bluuebunny/crossref_metadata_2025 huggingface dataset:
Escherichia coli K1 Induces the Upregulation of CD47 in Myeloid Dendritic Cells
Authors: Mittal Rahul , Prasadarao Nemani V
Published: June 2021

Escherichia coli K1 is one of the most common pathogens that cause meningitis in neonates. Studies have shown that a high degree of bactremia is required for E. coli K1 to cross the blood‐brain barrier, indicating that the bacterium must evade host immune defense mechanisms and survive in the blood. Dendritic cells (DCs) are the most potent antigen presenting cells that play a crucial role in initiation and modulation of specific immune responses. Nonetheless, very little is known about the interaction of E. coli K1 with DCs. Here, our studies demonstrated that the interaction of OmpA+ E. coli K1 with DCs enhances the expression of CD47, an integrin associated molecule reported to induce tolerogenicity in immune cells like T cells. Significant increase in the expression of TSP‐1, a natural ligand for CD47, on the surface of DCs was also observed. In contrast, OmpA− E. coli, which promotes the maturation of DCs, failed to upregulate the expression of CD47 and TSP‐1 in DCs. Pretreatment of DCs with CD47 blocking antibodies led to maturation of DCs substantiating the role of CD47 in OmpA+ E. coli K1 mediated tolerogenicity. Furthermore, OmpA+ E. coli K1 infected DCs failed to present antigen to T cells. The present study identified an important and novel mechanism by which E. coli K1 makes DCs tolerogenic by upregulating the expression of CD47. Targeting CD47 might help in restoring maturation of DCs in response to E. coli K1 leading to efficient antigen presentation thus promoting clearance of bacteria.
PKR Regulates Immunoglobulin Class Switching during RSV infection In Vivo
Authors: Thakur Sheetal A. , Zalinger Zachary , Johnson Teresa R , Corn Minor Radiah A. , Graham Barney S , Imani Farhad
Published: June 2021

Effective antibody responses play a critical role in protection against many pathogenic viruses including respiratory synctial virus (RSV). The humoral response during RSV infection is comprised of a combination of immunoglobulin (Ig) isotypes such as IgG, IgA and IgE. Previous studies from our laboratory have established protein kinase R (PKR) as an important mediator of RSV‐induced innate immunity and immunopathology. In this report the role of PKR in regulation of RSV induced Ig class switching in a mouse model was investigated. Wild type (WT) and PKR‐deficient (PKR−/−) mice were infected with RSV intratracheally (107 pfu/mouse) and serum Ig levels were determined at 3, 7 and 14 days post‐infection. Increased serum levels of IgA, IgG and IgE were observed in WT mice compared to the PKR−/− mice. Furthermore, western blot analysis on extracts from RSV infected cells, showed that serum from infected WT mice contained significantly greater levels of RSV‐specific IgG and IgA than serum from PKR−/− mice. Several immunoregulatory cytokines including TGF‐[beta], IL‐6 and IFN‐[gamma] were significantly lower in PKR−/− mice compared to the WT mice. Taken together, these results demonstrate that PKR plays an important role in virus‐specific Ig responses.
Yin and Yang: CCL2 and IL‐10 in Otitis Media‐induced Inner Ear Inflammation
Authors: Moon Sung K , Woo Jeong‐Im , Pan Huiqi , Li Xin , Lim David J
Published: June 2021

Otitis media (OM) may lead to inner ear inflammation resulting in sensorineural hearing loss. Here, we aim to determine the role of spiral ligament fibrocytes (SLFs)‐derived CCL2 and exogenous IL‐10 in inner ear inflammation, secondary to nontypeable H. influenzae (NTHI)‐induced OM. THP‐1 cells actively migrated in response to the conditioned medium of NTHI‐exposed SLFs. This migratory activity was inhibited by the CC chemokine inhibitor and by the deficiency of CCL2 in SLFs. The CCR2 inhibitor and CCR2 deficiency inhibited migration of monocyte‐like cells of the splenocytes in response to NTHI‐induced SLF‐derived CCL2. qRT‐PCR showed that recombinant IL‐10 inhibits SLF's NTHI‐induced up‐regulation of CCL2 expression. Luciferase assays showed that silencing of IL‐10RA in SLFs suppressed the inhibitory effect of IL‐10 on NTHI‐induced CCL2 up‐regulation. In contrast, IL‐10 deficiency of SLFs hardly affected NTHI‐induced CCL2 expression. Transtympanic inoculation of live NTHI led to inner ear inflammation in 10 out of 14 murine ears (71%). Unexpectedly, CCL2 deficiency equivocally decreased the incidence of OM‐induced inner ear inflammation (60%), indicating a compensatory effect of other chemokines in vivo. Taken together, we suggest that NTHI‐induced SLF‐derived CCL2 contributes to OM‐induced inner ear inflammation, which is negatively regulated by IL‐10. [Supported in part by DC008696 and DC006276]
Commensal‐epithlial signaling mediated via Formyl Peptide Receptor
Authors: Wentworth Christy Colleen , Jones Rheinallt , Nusrat Asma , Neish Andrew
Published: June 2021

Commensal bacteria and/or their products engender beneficial effects to the mammalian gut, including stimulating homeostatic cellular turnover and enhancing wound healing, without activating innate immune pathways. In the present study we observed commensal bacterial mediated activation of the pro‐proliferative ERK MAPK signaling pathway in gut epithelial cells and describe a mechanism for commensal‐host crosstalk. A range of commensal bacteria tested induced ERK phosphorylation without JNK or NF‐κB signaling. ERK‐specific phosphorylation was recapitulated using a purified small peptide, N‐formyl‐Met‐Leu‐Phe (fMLF), a bacterial product known to stimulate signaling in mammalian phagocytes. The fMLF tripeptide is recognized via the recently characterized epithelial formyl peptide receptors (FPR) located on the apical surface. Both commensal bacteria and fMLF application to apical surfaces of polarized gut epithleilal cells resulted in FPR activation. Also, pretreatment of model epithelia and the murine gastrointestinal tract with Boc2 (specific fMLF peptide antagonist) or pertussis toxin (Gi‐protein inhibitor) abolished commensal‐mediated ERK phosphorylation. Together, these data show commensal bacteria specifically activate the pro‐proliferative ERK pathway in an FPR‐dependent manner, and suggest a mechanism by which commensal bacteria contribute to gut epithelia homeostasis.
Effect of trichostatin A on human diabetic fibroblast migration and matrix metalloproteinase expression
Authors: Lindblad William John
Published: June 2021

