling inhibitor DAPT, raising the possibility that Notch3 acts through an autoregulatory loop. Our study provides compelling evidence that during blood vessel formation the expression of Notch3 in pericytes and smooth muscle cells is modulated by endothelial cells, and this serves to facilitate the differentiation of vascular support cells leading to stable blood vessels. Supported by NIH HL076428.
Defect matrix induces epithelial mesenchymal transition
Authors: Weihrauch Dorothee , Jones Deron Wade , Warltier David C , Pritchard Kirkwood Jr
Published: June 2021

One of the hallmarks of systemic scleroderma (SSc) is the increase in the production of extracellular matrix protein. We have previously shown that D4F, an ApoA1 mimetic, reduces the extent of fibrosis in the heart and decreases proinflammatory HDL and angiostatin levels in tight skin mice (Tsk−/+ mice), a model for SSc. To further understand the mechanisms of the process of fibrosis and lastly development of heart failure in SSc we studied the involvement of epicardial cells transitioning into fibroblasts, a process called epithelial mesenchymal transition (EMT). We performed immunohistochemistry using an antibody against the fibroblast marker FSP‐1 and an antibody against the transcription factor twist which is described to be involved in EMT. Epithelial transitioned into fibroblasts in vitro when grown on microfibers harvested from skin from Tsk−/+mice compared to epithelial cells grown on microfibers from C57/BL6 mice. D4F treatment reduced the number of transitioning, and FSP‐1 positive epithelial cells but increased the expression of twist. In vivo we found epicardial cells in transition characterized by expressing higher levels of FSP‐1 in myocardium of TSK−/+ mice. In TSK−/+ myocardium twist protein levels were increased These data show that one of the contributors to fibrosis in the heart in SSc are epicardial cells transitioning into fibroblasts and that D4F reverses this process by increasing twist levels and promoting cell migration and tissue remodeling.
The influence of the cellular environment on protein folding and stability
Authors: Gierasch Lila M , Ignatova Zoya , Krishnan Beena , Hong Jiang , Wang Qinghua , Sahoo Harekrushna , Budyak Ivan
Published: June 2021

The complex cellular environment in which proteins fold presents many physicochemical challenges. Macromolecular concentrations in the cell are 200 to 400 g/l, which may restrict free movement of folding chains. The volume elements accessible to a polypeptide chain are also heterogeneous, and their impact will depend on the size, shape, degree of compactness, and surface properties of folding chains, all of which change as folding proceeds. In addition, vectorial synthesis of polypeptide chains and potential interactions with chaperones influence the folding landscape. We are developing methods to explore protein folding in the cell, both by observing folding in vivo and by mimicking cellular conditions in vitro. We engineered a FlAsH‐binding site into CRABP, a β‐sheet protein extensively studied in our lab. The quantum yield of bound FlAsH was higher for denatured tetra‐Cys CRABP than native, so that we could follow an unfolding transition via a urea melt in vivo. We interpret the resulting thermodynamic data with caution, but they showed decreased stability and a higher m‐value for the protein in vivo, contrary to expectations based only on crowding. Importantly, the quantum yield of FlAsH‐labeled aggregated protein was also high, enabling us to follow aggregation in vivo. We will discuss our ongoing efforts to understand factors affecting protein folding and susceptibility to aggregation in the cell.
Tracking enzymes in a bivalve, Mercenaria mercenaria: A biochemical laboratory exercise in the study of proteins
Authors: Hamilton Mary G
Published: June 2021

Gel electrophoresis is a valuable technique for teaching protein chemistry and enzymology. While students have encountered the use of agarose gel electrophoresis in analyzing DNA digests, few have used the technique to study proteins. Marine invertebrates are readily‐available. When supplemented by column chromatography and spectrophotometry, quantitative data can be obtained about specific enzymes. Data analysis requires densitometry. We are studying three enzymes in the tissues of the “steamer” clam: superoxide dismutase (SOD), esterase(s), and acetylcholine esterase (AChE). While the main methods for assaying enzymes must be non‐denaturing, SOD can be renatured on an SDS‐PAGE gel. Non‐denaturing agarose gels illustrate net charge properties in contrast to sieving in PAGE. Molecular size can be obtained directly for SOD in an SDS‐gel, but two‐dimensional gels must be used to estimate the sizes of the enzymes. We have found similar SOD's in the gill and mantle extracts of soluble proteins, but different ones in hemolymph (HL). An esterase in HL differs from those in the tissues. AChE is present in hemolymph, but absent from the tissues. The next step is Western blotting with specific antibodies.
Medical Student Beliefs in Human Origins
Authors: Norton James M
Published: June 2021

