bluuebunny/crossref_metadata_2025 huggingface dataset:
Beta‐Carotene Inhibits Expression of Tumor Necrosis Factor Receptor‐Associated Factor 1 and Hyper‐Proliferation in Helicobacter pylori‐Infected Gastric Epithelial Cells
Authors: Park Yong Chae , Lim Joo Weon , Kim Hyeyoung
Published: November 2023

Hyper‐proliferation is observed in the mucosa of Helicobacter pylori‐(H. pylori) infected patients with adenocarcinoma. Tumor necrosis factor receptor‐associated factor 1 (TRAF1) is involved in cell survival and regulated by redox‐sensitive transcription factor NF‐kappa B. Overexpression of TARF1 by H. pylori promotes proliferation of gastric mucosal cells. The complex formed by TRAF1 interacts with inhibitor‐of‐apoptosis proteins (IAPs) such as cIAP1 and cIAP2, and thus mediates the anti‐apoptotic signals. Previously, we found that H. pylori activates NADPH oxidase to produce reactive oxygen species (ROS) and activate NF‐kappa B in gastric epithelial cells. We previously showed that beta‐carotene inhibits the expression of cyclooxygenase‐2 and inducible nitric oxide synthase by reducing ROS levels and NF‐kappa B activation in H. pylori‐infected gastric epithelial cells. The purpose of the study is to determine whether beta‐carotene inhibits expression of TRAF1 by reducing ROS levels and suppresses cell proliferation in gastric epithelial cells infected with H. pylori treated with or without beta‐carotene.

Human gastric epithelial AGS cells were infected with H. pylori strain NCTC11637 at the ratio of 1:50 (cell/bacterium). Beta‐carotene was dissolved in tetrahydrofuran and treated to the cells at final concentration of 0.5 and 1 microM. Cell proliferation was determined by viable cell numbers. mRNA and protein levels were determined by real time PCR analysis and western blotting. ROS levels were measured using DCF‐DA assay.

As a result, H. pylori increased ROS levels and induced expression of TRAF1, cIAP1 and cIAP2 in AGS cells. H. pylori induced hyper‐proliferation of AGS cells. Beta‐carotene inhibited NADPH oxidase activation, hyper‐proliferation, and expression of TRAF1, cIAP1, and cIAP2, and reduced ROS levels in H. pylori‐infected cells. In addition, both a known antioxidant N‐acetyl cysteine and an NADPH oxidase inhibitor diphenyleneiodonium inhibited H. pylori‐induced TRAF1.

In conclusion, consumption of beta‐carotene‐rich foods may be beneficial for inhibiting H. pylori‐induced hyper‐proliferation of gastric epithelial cells by suppressing ROS‐mediated expression of TRAF1.
Ferroportin Deficiency Decreases Manganese Accumulation in the brain after Olfactory Manganese Exposure
Authors: Qutob Haya , Goodman Lenora , Seo Young Ah
Published: November 2023