Fibroblast migration is essential for the repair of damaged tissues, and we have shown that fibroblasts from diabetic patients show aberrant migration possibly secondary to enhanced secretion of matrix metalloproteinases (MMPs). We hypothesized that an HDAC inhibitor could reverse the aberrant migration by reducing this increased expression of MMPs. Control and diabetic, human non‐transformed fibroblast cell lines were examined for cell migration and MMP secretion, with and without treatment with Trichostatin A. Cells were maintained in DMEM containing 10% FCS, antibiotics, and either 450 or 100 mg/dL glucose, until assayed for migration and MMP secretion under serum‐free conditions. Migration was measured as the uniform outward movement of cells plated onto a type I collagen‐coated cell culture dish using a cloning ring. Medium was collected for MMP analysis by enzymatic assay and gelatin zymography; with mRNA levels determined for selected MMPs. Cultures were treated with Tricostatin A at 50, 100 and 500 nM for 24 hours prior to assay plating and was included in selected cultures during the next 72 hours. Fibroblasts isolated from controls migrated as a halo of cells (0.120 ± 0.0012 mm/hr) whereas diabetic derived cells failed to form a uniform halo. Soluble MMP activity in the diabetic cultures was significantly elevated (~ 5 fold) compared to controls; with gelatin zymography and mRNA levels showing significant elevation of MMP‐1 and ‐9. Increased levels of MMP activity were normalized by treatment with 500 nM Trichostatin A. These results suggest that modification of histone acetylation by the diabetic state may be associated with altered fibroblast migration.
Fibrocytes express several matrix metalloproteinases. Possible role in migration and homing
Authors: de Alba Carolina Garcia , Becerril Carina , Reyes Silvia , Selman Moises , Pardo Annie
Published: June 2021

Circulating fibrocytes are mesenchymal progenitor cells that participate in tissue remodeling although the mechanisms involved in the migration and subsequent homing remain unclear. We hypothesized that fibrocytes should express matrix metalloproteases that facilitate their capacity for mobilization and homing. Fibrocytes were purified from CD14+ monocytes; purity of fibrocytes was ≥95% determined by flow cytometry using fibrocyte markers (Collagen I, CD45, CXCR4). Our results showed that fibrocytes expressed MMP‐2, ‐7, ‐8 and ‐9 mRNA while MMP‐1 was not detected. At protein level we found a marked release of MMP‐2, MMP‐8 and MMP‐9 to the medium and moderate amounts of MMP‐7. MMP‐2 and MMP‐9 activities were revealed by gelatin zymography. Confocal microscopy demonstrated co‐localization of MMP‐8 with type I collagen in fibrocytes. Migration toward PDGF‐B or SDF‐1α, was significantly reduced by a specific MMP‐8 inhibitor. These findings reveal for the first time that fibrocytes, express a variety of MMPs including MMP‐8 and MMP‐7, metalloproteases commonly expressed by specific cell types such as neutrophils and epithelial cells respectively. These enzymes may play a role in the process of fibrocyte migration throughout the basement membranes and the interstitial collagen matrix, and may also be implicated in fibrotic tissue remodeling.
Oligonucleotide‐based array CGH analysis of diffuse large B‐cell lymphomas identifies gain of 7q22 as the most common genomic alteration
Authors: Medina Edward A. , Bolla Aswani R , Bandi Rajya L , Robetorye Ryan S
Published: June 2021

While 40–50% of patients with diffuse large B‐cell lymphoma (DLBCL) respond well to therapy, for the remainder, this malignancy is rapidly fatal. The varied clinical response is likely due to the underlying molecular heterogeneity in DLBCL. Gene expression analyses have revealed clinically relevant subtypes and potential signaling pathways for therapeutic targeting. Nevertheless, there is a need to identify genes that have a critical function in DLBCL in order to broaden the number of therapeutic targets to treat this heterogeneous malignancy. We analyzed 60 nodal and extranodal DLBCLs and 28 DLBCL cell lines using Agilent Human Genome 44K or 105K CGH oligonucleotide arrays (Agilent Technologies). Slides were scanned with an Agilent Scanner, and the data were analyzed with Agilent Feature Extraction, Agilent CGH Analytics, and Nexus Copy Number software (Biodiscovery, Inc.). 60% of cases exhibited a gain in chromosomal material from 7q22. Patients whose malignancies exhibited this particular abnormality showed an increased survival of approximately 30% compared with those that did not. The smallest common region of gain consisted of a 150 kb stretch of DNA that contains a gene that encodes the zinc finger transcription factor protein ZKSCAN1. ZKSCAN1 protein expression was increased in cases that exhibited gains of 7q22. ZKSCAN1 has previously been implicated in the regulation of cell proliferation. It also appears to be regulated by the serine/threonine kinase ataxia‐telangiectasia mutated (ATM). ATM is activated in response to genotoxic insults and regulates proteins involved in the DNA damage checkpoint, cell cycle arrest, DNA repair, and apoptosis. Thus, ZKSCAN1 may play a key role in regulating the proliferation and/or survival of lymphoma cells in DLBCLs that exhibit a gain of 7q22.
Immunoexpression of endoglin and CD‐34 in various human pituitary adenoma types
Authors: Rotondo Fabio , Sharma Soniya , Scheithauer Bernd W , Cusimano Michael , Kovacs Kalman
Published: June 2021