An online survey about beliefs in human origins sent to 242 UNECOM students generated 201 responses (58% female, 42% male; mean age, 26.8 years). Of all respondents, 75.4% believed humans developed from earlier species, 80% believed plants and animals have evolved from earlier species, and 77.4% believed humans and apes have a common ancestry. When asked to choose which came closest to their belief about human origins, 39.5% chose either direct creation or intelligent design, and 60.5% chose evolution. When asked whether Darwin's theory of evolution is proven by fossil discoveries, 36.4% strongly agreed, 45.6% somewhat agreed, and 18% strongly or somewhat disagreed. When asked to indicate topics that should definitely or probably be included in medical school curricula, students responded as follows: evolution, 81%; creationism, 17%; intelligent design, 30%; comparisons between human and animal structure and function, 79%; genetic basis for human disease, 100%; genetically‐based differences among human populations in disease incidence or response to therapy, 100%; evolutionary basis for certain chronic human diseases, 96%; and evolution of bacterial antibiotic resistance, 99%. UNECOM students believing in evolution were less likely to attend organized religious services, and were more likely to be Democrat and from families with post‐baccalaureate degrees. Sampling error for this survey was +/− 6.4%.
Total urinary bladder regeneration with restoration of native structure and pharmacological response
Authors: Bertram Timothy A , Christ George J , Wagner Belinda J , Jain Deepak , Aboushwareb Tamer , Ludlow John , Payne Richard , Jarajapu Yagna , Turner Chanda , Jayo Manuel J
Published: June 2021

Neo‐bladder histology and pharmacological response were investigated in 7 dogs at 51 (n=1), 89 (n=3), and 179 (n=3) days (d) after radical cystectomy and implantation of a Neo‐Bladder Replacement Construct (Construct). Pharmacological responses of neo‐bladder tissue strips (51, 89, and 179 d; 3, 18, and 27 strips, respectively) were compared to age‐matched native bladder (n=13; 49 strips).

By 51 d post‐implantation (p.i.) regenerated bladder was histologically consistent with native bladder including mucosal and serosal linings, detrusor muscle, vascular, and nerve composition. Logistic analysis revealed similar EC50 and slope factor values for contraction of bladder tissue strips derived from all groups induced by carbachol (Car) and electrical field stimulation (EFS). A statistically significant and progressive increase in the mean Emax values occurred in response to both Car and EFS over time. While contractile responses to Car and EFS increased over time, they were lower than native bladder responses at 51 and 89 d p.i. Car‐induced contractions, but not EFS‐induced contractions, were equivalent to native tissue at 179 d p.i.

A Neo‐Bladder Replacement Construct is capable of regenerating urinary bladders in vivo with histological and pharmacological responses similar to native bladder by 179 d p.i. There was no evidence of abnormal cell growth, immune response, or adverse systemic effects.
Tissue Engineered Tubularized Urethra for Surgical Reconstruction: A Pre‐Clinical Study
Authors: Orabi Hazem Osman , Aboushwareb Tamer , Yoo James J. , Atala Anthony
Published: June 2021

Introduction

Various urethral conditions often require extensive surgery. The treatment options for patients requiring repair of a large segment of tubularized urethra are limited by the availability of autologous tissues. In this study we explored the feasibility of engineering clinically relevant long urethras for surgical reconstruction in a canine pre‐clinical model.

Materials and Methods

Autologous bladder epithelial and smooth muscle cells were grown and seeded onto collagen based tubular matrices (6 cm in length). The urethral segment of approximately 6 cm in length was removed in 18 male dogs and replaced with the cell seeded collagen scaffolds in 12 animals, and without cells in 6 animals. The animals were sacrificed at 1, 3, 6 and 9 months for analyses.