Manganese (Mn) is both essential, yet toxic when present in excessive amounts. In particular, accumulation of Mn in the brain produces neurotoxic effects, disrupting motor function and behavior. A significant route of entry is uptake of airborne Mn, since this metal is efficiently transported into the brain through the nasal epithelium. Ferroportin (Fpn; SLC40A1) can function in the transport of Mn in addition to iron (Fe). Our previous studies have shown that Fpn deficiency impaired Mn metabolism in flatiron (ffe/+) mice, a genetic model of human “ferroportin disease”. Our recent studies have further shown that Fpn deficiency alters body Mn levels, and promotes brain Mn accumulation in flatiron mice, suggesting that toxicity related to Mn exposure could be modified by deficiency of Fpn export function. Therefore, we characterized the impact of Fpn deficiency on Mn accumulation in the brain after olfactory Mn exposure. Ffe/+ mice and wild‐type control (+/+) mice were intranasally instilled with MnCl2 (5 mg/kg) daily for 3 weeks. Mn and Fe levels in the blood, liver, and microdissected brain tissues were determined using inductively coupled plasma mass spectrometry (ICP‐MS). Flatiron mice had reduced blood Fe levels (532.3 vs. 475.5 mg/kg; P=0.0107) and higher Fe levels in liver (44.54 vs. 56.91 mg/kg; P=0.0481) compared to +/+ mice. These data confirm the Fe deficiency and loading phenotype of flatiron mouse associated with ferroportin disease. Blood Mn levels were significantly reduced in flatiron mice compared to +/+ mice (0.0315 vs. 0.0353 mg/kg, P=0.0332). These data agree with our previous findings of the Mn deficiency phenotype of flatiron mouse. Mn instillation increased Mn levels in the liver, which were not affected by Fpn deficiency (Mn X FFE interaction effect; P=0.0369). Flatiron mice showed higher Mn levels in most brain regions, including the olfactory bulbs (0.224 vs. 0.001 mg/kg, P < 0.0001), prefrontal cortex (0.273 vs. 0.163 mg/kg), hippocampus (0.230 vs. 0.180 mg/kg), cerebellum (0.196 vs. 0.172 mg/kg), and brain stem (0.253 vs. 0.174 mg/kg) compared to +/+ mice. Mn instillation increased Mn levels in brain regions, including olfactory bulbs (Mn effect, P=0.0013), cortex (Mn effect, P=0.0092), and midbrain (Mn effect, P=0.0033) compared with vehicle control. Interestingly, Mn‐instilled flatiron mice had significantly reduced Mn levels in the olfactory bulbs (FFE effect, P=0.0013), and most brain regions including the cortex, midbrain, hippocampus, and cerebellum compared to Mn‐instilled +/+ mice. Furthermore, Mn‐instilled flatiron mice displayed reduced Fe levels in the olfactory bulbs (FFE effect, P=0.0013) and elevated Fe levels in prefrontal cortex (FFE effect, P=0.0078) compared to Mn‐instilled +/+ mice. Taken together, these results indicate that olfactory Mn exposure decreases brain Mn accumulation in flatiron mice. These findings further suggest that Fpn deficiency reduces susceptibility to neurotoxicity induced by inhaled Mn.
Developmental Innervation of Rat Palatine Glands
Authors: Hakami Zaki , Wakisaka Satoshi
Published: November 2023

Objectives

Salivary glands maturation and function are controlled and influenced by the nervous system. Nevertheless, little is known about salivary glands innervation during development, particularly minor salivary glands. Therefore, this study investigated the development of the innervation of the palatine glands of rat.

Materials and methods

Frozen sections of rat palatine glands at different stages were immunohistochemically labeled for detection of two general nerve markers; protein gene product 9.5 (PGP 9.5) and growth‐associated protein‐43 (GAP‐43), and two neuropeptides; calcitonin gene‐related peptide (CGRP) and neuropeptide Y (NPY).

Results

PGP 9.5 and GAP‐43‐immunoreactive fibers (IRF) were present in the mesenchyme and in association with developing acini, ducts and blood vessels. GAP‐43‐IRF were more abundant and diffuse than PGP 9.5‐IRF at early stages, but showed similar distribution with growth, ramifying out from thick bundles in connective tissues until encircling the secretory units observed around postnatal day 21 (PN21). CGRP‐IRF were detected in the mesenchyme at embryonic day 20 (E20) and PN0. CGRP‐IRF became numerous around PN7 and PN10. They then decreased to the adult level at PN21, mainly located around ducts and infrequently blood vessels. NPY‐IRF were sparsely detected in the mesenchyme at E20, then detected in close proximity to acini in addition to blood vessels at PN3. NPY‐IRF increased till reaching the adult stage, and were mainly associated with blood vessels and around mucous cells and some serous demilunes.

Conclusion

The findings indicated a developmental modification of the sensory and autonomic innervation which may play a role in the functional maturation of the palatine salivary glands. Moreover, they may indicate their roles in regulating the secretory mechanisms and local blood flow of these glands.

Support or Funding Information

None
Associations between Depression and Cognitive Function in Turkish Elderly
Authors: İlhan Azad , Bilgic Pelin , Varli Murat , Rakıcıoğlu Neslişah , Hongu Nobuko
Published: November 2023

Introduction

The burden of depression among older individuals is an important public health issue, considering the increasing number of elderly population in Turkey. The prevalence of obesity has also grown in Turkish older adults. Therefore, the aim of this study was to investigate the relationship between depression and cognitive function in community‐dwelling older adults living in Ankara, Turkey.

Materials and Methods

A sample of 95 functionally independent patients (53 women and 42 men) aged 65 years or older (mean age: 74.01±7.4) was selected from Medicine Faculty of Ankara University. Standardized Mini Mental State Examination (S‐MMSE) was used to evaluate cognitive function. Geriatric Depression Scale (GDS) was used to assess depression status for all patients. Patients with advanced visual and auditory impairment, delirium, cancer and disabled were excluded.