Angiogenesis is a crucial event in tumor growth, invasion and development of metastasis. Endoglin (CD‐105), a disulfide‐linked homodimeric cell membrane glycoprotein of 180 kDa, located on chromosome 9q and binds to several components of the TGF beta family. In contrast to other endothelial cell markers, endoglin can be demonstrated only in proliferating endothelial cells. We investigated endoglin immunoexpression in various surgically removed human pituitary tumor types and compared the results with CD‐34 immunoexpression. For immunohistochemistry, the streptavidin‐biotin‐peroxidase complex method was used and the number of vessels were counted in 10 low power fields. Results show that endoglin immunostains fewer vessels than CD‐34 in every pituitary adenoma type. The number of endoglin immunopositive vessels is highest in untreated prolactinomas and subtype 3 silent pituitary adenomas and lowest in subtype 1 silent corticotroph adenomas and GH adenomas exposed to somatostatin analogs and prolactinomas exposed to dopamine agonists. Our findings are consistent with the view that endoglin is upregulated in proliferating endothelial cells and its expression can be used as an indicator of vascular neoformation in pituitary tumors. The low levels of endoglin expression in pituitary tumors exposed to somatostatin or dopamine agonists suggests that these therapeutic drugs can inhibit angiogenesis.
Molecular Histology of Tissue using Imaging Mass Spectrometry and Classification Analysis
Authors: Cornett Dale Shannon , Schwamborn Kristina , Wild Peter , Caprioli Richard M
Published: June 2021

Introduction

Imaging mass spectrometry (IMS) applied to fresh frozen or formalin‐fixed tissues enables the visualization of tissue/cancer specific protein/peptide distribution without prior knowledge of the target compounds. Classification analyses of these ion images exhibit strong correlation to high resolution histological images.

Method

Serial sections from a tissue microarray (TMA) with formalin‐fixed and paraffin‐embedded (FFPE) normal tissue samples (n=12), cancer samples (n=30) and cell lines (n=16) from different organ sites were analyzed by on‐tissue digestion followed by MALDI imaging and MS/MS analysis.

Preliminary data

On average, 210 high abundance peptides were observed in the mass range from m/z 600–4000. Analyses of serial sections performed on different days and two different instruments showed reproducible patterns. Spectra from various samples, when compared with high resolution histology, showed distinctive differences in peak patterns. For example, peptides detected at m/z 1650.0 and 2089.2 were at significantly higher expression in samples with leiomyosarcoma. Similarly, peptides at m/z 1506.7 and 1177.7 were also seen with significantly higher abundances in pancreas and astrozytoma, respectively.
Effect of cancer hereditary susceptibility on the prognosis of esophageal cancer patients from high esophageal cancer incidence area in China
Authors: Li Xiumin , Zhao Zhi‐Min , Zhang Yuan‐Zhi , Zhou Fu‐You , Hou Xiao‐Hua , Ji Hong‐Long
Published: June 2021

Objective

To investigate the impact of hereditary susceptibility on prognosis in patients with esophageal cancer.

Methods

A total of 742 cases of esophageal cancer from high risk esophageal cancer regions were followed up successfully. Student's t‐test was used to compare the average age of esophageal cancer onset. Survival curves were plotted by Kaplan‐Meier methods.

Results

The average onset age in patients with esophageal cancer was not related to the cancer hereditary susceptibility; the survival curve of esophageal cancer cases with positive esophageal cancer family history was significantly higher than negative group (59.83±3.60 VS 37.47±1.95 months, P=0.002); Within both the positive family history of esophageal cancer and malignant tumor groups, cases under 60 years old had significant higher survival rates compared with those of older (P <0.01). In contrast, neither within the negative family history of esophageal cancer nor malignant tumor groups, there was similar difference.

Conclusions

Esophageal cancer patients with positive but not negative family history have a better prognosis. Moreover, the younger and middle‐aged patients within both positive family history of esophageal cancer and malignant tumor groups had a better prognosis. These clinical studies indicate that family history and age possess prognostic relevance.
Myogenic constriction occurs in the absence of a detectable increase in pLC20 in the presence of agonist‐induced tone
Authors: EL‐Yazbi Ahmed Fawzy , Walsh Emma J , Walsh Michael P , Cole William C
Published: June 2021

Force generation in resistance arteries in response to increased transmural pressure, the myogenic response, is thought to involve increased cytosolic [Ca2+], activation of myosin light chain kinase, phosphorylation of myosin regulatory light chain subunits (LC20), actomyosin ATPase activation and cross‐bridge cycling, as well as Ca2+ sensitization owing to Rho‐associated kinase‐mediated inhibition of myosin light chain phosphatase activity (Johnson RP et al., J Physiol. 2009, 587:2537). Here, we studied the interplay between pressure‐ and agonist‐induced mechanisms of vasoconstriction in rat cererbal arteries. Elevating pressure from 10 to 60 mmHg caused myogenic constriction and an increase in LC20 phosphorylation. Subsequent exposure to serotonin (1 μM) at 60 mmHg evoked a further increase in pLC20 content and constriction. In contrast, a further increase in pLC20 was not detected when pressure was elevated after serotonin pretreatment despite the presence of a clear myogenic response. These results suggest that: 1) altered wall tension owing to agonist pretreatment affects the mechanism underlying pressure‐induced force generation, and 2) myogenic force generation may involve LC20‐independent mechanisms or require minimal additional (undetectable) LC20 phosphorylation when activated in the presence of agonist. (Funded by CIHR MOP‐97988, CIHR & AHFMR Fellowships)
Evidence for the role of A2B adenosine receptor in the regulation of vascular tone using A2B KO mice
Authors: Sanjani Maryam Sharifi , Ponnoth Dovenia , Roush Kevin , Tilley Stephen , Mustafa S. Jamal
Published: June 2021