Results and Discussion

The CT scan showed wide caliber urethras in the animals implanted with the cell seeded matrices. The urethral segments replaced without cells had tissue breakdown and scaffold collapse. The urethral implants seeded with cells showed normally appearing tissue without evidence of fibrosis grossly. Histologically, formation of an organized urethral tissue structure was evident in the cell seeded constructs.

Conclusions

These findings show that long segments of cell seeded tubularized scaffolds can be used to repair large urethral defects. This study demonstrates that tissue engineered tubularized long urethras may be used for surgical reconstruction in patients requiring urethral tissue repair.

Please acknowledge NIDDK P01 DK65298‐01
Targeting quantum dots to surface proteins in living cells
Authors: Yoo Young‐Mi , Kim Yong‐Jung , Yoo Hyun‐Tae , Lee Uhn
Published: June 2021

Quantum dots (QDs) are semiconductor nanoparticles that greatly expand the possibilities for fluorescence imaging of cells and living animals. Compared with small molecule dyes, the intense fluorescence emission of QDs makes it easier to track single protein molecules, they are remarkably resistant to photobleaching, their narrow emission spectrum facilitates imaging of many proteins simultaneously, and their large

The size of this QD complex (≈50 nm) can affect membrane protein trafficking and can reduce accessibility to crowded locations in cells. Our aim was to develop a method to target QDs to cell surface proteins that eliminated the bulky antibodies and provided a stable linkage between the QD and the protein of interest.

The speed, specificity, and simplicity of labeling with Qtracker should make it a common tool for the study of cell surface proteins.
Oxidative stress and release of bioactive lipid peroxidation by‐products following herpes simplex virus infection of neural cell cultures
Authors: Valyi‐Nagy Tibor , Kavouras Jerry H. , Prandovszky Emese , Kovacs Krisztian , Shukla Deepak , Valyi‐Nagy Klara
Published: June 2021

Herpes simplex virus type 1 (HSV‐1) encephalitis is associated with oxidative tissue damage. The relative contributions of direct viral toxic effects and that of reactive inflammation to this damage are unknown. To determine whether HSV‐1 infection causes oxidative stress and lipid peroxidation in neural cells, mouse P19N neural cell cultures were infected with HSV‐1 and cellular levels of reactive oxygen species (ROS) and the release of lipid peroxidation by‐products malondialdehyde (MDA) and hydroxyalkenals [4‐HAE (predominantly 4‐hydroxy‐2‐nonenal, HNE)] into the tissue culture medium were then measured. HSV‐1 infection increased ROS levels in neural cells as early as one hour post infection (p.i.) and ROS levels remained elevated at 24 hours p.i. HSV‐1 infection also was associated with increased levels of MDA/HAE in the culture medium at 2 and 4 hours p.i. HSV‐1 replication in P19N cells was inhibited by the antioxidant compound ebselen and high concentrations of HNE, but was increased by low concentrations of HNE. These findings suggest that viral effects may directly contribute to oxidative tissue damage caused by HSV‐1 encephalitis. Furthermore, these data suggest that HSV‐1 infection‐induced soluble, bioactive lipid peroxidation by‐products may be mediators of HSV‐1‐induced tissue damage of infected and bystander cells as well as regulators of HSV‐1 replication in the nervous system.
The glutamate effect on DNA binding by DNA polymerases from Thermus aquaticus and Escherichia coli
Authors: Deredge Daniel Joseph , Thompson Gregory S , Jiang Ke , Patel Shree H , Baker John T , LiCata Vince J
Published: June 2021