Results

18 of 95 (%18.9) patients were diagnosed with dementia. Those with dementia and without dementia were divided into two groups. The dementia group's median scores of S‐MMSE and GDS were 16±4.25 and 16±8.25, respectively. Non‐demented group's median scores of S‐MMSE and GDS were 27±3 and 5±4, respectively. In all groups; S‐MMSE scores of men were significantly higher than women (p<0.05) and mean values of BMI were 26.95±5.1. When we analyzed the relationship between S‐MMSE values and the results of the BMI and GDS, we found significant negative correlations for S‐MMSE versus BMI (n=95, r= −0.39, p< 0.001) and S‐MMSE versus GDS (n=95, r= −0.65, p<0,001).

Discussion and conclusion

The study revealed that the cognitive impairment was significantly associate with depression, particularly in overweight both men and women. Therefore depression may have been reduced by both healthy eating and physical activity program in elderly.
Process Evaluation of a Youth‐Mentor Led Childhood Obesity Prevention Intervention at Urban Recreation Centers
Authors: Schmall Alexandria M , Trude Angela , Gittelsohn Joel
Published: November 2023

Urban recreation centers offer potential venues for youth‐led childhood obesity prevention programs in low‐income African‐American (AA) communities. However, little is known about how well such programs have been implemented. Thus, we aimed to examine the implementation of the B'more Health Communities for Kids (BHCK) peer‐mentoring nutrition program through a detailed process evaluation. We used a train‐the‐trainers approach to train Baltimore college and high school students as BHCK youth‐leaders (n=17), able to deliver nutrition education and cooking demonstrations to children (10–14 years old) attending seven recreation centers located in low‐income, food desert neighborhoods in Baltimore, MD. Each nutrition session (n=98) was evaluated through multiple process measures to ensure adequate reach, dose delivered, and fidelity, based on standards set a priori. Dose was determined based on the number of intervention materials (e.g. taste tests, educational handouts) delivered to the target population (4 standards). Fidelity was defined as the number of BHCK intervention sessions at recreation centers and youth‐leaders attending each session (5 standards). Reach identified the extent that the target population was exposed to intervention activities (2 standards). Process evaluation was analyzed using Microsoft® Excel and was scored as a “high” if the process domain met ≥100% of the high standard set, “medium” (50–99.9% of high standard) and “low” (<50% of high standard). Overall, the BHCK youth‐leader program was delivered with moderate to high reach and dose, and moderate fidelity. On average, 9 children attended each interactive session (total youth interaction, n= 912), over 1000 taste tests and giveaways were provided at sessions, and seven youth leaders completed the BHCK program. Given the success of the BHCK peer‐mentoring program, youth‐led programs can offer a feasible approach to addressing childhood obesity in low‐income African American neighborhoods.

Support or Funding Information

This research was supported by the Johns Hopkins Global Obesity Prevention Center (NIH U54HD070725) and NE‐Regional Nutrition Education Center of Excellence.
Maternal low protein diet decreases leptin expression in the brains of the neonatal rat offspring
Authors: Marwarha Gurdeep , Claycombe Kate , Ghribi Othman
Published: November 2023

Maternal low protein (LP) diets during gestation cause learning and memory impairment as well as cognitive deficits in the neonatal and adult offspring. The cellular and molecular mechanism that mediate the deleterious effects of prenatal exposure to a maternal LP diet on cognitive function is egregiously understood. Leptin, an adipocytokine expressed endogenously in the brain, exhibits pleiotropic effects in the brain and plays an indispensable role in learning and memory, as well as neuronal survival. In this study, we show that prenatal exposure to a maternal LP diet results in the mitigation of basal expression of leptin in the brains of the neonatal rat offspring. Sprague‐Dawley female rats were fed either a 20% normal protein diet (NP) or an 8% low protein (LP) diet inducted at three weeks before breeding and continued for the entirety of the gestation period. Prenatal exposure to a maternal LP diet resulted in reduced leptin expression in the brains of the neonatal offspring that was ascribed to a reduction in the C/EBPα and C/EBPβ – mediated transactivation of the leptin promoter. We further delved into and delineated the upstream molecular mediators and elucidated the role of lower CREB‐mediated transcription of C/EBPα and C/EBPβ that resulted in a reduction of the basal expression of C/EBPα and C/EBPβ leading to a significant attenuation in their transcriptional activities in the brains of the neonatal progeny prenatally exposed to a maternal LP diet. This decrease in transcriptional activity of C/EBPα and C/EBPβ results in the attenuation of the basal leptin promoter activity. Furthermore, maternal LP diet also induces a reduction in the enrichment of the acetylated histones in the nucleosomes that envelope the leptin promoter thereby making it refractory to trans‐activation. Our study unveils a mitigation of leptin expression in the brain as a novel mechanistic insight and a conduit into the deleterious effects evoked by prenatal exposure to a maternal LP diet on cognitive function.