Adenosine, an endogenous nucleoside, exerts its effects through its four receptor subtypes (AR): A1, A2A, A2B, and A3. Adenosine is involved in the regulation of vascular tone where its effects are subtype‐dependent. The contribution of ARs needs to be further investigated in order to better understand the heterogeneity of vasculature responses. In mice aorta, A2A AR is known to be involved in vascular relaxation, however, the role of A2B AR has not been studied in this tissue. To investigate this, we performed organ bath experiments. Tissues with <40% ACh relaxation to PE (10−6M) contraction were excluded (endothelial integrity). NECA, a non‐selective AR agonist, induced a significantly higher contraction in A2B KO compared to WT (21.8±3.2% contraction vs 21.9±8.5% relaxation at 10−5M, p<0.01) showing that activation of A2B AR may induce relaxation. CCPA, a selective A1 agonist, induced contraction (38.9±6.3% at 10−6M) was significantly reduced in A2B KO (5.9±7.8% at 10−6M, p<0.01) suggesting the down‐regulation of A1 AR. Cl‐IBMECA, a selective A3 agonist, showed significantly lower contraction in A2B KO compared to WT (14.6±3.3% vs 42.9±5.3% at 10−6M, respectively) further suggesting the down‐regulation of A3 AR in A2B KO. These data show a relationship between A2B and A1 and A3 ARs which must be taken into account in the control of vascular tone. Supported by HL027339, HL094447, HL071802, and T‐32 HL090610.
Modulation Of Cyclosporine‐Induced Hypertension By Central Endothelial And Neuronal Nitric Oxide Synthases
Authors: El‐Mas Mahmoud M , Omar Amal G , Helmy Mai M , Mohy El‐Din Mahmoud M
Published: June 2021

We recently reported an intermediary role for central sympathoexcitatory neurons in the hypertensive action of acute cyclosporine (CSA) in conscious rats. Here, we report on the role of central nitrergic pathways, which negatively correlate with sympathetic outflow, in CSA hypertension. CSA (20 mg/kg i.v.) produced an abrupt increase in blood pressure that was maintained for at least 45 min. Intracisternal (i.c.) administration of Nω‐nitro‐L‐arginine methyl ester (L‐NAME, nonselective NOS inhibitor), or 1H‐[1,2,4] oxadiazolo[4,3‐a] quinoxalin‐1‐one (guanylate cyclase inhibitor), or L‐arginine (NOS substrate) abolished the hypertensive action of CSA, suggesting a role for inhibition of NO/GC signaling in CSA response. CSA hypertension was also reduced after selective inhibition of endothelial or neuronal NOS by i.c. N5‐(1‐iminoethyl)‐L‐ornithine and Nω‐propyl‐L‐arginine, respectively, implicating both constitutive NOS isoforms in the hypertensive response. Consistent with the sympathoinhibitory role of central NOS, i.c. L‐NAME attenuated the hypotensive action of the central sympatholytic drug moxonidine. It is concluded that the inhibition of constitutive NOS isoforms and downstream GC/cGMP underlies the acute hypertensive, and perhaps the causally related sympathoexcitatory, action of CSA.
p38 MAPK inhibition prevents angiotensin II‐induced elevation of arginase (ARG) activity and impaired vasorelaxation
Authors: Shatanawi Alia , Romero M J , Chandra S , Yao L , Toque H Flores , Iddings J A , Caldwell R B , Caldwell R W
Published: June 2021

Elevated angiotensin II (Ang II) levels contribute to vascular endothelial dysfunction (ED) in a variety of diseases. Enhanced ARG activity contributes to ED by decreasing L‐arginine availability to NO synthase, thus decreasing NO production and increasing reactive oxygen species. We have shown previously that Ang II elevates ARG activity and reduces NO formation in bovine aortic endothelial cells (BAECs). Here we show that Ang II also increases ARG activity in vivo and that activation of p38 MAPK is involved. In aortas from mice given s.c. infusion of Ang II (42 μg/kg/h, 2 wks), ARG activity was elevated compared to controls and was accompanied by impaired EC‐dependent vasorelaxation. Treatment of the mice with the p38 MAPK inhibitor SB 202190 (5 μg/kg/d, s.c.) significantly inhibited the Ang II‐induced increase in ARG activity and improved vasorelaxation. Studies in BAECs exposed to Ang II (0.1 μM, 24h) showed elevated ARG activity/expression (40%) and decreased NO production (25–30%). These effects were prevented by pretreatment with SB 202190 (2 μM) or the Rho kinase inhibitor Y‐27632 (10 μM), which also prevented phosphorylation of p38 MAPK. Collectively, our results indicate involvement of Rho kinase and p38 MAPK in the Ang II signaling pathway for ARG elevation and ED. Inhibiting p38 MAPK might be a beneficial therapeutic target for ED associated with Ang II. Support: NHLBI 70215 and 76146.
Impaired L‐arginine recycling induces compensatory mechanisms in NO‐dependent vessels
Authors: Chennupati Ramesh , Janssen Ben J.A. , Lamers Wout H , De Mey J.G.R , Koehler SE
Published: June 2021

Introduction

Impaired endothelial nitric oxide (NO) is a hallmark of endothelial dysfunction. Deficiency of the nitric oxide synthase (NOS) substrate arginine (Arg) may be even more detrimental than NOS deficiency.

Objective

We assessed whether impaired Arg recycling inhibited endothelium‐dependent relaxation (EDR).

Methods

We generated endothelium‐selective ASS knockout mice (ASS‐KO) with the loxP/Cre approach, using Tie2Cre for gene ablation. Femoral artery (FA) and saphenous resistance arteries (SRA) were investigated using wire‐myography.

Results

Immunohistochemical stainings on FA and SRA showed specific and complete deletion of ASS in endothelial cells (EC). EDR of FA is dependent on both NO and endothelium‐derived hyperpolarizing factor (EDHF) and was identical in ASS‐KO and WT vessels. In SRA, EDR is NO‐dependent and can be completely blocked by L‐NAME (100μM) in WT vessels. In ASS‐KO vessels, however, L‐NAME inhibits EDR only by 50%.