DNA binding by Klenow (E. coli) and Klentaq (T. aquaticus) DNA polymerases has been characterized as a function of monovalent salt concentration, pH, and osmotic pressure. Nanomolar DNA binding by Klenow persists into much higher KCl concentration than Klentaq and results in the net release of 1–2 more ions. Both proteins exhibit a “glutamate effect,” wherein replacement of Cl− by glutamate (Glu−) as the primary anion results in a significant increase in DNA binding affinity. However, the linked ion release upon binding of Klentaq remains the same in Cl− versus Glu−, while it is reduced by >50% in Glu− for Klenow. Furthermore, the presence of glutamate also affected proton linkages of the two proteins differently. Salt displacement titrations suggest that Glu− is more excluded than Cl− from the surface of both proteins. Such exclusion could result in decreased electrostatic competition at low salt coupled with an enhanced osmotic effect at high salt which could explain the different character of the glutamate effects on the two proteins. Preliminary data suggests that the presence of Cl− versus Glu− can dramatically influence the osmotic stress effects on DNA binding by the polymerases. NSF.
SSB: Organizational center and Achilles heel
Authors: Shereda Robert D. , Lu Duo , Bernstein Douglas A. , Reiter Nicholas J. , Butcher Samuel E. , Keck James L.
Published: June 2021

The C‐terminus (Ct) of the single‐strand DNA binding protein (SSB) from Escherichia coli forms a conserved binding site for numerous proteins involved in DNA replication, recombination, and repair. SSB stimulates the enzymatic activities of several of its partner proteins, notably Exonuclease I and RecQ helicase. This stimulation is thought to be a result of the physical interaction between the proteins, which is mediated by the SSB‐Ct. We have utilized NMR and X‐ray crystallography to map the sites of interaction for the SSB‐Ct in RecQ and Exonuclease I, respectively. Mutation of SSB‐Ct binding site residues in either protein results in loss of stimulation and/or inhibition by SSB. In addition to these biochemical studies, we have identified small molecule compounds that disrupt SSB interaction with Exonuclease I. With the essential nature of SSB protein interactions in bacteria, we hypothesized that these molecules could provide novel lead antibacterial compounds. Consistent with this hypothesis, the compounds are potent anti‐bacterial agents with little or no activity against eukaryotic cells.
Dumbbell‐shaped Circular Oligonucleotides as Inhibitors of Human Topoisomerase I
Authors: Ng Magdeline Tao Tao , Li Tianhu
Published: June 2021

The interaction of human DNA topoisomerase I (topo 1) with specific sequence oligodeoxynucleotides (ODNs) of different structures was investigated. Certain dumbbell‐shaped circular oligonucleotides containing topoisomerase I‐binding sites and mismatched base pairs in the sequence were designed and synthesized. It was demonstrated that some ODNs used during our investigation inhibited the topo I catalyzed relaxation of supercoiled DNA in an irreversible manner. Our studies demonstrated that this particularly designed oligonucleotide displays an IC50 value of 9 nM in its inhibition on the activity of human topoisomerase I, a magnitude smaller than that of camptothecin, an anticancer drug currently in clinical use.
An emerging role for thioester‐linked polyubiquitin chains in protein degradation
Authors: Ravid Tommer , Hochstrasser Mark
Published: June 2021

The mechanisms of ubiquitin chain assembly are incompletely understood. The classical model proposes that ubiquitin is first added to a lysine residue within a substrate and then to the distal end of the growing ubiquitin chain. This model becomes less intuitively appealing when applied to substrates with long chains, because the newly added ubiquitin becomes structurally remote from original substrate. One hypothesis that tackles this structural dilemma proposes that ubiquitin chains are pre‐assembled on the catalytic cysteine of an E2 enzyme, prior to transfer onto a substrate. However, until recently no experimental evidence supported the existence of cysteine‐linked polyubiquitin chains in living cells.

We found that Ubc7, a yeast E2 enzyme of the endoplasmic reticulum (ER) protein quality control system, is polyubiquitylated exclusively on its catalytic cysteine. The polyubiquitin polymer can then be transferred to a nearby lysine side chain. Moreover, thioester‐linked polybiquitylated Ubc7 undergoes proteasomal degradation when its levels exceed that of its binding partner Cue1, a transmembrane protein that tethers Ubc7 to the ER membrane. Unassembled, and thus mislocalized, Ubc7 is targeted to the proteasome by Ufd4, a HECT‐class E3 ligase. This ensures that no Ubc7 activity persists beyond the ER. Thus, cysteine‐linked polyubiquitin chains can function as an additional degradation signal.