Support or Funding Information

This work was supported by National Institutes of Health, Grant # R01AG0145264, to Dr. Othman Ghribi.
Neither Linoleic Acid nor Arachidonic Acid Promote White Adipose Tissue Inflammation in Fads2−/− Mice Fed Low Fat Diets
Authors: Suitor Katherine , Payne George W , Abdelmagid Salma , Mutch David M , Nakamura Manabu T , Ma David WL
Published: November 2023

Dietary n‐6 polyunsaturated fatty acids (PUFAs) are widely perceived to promote inflammation and contribute to the development of cardiometabolic disease. However, emerging evidence casts doubt on the relationship between n‐6 PUFAs and inflammation, and suggests that different n‐6 PUFAs may influence this relationship differently. Establishing the roles of individual n‐6 PUFAs in vivo is challenging because linoleic acid (LA, 18:2n6) is endogenously and continuously converted into arachidonic acid (AA, 20:4n6). The fatty acid desaturase 2 gene (Fads2) encodes the rate‐limiting delta‐6 desaturase enzyme necessary for converting LA into AA. The goal of this study was to elucidate the independent effects of the two predominant dietary n‐6 PUFAs, LA and AA, on inflammatory signalling in white adipose tissue (WAT) using Fads2 knockout (Fads2−/−) mice. We hypothesized that LA would not induce WAT inflammation unless it is converted into AA. Male C57BL/6 wild‐type (WT) and Fads2−/− mice (n=8 per diet per genotype group) were fed low‐fat isocaloric diets containing either 7% corn oil w/w (containing ~42% LA) or 7% ARASCO oil w/w (containing ~27% AA) for 9 weeks. WAT gene expression, protein levels, and triacylglycerol (TAG) fatty acid composition were analyzed by qRT‐PCR, Western blots, and gas chromatography, respectively. Fads2−/− mice fed the corn diet had no downstream AA, and reduced GLA (18:3n6) and DGLA (20:3n6), in TAGs compared to their WT counterparts. WT and Fads2−/− mice fed the ARASCO diet had significantly higher levels of AA, DGLA, and GLA in TAGs compared to WT mice fed the corn diet. Estimated delta‐5 desaturase enzyme activity (AA: DGLA ratio) in WAT was reduced in Fads2−/− mice fed the corn diet compared to their WT counterparts (p<0.05), and this was reflected by a 1.6‐fold reduction in FADS1 protein content (p<0.04). No differences in gene expression for common cytokines (e.g. Mcp‐1, Ccl5, Arg1, Nos2) and key regulators of oxylipin production (e.g. Cox‐2, Alox12, Alox15) were detected between WT and Fads2−/− mice fed either diet. WT and Fads2−/− mice fed both corn and ARASCO diets showed no difference in total or phosphorylated STAT3 and p38 proteins. These results suggest that in an unstimulated model neither LA nor AA promote WAT inflammation when consumed as part of a low‐fat diet.

Support or Funding Information

This work was supported by a grant from NSERC.
Effects of α‐Lipoic on mitochondrial function in HepG2 cells under H2O2‐induced oxidative stress
Authors: Tang Xue , Xing Xingan , Xiao Hang
Published: November 2023

α‐Lipoic acid (LA) is an antioxidant that protects against oxidative stress conditions in variety of cells including HepG2 cells. In the present study, we investigated the effects of LA in the mitochondrial DNA (mtDNA) copy number and energy metabolism in H2O2‐injured HepG2 cells. Cells were pretreated with/without 100 μM/L LA for 96 h, then incubated with 800 μmol/L H2O2 for 4 h. The relative mtDNA copy numbers were measured by Real‐time PCR and corrected by simultaneous measurement of the nuclear DNA. Lactate and NADH‐NAD+ were monitored by transfecting FRET nanosensor Laconic and Peredox. Cellular ATP and acetyl coenzyme A contents were also measured. Additionally, Western blot was used to determine the protein expression of mitochondrial transcription factor A (TFAM) and peroxisome proliferator‐activated receptor‐γ coactivator‐1α (PGC‐1α) protein levels. The results showed that H2O2 treatment decreased mtDNA copy number and damaged mitochondrial energy metabolism for decreasing the generation of acetyl coenzyme A, NADH‐NAD+ and ATP level, and increasing lactate content. 100 μmol/L LA treatment significantly increased mtDNA copy number and cellular ATP content compared with H2O2 group. Moreover, acetyl coenzyme A and NADH‐NAD+ were also significantly enhanced by LA. Furthermore, western blot results indicated that LA upregulated the level of TFAM and PGC‐1α protein. Our findings show that LA inhibited glycolysis and improved tricarboxylic acid cycle and oxidative phosphorylation. LA improved mitochondria energy metabolism may be related to its effect on mtDNA copy number.