Conclusion

Reduced arginine recycling in EC of SRA resulted in induction of EDHF‐dependent relaxation in NO‐dependent arteries.
A rapid dietary assessment tool to estimate erythrocyte omega‐3 fatty acid levels
Authors: Harris Kristina Arline , Kris‐Etherton Penny , Hartman Terryl , Mitchell Diane , Collison‐Schmidt Brenda , Harris William Stephen
Published: June 2021

The omega‐3 index (O3I), the percentage of long chain omega‐3 fatty acids (LC n‐3 FAs) in erythrocyte membranes, is an emerging risk factor for sudden cardiac death. A dietary tool to assess LC n‐3 FA intake validated by the O3I would be useful in clinical settings. We hypothesized that a single‐question tool measuring the frequency of LC n‐3 FA‐rich fish consumption would correlate more strongly with the O3I than dietary recall data due to the episodic nature of fish consumption. Healthy participants (n=29) were asked: “How often do you eat—as a main course—tuna or other non‐fried fish? a) > 1x/month, b) 2–3x/month, c) 1x/week d) 2x/week, e) >2x/week.” A blood sample was taken, and 3, unannounced 24‐hour recalls were taken over 2 wks. The O3I's for each fish intake level were: 3.76±0.15%, 5.04±0.01%, 4.08±0.14%, 5.65±0.08%, and 5.94±0.36% (respectively; a, b vs e, p<0.05). LC n‐3 FA intakes by recall (mg/d/1000 kcal) were: 18±2, 80±25, 388±110, 205±30, and 309±48 (respectively; a vs e, p>0.05). The Spearman correlation between O3I and LC n‐3 FA intake was 0.38 (p=0.04). The model predicting the O3I using ordinal frequency categories had R2 of 0.31 (p=0.002), and using LC n‐3 FA intake as a continuous variable had an R2 of 0.15 (p=0.04). We conclude that a single question focusing on the habitual intake of n‐3‐rich fish provided a better estimate of the O3I than dietary recalls. (Self‐funded).
Predictive Validity of Dietary Assessment Questionnaires
Authors: Arab Lenore , Tseng Chi‐hong , Cambou Mary Catherine
Published: June 2021

Traditionally correlation analyses and underreporting have been used for determining the value of a dietary assessment tool. We explored the use of predictive validation using log regression models with and without covariates and interaction terms to compare 6 days of 24‐hour recalls with food frequency questionnaires (DHQ). The data derives from a population of African‐Americans and Caucasians from Los Angeles participating in the Energetics Study, designed to test the validity of a web‐based automated 24‐hour recall (DietDay) using doubly labeled water as a marker of true energy intake. We observed that underreporting was considerably lower, with the recalls capturing 84% of energy expenditure as compared with 73% by the DHQ, while the correlation analyses showed similar correlations for both dietary assessment tools. We used Akaike's Information Criterion (AIC) to compare models and Bayesian Information Criterion (BIC) as a form of regularization to prevent over fitting. With the backward model selection procedure the adjusted R‐square of the final model was 0.511 for DietDay with covariates, compared to 0.103 without covariates. The Adjusted R‐squares for DHQ was 0.509 with and 0.118 without covariates. This quantitative approach provides more robust comparisons of the predictive validity of dietary assessment tools. Supported by NIH R01CA105048.
Norepinephrine decreases ejection fraction and downregulates sepsis‐induced elevated antiapoptotic genes in a hyperdynamic polymicrobial septic rat model
Authors: Chopra Mani , Das Padmalaya , Golden Honey B. , Dostal David E. , Sharma Avadhesh C.
Published: June 2021

The present study was conducted to elucidate the signaling cascade targets and echocardiographic alterations following NE‐infusion during sepsis. Male Sprague‐Dawley rats (350–00g) were made septic using 200mg/kg cecal inoculum, ip. Echoacardiography was performed at the baseline and 3 and 7 days post sepsis. NE was infusedfor 2 hr, before the end of 3rd day of echocardiographic measurements. On the 7th day, each rat was euthanized and heart tissues were harvested for isolation of RNA. The PCR reactions were performed using RT2 profiler ™PCR array PARN‐012 on a ABI Fast 7900 using RT2 Real‐Time™ SYBR Green PCR master mix PA‐012. Echocardiography demonstrated that ejection fraction (EF) and fractional shortening (FS) were significantly decreased as the sepsis progressed from day 1 to day 7. The NE‐infusion further deteriorated the EF in sepsis at day 7. NE failed to improve sepsis‐induced decreased FS at days 3 and 7. Super array data revealed that during sepsis TNF‐alpha and anti‐apoptotic genes (Prok2 and IL‐10) were upregulated at day 1, while Bid 3 was uppregulated at day 3 compared to the sham group. At 7 day post sepsis, CD40lg, Bid 3 and Birc1b were upregulated compared to the sham group. The echocardiography alterations demonstrated that NE exacerbates sepsis‐induced depressed EF, which was associated with the reversal sepsis‐induced elevated expression of antiapoptotic genes during hyperdynamic sepsis.
Effect of alcohols on a soluble form of adenylyl cyclase type 7 expressed in bacteria
Authors: Dokphrom Usa , Yoshimura Masami
Published: June 2021

Cyclic AMP (cAMP) signal transduction has been postulated to play an important role in the development of alcoholism in humans. Our previous studies showed that generation of cAMP by mammalian membrane‐bound adenylyl cyclase (AC) is enhanced in an isoform specific manner and that alcohol‐cutoff phenomena are also isoform‐specific. We hypothesized that alcohols exhibit their effect on AC activity by direct interaction with AC proteins. However, experimental systems employed in the past such as intact cells and membrane preparations are too complex and do not allow us to unequivocally test this hypothesis. We decided to study the effect of alcohols on AC recombinant proteins expressed in bacteria. A type 7 AC (AC7) recombinant protein consisting of the C1a and C2 domains was designed and expressed in E. coli. The AC activity of the soluble form of AC7 recombinant protein was significantly increased by manganese and forskolin. In addition, n‐alkanols (ethanol to hexanol) significantly enhanced manganese‐stimulated activity of the AC7 recombinant protein. The alcohol cutoff for this recombinant protein is pentanol, which is same as that of wild type observed in mammalian system. These results suggest that the AC7 recombinant protein retains alcohol response similar to that of the parent enzyme. The results are consistent with our working hypothesis. Supported by NIH Grant AA13148 (MY).
SH3 domain of βPix interacts with Sprouty2
Authors: Ying Chunyi , Anderson Kimberly M , Patel Tarun B
Published: June 2021

Sprouty2 (Spry2) is an important modulator of receptor tyrosine kinase signaling and, therefore, regulates a number of growth factor‐mediated processes such as cell proliferation, cell migration and tubular morphogenesis. Previously, we reported that Spry2 inhibits the migration of a number of different cell types. In rat intestinal epithelial cells (IEC‐6) Spry2 inhibits cell migration by attenuating the activation of Rac1. However, the mechanism(s) by which Spry2 inhibits Rac1 activation remain unknown. βPix, a GEF protein, binds to Rac1 and targets it to membrane ruffles and focal adhesions, which is necessary for Rac1 activation. Using immunoprecipitation and pull‐down assay, we found that Spry2 constitutively interacts with βPix in Spry2 expressing Hela cells. The SH3 domain of βPix is necessary and sufficient for binding Spry2, and mutation in the c‐Cbl binding site (Y55F) of Spry2 does not alter the interaction between Spry2 and βPix. These data demonstrate that the Spry2/βPix interaction is independent of c‐Cbl binding to Spry2. Mutation of a non‐canonical proline‐rich domain (P86–P92, PEHRQPP) on Spry2 attenuates its interaction with βPix. In addition, βPix specifically associates with Spry2, not other Spry isoforms which lack the P86–P92 domain. These data suggests that the Spry2/βPix interaction is important in Spry2‐mediated inhibition of Rac1 activation.
Heterologous sensitization of human AC5: dependence on heterotrimeric G proteins
Authors: Ejendal Karin F K , Sadana Rachna , Vidi Pierre‐Alexandre , Dessauer Carmen W , Watts Val J
Published: June 2021

We investigated the role of heterotrimeric G proteins in heterologous sensitization of adenylyl cyclase type 5 (AC5). Although acute stimulation of Gαi‐coupled receptors inhibits AC5 activity, persistent receptor activation enhances AC5 activity. This phenomenon is known as heterologous sensitization of AC, and has been implicated in drug dependence.

Previous work revealed an absolute requirement for Gαs in heterologous sensitization of AC5. To further elucidate the mechanism, we assessed sensitization in cells devoid of endogenous Gαs, co‐expressing the D2L receptor, AC5, and Gαs mutants. Our data showed that AC5‐Gαs, but not Gβγ‐Gαs interactions or palmitoylation of Gαs, were required for heterologous sensitization of AC5.

Next, we explored the role of Gβγ‐AC5 interactions in heterologous sensitization. Sequestering Gβγ with βARK‐ct partially attenuated heterologous sensitization. Deletion of a novel N‐terminal Gβγ interacting domain of AC5 abolishes Gβγ binding, but does not greatly alter other forms of acute regulation. However, the AC5 deletion mutant was selectively impaired in heterologous sensitization. Together, these studies support a complex model of heterologous sensitization of AC5 where Gαs is directly interacting with AC5. In contrast, Gβγ appears to have multiple and indirect roles in heterologous sensitization of AC5.

Supported by P.U. and grants VJW‐MH060397 and CWD‐GM060419.
Inhibition of fMLP‐stimulated respiratory burst in human neutrophils by 5‐hydroxy‐2‐(4‐hydroxy‐3‐methoxyphenyl)‐3,7‐dimethoxy‐4H‐chromen‐4‐one (MSF‐2), a natural lignan extract from Melicope Semecarpifolia: specific inhibition of phosphatidylinositol 3‐kinase activity
Authors: Liao Chang‐Hui , Liu Chia‐Hsin , Chen Jih‐Jung , Tseng Ching‐Ping , Day Yuan‐Ji
Published: June 2021

We examined the underlying inhibitory mechanisms of a lignan, MSF‐2, extracted from the fruit of Melicope. Semecarpifolia in human neutrophils.To clarify the effect of MSF‐2 on fMLP‐induced neutrophil activation, intracellular signals induced by fMLP were evaluated. MSF‐2 inhibited the fMLP‐induced superoxide anion production and cathepsin G release in a concentration‐dependent manner with respective IC50 values of 22.8±4.8 and 14.2±1.7 μM. MSF‐2 was found to suppress fMLP‐induced phosphorylation of on Akt and intracellular calcium mobilization, however, it was not observed to inhibit the phosphorylation of ERK and p38 induced by fMLP. Moreover, MSF‐2 inhibited the PDK1, which subsequently phosphorylated on Akt. This could be its underlying inhibitory mechanism of phosphorylation because. In addition, MSF‐2 demonstrated inhibitory effects on PI3K activity and it was found to inhibit phosphorylation of PLCγ2 which was activated by PI3K. MSF‐2 inhibited fMLP‐induced superoxide anion production by inhibiting fMLP‐induced Akt activation.
Modulation of Epidermal Growth Factor stimulated ERK phosphorylation and cell motility by inositol trisphosphate kinase
Authors: Sekar M. Chandra , Shahiwala Kirat , Leloup Ludovic , Wells Alan
Published: June 2021

Epidermal growth factor [EGF] mediated stimulation of its receptor in endothelial cell [EC] is accompanied by phosphorylation of the EGF‐receptor [EGFR] and activation of phospholipase C‐γ, resulting in the breakdown of phosphatidylinositol(4,5)‐bisphosphate and generating inositol (1,4,5)‐trisphosphate [IP3] and diacylglycerol. IP3 thus formed can be further converted to inositol (1,3,4,5)‐tetrakisphosphate [IP4] by an enzyme called IP3‐kinase [IP3K]. In this study we have investigated the effect of modulation of intracellular IP3K activity by the use of an inhibitor, 2‐trifluoromethyl [6‐(4‐nitrobenzyl)‐purine] [IP3KI] and siRNA against IP3KB on EGF‐induced ERK‐phosphorylation and cell motility. EC cells were stimulated with EGF in presence or absence of IP3KI. Cells were lysed, and proteins separated by SDS gel electrophoresis; the western blots were probed with polyclonal antibody to designated proteins. IP3KI, at 10 μM and 20 μM concentrations inhibited both EGF‐stimulated ERK phosphorylation and motility in a dose‐dependent manner. Interestingly, at the above concentrations, IP3KI also inhibited EGFR phosphorylation at Y1173. To further establish whether any of the above effects of IP3KI can be attributed to the IP3K activity rather than the direct inhibition of EGFR kinase by IP3KI, we downregulated intracellular IP3KB with siRNA. Lipofectamine transfection of the EC with 50 pmole and 75 pmole siRNA against IP3KB was accompanied by decreased level of IP3KB [as determined by western blot], as well as inhibition of phosphorylation of both EGFR [at Y1173] and ERK1/2. Based on these data, we conclude that IP3KB, and therefore its resulting product IP4, is capable of modulating EGF stimulated response at the receptor level.
Gβγ Signaling Promotes Breast Cancer Cell Migration and Invasion
Authors: Kirui Joseph K , Xie Yan , Wolff Dennis W , Jiang Haiong , Abel Peter W , Tu Yaping
Published: June 2021

Signaling through G‐protein coupled receptors (GPCRs) promotes breast cancer metastasis. G‐proteins convey GPCR signals by dissociating into Gα and Gβγ subunits. The aim of this study was to determine whether blockade of Gβγ suppresses breast cancer cell migration and invasion, critical steps of metastasis. Transwell chamber assays showed that expression of a Gβγ scavenger peptide derived from β‐adrenergic receptor kinase, attenuated migration and invasion of both breast cancer MDA‐MB‐231 and MDA‐MB‐436 cells by 50%. Migration and invasion of those cells were also blocked by M119K, an inhibitor of Gβγ, with maximum inhibition of 80% and IC50 of 1–2 μM. Activation of Gi‐protein coupled CXC chemokine receptor 4 (CXCR4) promotes breast cancer metastasis. M119K suppressed CXCR4‐dependent MDA‐MB‐231 cell migration by 75% with IC50 of 0.9 μM. Immunostaining analysis indicated that 10 μM M119K attenuated CXCR4‐stimulated, Rac‐dependent formation of lamellipodia, a key structure required for metastasis. In contrast, CXCR4‐dependent inhibition of adenylyl cyclase, a Giα‐mediated response, was not blocked by M119K but was blocked by pertussis toxin, which inactivates Giα. This is the first to directly demonstrate the role of Gβγ in breast cancer metastasis and suggests that targeting Gβγ signaling pathways may provide a novel strategy for suppressing breast cancer metastasis. Supported by Nebraska State LB595.
Preparation of a claudin‐4‐targeted anti‐tumor molecule
Authors: Kondoh Masuo , Saeki Rie , Kakutani Hideki , Hamakubo Takao , Watari Akihiro , Yagi Kiyohito
Published: June 2021

The majority of malignant tumors are derived from epithelial tissues. Tight junctions (TJs) are typical structure of epithelium, and deregulation of TJs is often observed during neoplastic transformation. Claudins are key molecules in formation of TJs; proteins in the 24‐member claudin family contain four transmembrane domains. Claudin‐4 is overexpressed in some human neoplasias, including breast, pancreatic and prostate cancers. These findings indicate that the targeting of claudin‐4 may be a novel strategy for tumor therapy, whereas there are few successes to prepare claudin‐4‐targeting molecule because of its low antigenicity. We previously found that the claudin‐4 binder (C‐CPE) is a novel type of absorption‐enhancer of drug (Mol. Pharmacol., 2005). In the present study, we prepared a claudin‐4–targeting molecule by fusion of C‐CPE with the protein synthesis inhibitory factor (PSIF) derived from Pseudomonas exotoxin. C‐CPE‐PSIF was cytotoxic to claudin‐4‐expressing cells, and pretreatment of the cells with C‐CPE attenuated C‐CPE‐PSIF‐induced cytotoxicity. Mutation of the claudin‐4‐binding domains also attenuated its cytotoxicity. Intratumoral injection of C‐CPE‐PSIF significantly suppressed tumor growth. In contrast, C‐CPEmutant‐PSIF lacking claudin‐4‐binding activity had no effect on tumor growth. These data indicate that a claudin‐4‐targeting may be a potent anti‐tumor strategy.
Effect of Resveratrol and Curcumin on Tumor Volume and Survival in Ehrlich Ascites Carcinoma‐Bearing Mice
Authors: El‐Azab Mona Farag , Hishe Hailemichael , Moustafa Yasser , El‐Awady El‐Sayed
Published: June 2021

Solid tumors were induced by a subcutaneous injection of Ehrlich ascites carcinoma (EAC) cells (2.5x106 cells/mouse) in the right flank of Swiss albino mice. These tumors were used to evaluate the antitumor effect of resveratrol (10mg/kg, i.p), or curcumin (10mM, 100μl/mouse, i.p.) as individual treatments or in combination with carboplatin (5mg/kg, i.p). Tumor size was measured every other day for 21 days. Mortality was recorded daily along the study period to calculate the percentage survival of animals, mean survival time (MST) and percentage increased life span (%ILS). Individual treatments with resveratrol or curcumin produced a significant reduction (p≤0.05) in tumor volume, which tended to be enhanced progressively from the individual treatments to the combinations with carboplatin. The treatment with carboplatin, curcumin and their combination could expand the life span of animals for 78, 107 and 105 days respectively. The combination of curcumin with carboplatin could produce the highest increase in MST (68.20±6.70) and %ILS (82.4%). Single and combined treatment of resveratrol produced an enhancement of the life span by 34% and 57.2%, respectively.

These data show that resveratrol and curcumin produced antitumor effect on EAC solid tumor in Swiss mice. The results suggest the beneficial role of the phytochemicals as adjuvants to chemotherapy in the treatment and/or prevention of cancer.
Cytotoxic activity of Taraxacum officinale in OV2008 human ovarian cancer cell line is mediated by caspase 8 activity
Authors: Levy Arkene Stacy‐Ann , Stupack Dwayne G
Published: June 2021

The plant Taraxacum officinale, commonly known as dandelion belongs to the family Asteraceae. An aqueous extract of dried Taraxacum officinnale (TO) leaves was evaluated for its cytotoxic activity in OV2008 ovarian cancer cells. Cells were cultured in DMEM media supplemented with 10% FBS. OV2008 cells were seeded in 96 well plates and exposed to TO extract at concentrations ranging from 120–180ug/ml for 72 hours in triplicate. Cytotoxicty was measured using the MTT assay. Significant cytotoxic activity of TO extract was observed in OV2008 cells (IC50 = 140ug/ml). Staining of TO treated cells with propidium iodide revealed the presence of apoptotic bodies. To examine the role of caspases in the apoptotic death of these cells, Caspase 3, 8 and 9 deficient OV2008 cells were prepared via shRNA‐based knockdown of the caspase‐8 transgene. The cytotoxic activity of TO was abolished selectively in caspase‐8 and ‐3 negative OV2008 cells, suggesting that caspase 8 promotes the cytotoxic activity of TO in OV2008 cells. This project was funded by the Fulbright organization and NCI grant CA107263 to DGS.
Involvement of NSAID‐activated gene‐1 in a novel synthetic hexahydrocannabinol analogue‐induced growth inhibition and apoptosis of colon cancer cells
Authors: Thapa Dinesh , Babu Dinesh , Kwak Mi‐Kyoung , Lee Yong‐Rok , Kim Jung‐Ae
Published: June 2021

Nonsteroidal anti‐inflammatory drug (NSAID)‐activated gene‐1 (NAG‐1) has received greater attention as a novel molecular target for various cancers including colorectal cancer. In this study, we identified a novel synthetic hexahydrocannabinol analogue LYR‐8 as a potent NAG‐1 inducer which inhibited growth and induced apoptosis in a panel of human cancer cells irrespective of p53‐status. In colon cancer cells, LYR‐8 dramatically induced NAG‐1expression in a concentration‐ and time‐dependent manner. The induction of NAG‐1 protein expression was independent of p53‐activation even in wild‐type HCT116 cells which had increased nuclear p53 after LYR‐8 treatment. The induction of NAG‐1 promoter activity by the LYR‐8 was strongly associated with increased Sp1 activation as noted in various luc‐promoters activities. Furthermore, pre‐treatment of specific Sp1 inhibitor Mithramycin A completely reversed the LYR‐8‐induced NAG‐1 expression. Genetically knockdown of NAG‐1 using siRNA significantly reversed cell death induced by LYR‐8 in both wild‐type as well as mutant p53‐expressing colon cancer cells, thus suggesting the role of p53‐independent NAG‐1 activation. These results suggest that p53‐indepenent induction of NAG‐1 via Sp1 activation is a promising therapeutic approach and novel compound like LYR‐8 could be a potent chemotherapeutic agent targeting colon cancers irrespective of p53‐mutation.
Novel Small‐molecule Agonists of Human Formyl Peptide Receptors and Pharmacophore Models of their Recognition
Authors: Kirpotina Liliya N , Khlebnikov Andrei I , Schepetkin Igor A , Ye Richard D , Rabiet Marie‐Josèphe , Jutila Mark A , Quinn Mark T
Published: June 2021

N‐formyl peptide receptors (FPR) are G protein‐coupled receptors involved in host defense and sensing cellular dysfunction. Our recent high‐throughput screening efforts have focused on the identification of unique non‐peptide agonists of FPR1/FPR2. In the present studies, we screened a chemolibrary of drug‐like molecules for their ability to induce intracellular calcium mobilization in RBL‐2H3 cells transfected with human FPR1 or FPR2. Screening resulted in the identification of novel and potent agonists that were specific for either FPR1 or FPR2 with EC50 values in the low micromolar range. Specificity of the compounds was supported by analysis of calcium mobilization in HL‐60 cells transfected with human FPR1 and FPR2. Additionally, all but one agonist activated intracellular calcium flux and chemotaxis in human neutrophils. Molecular modeling of the group of FPR1 and FPR2 agonists using field point methodology allowed us to create pharmacophore models for ligand binding sites and formulate requirements for these specific N‐formyl peptide receptor agonists. These studies further demonstrate that agonists of FPR1/FPR2 include compounds with wide chemical diversity and that analysis of such compounds can enhance our understanding of FPR ligand/receptor interactions. This work was supported in part by NIH grants P20 RR‐020185 and P01 AT0004986‐01, and contract HHSN266200400009C.
Ligand‐Induced Conformational Changes in Cytochrome P450 2B4: A Multi‐Technique Approach
Authors: Wilderman Paul Ross , Gay Sean , Roberts Arthur , Li Sheng , Liu Tong , Woods Virgil , Halpert James
Published: June 2021

Important structural information on the xenobiotic metabolizing cytochrome P450 2B4 (CYP2B4) has been provided by crystal structures either without a ligand or in the presence of a series of imidazole containing inhibitors [1,2]. While there is a wealth of information about static structures of CYP2B4 complexed with inhibitors, less is known about their dynamic solution behavior. Hydrogen‐deuterium exchange mass spectrometry (DXMS) was utilized to elucidate the solution behavior of CYP2B4 in the absence and presence of 4‐(4‐chlorophenyl)imidazole (4‐CPI), 1‐biphenyl‐4‐methyl‐1H‐imidazole (1‐PBI), and the drug ticlopidine [1,2]. The results revealed similar exchange rates for CYP2B4 alone and in the presence of 1‐PBI. In addition, the exchange rates were significantly slowed to a similar extent in the B‐C loop and the F‐G cassette of CYP2B4 in the presence of either 4CPI or ticlopidine. As predicted by DXMS, the crystal structure of the CYP2B4 complex with ticlopidine was very compact. Interestingly, the thiophene ring appeared to be oriented away from the heme iron, which was verified by T1 relaxation measurements from NMR. This would preclude metabolism at the thiophene ring in contrast to the closely related human CYP2B6. (Supported by ES003619 (JRH), GM066170 (VLW), and T32‐DK07233 (PRW)).