Support or Funding Information

The present study was supported by the 12th Five‐Year Plan for for Science and Technology Development (No. 2012BAD33B05).
Long‐term development and culture of artificial breast tumor tissue using natural de‐cellularized ECM scaffold materials and the adenocarcinoma cell line MCF‐7
Authors: Lyden Timothy , Topel Samantha , Thueson Hanna
Published: November 2023

Over the past decade our laboratory, the UWRF Tissue and Cellular Innovation Center has been engaged in a series of studies exploring the application of natural de‐cellularized ECM materials as scaffolds for the generation of 3D artificial tissues. In these studies, we have repeatedly observed the development of significant tissues with extremely long culture lives. In fact, we regularly generate and maintain both tumor and “normal” tissues from standard ATCC cell lines which establish a distinct “tissue homeostasis” leading to long‐term growth and maintenance of complex structures. In recent studies with breast cancer adenocarcinoma cells, MCF‐7 and normal breast ductal cells, MCF10A, viable artificial tissues have been established and maintained for longer than 10 months on a natural marine invertebrate scaffold. Following that time in continuous culture both tumor and normal artificial tissues are still shedding viable cells into their respective wells and generating new monolayers. In both cases these display significant evidence of spontaneous differentiation as well as strong growth characteristics. More significantly, in a related series of studies, MCF‐7‐based artificial tissues have been maintained on de‐cellularized and decalcified bovine trabecular bone scaffolds for longer than 3.5 years of continuous culture. Once again the tissues shed cells into the wells and formed new monolayers continuously throughout the entire culture period. These monolayers consistently displayed a phenotype which includes large numbers of cells generating ridges and potential duct‐like tube structures. Subcultures from regular intervals during this long culture period, 3.5 years, are undergoing further analysis and comparison to explore changes in cell population with an eye to the modeling of tumor progression over the culture period.
Modulation of human umbilical cord‐derived mesenchymal stromal cells immunophenotype through cellular priming
Authors: Cromer Adrienne , Zuniga Michael , Cohen Don , Smith J. Robert , Weiss Mark
Published: November 2023

The in vitro immunomodulation effects of bone marrow‐derived mesenchymal stromal cells (BM‐MSCs) can be drastically altered by stimulation of toll‐like receptor (TLR) 3 and 4 into diametrically different immunomodulatory phenotypes similar to activated macrophage type 1 (M1) and macrophage type 2 (M2) polarization. BM‐MSCs activated by TLR4 ligand lipopolysaccharide (LPS) display an enhanced pro‐inflammatory phenotype (MSC1). In contrast, BM‐MSCs stimulated by Poly I:C, a TLR3 ligand that simulates a viral infection, display an anti‐inflammatory phenotype, called MSC2. This phenomenon has been termed “MSC priming”. The objective of this study was to determine whether human umbilical cord‐derived (HUC) MSCs display enhanced immune suppression after MSC1 versus MSC2 priming. Four different media conditions: 1) the TLR4 ligand LPS, 2) the TLR3 ligand Poly I:C, 3) interferon gamma (IFN‐γ) which upregulates TLR4 receptors and 4) human platelet lysate (HPL) versus control (multipotent adult progenitor cell (MAPC) medium. Five independent MSC lines were tested. Immune properties were assayed by examining the cell's suppression on the proliferation of stimulated murine splenocytes and 2) cytokine production: PGE2 and indoleamine 2,3‐dioxygenase (IDO) via ELISA. Cells cultured in IFN‐γ, LPS and Poly I:C media showed significant upregulation of IDO compared to MAPC medium. The expression of PGE2 was not significantly altered. Compared with MAPC medium, HPL medium inhibited the immunosuppressive effects of MSCs. In contrast, IFN‐γ and MAPC medium had the greatest immunosuppressive effects. Umbilical cord‐derived MSCs did not undergo MSC1 priming by TLR3 ligand stimulation like bone marrow‐derived MSCs.
In ‐ vitro Anti‐Proliferative Analysis of Metformin Hydrochloride on Androgen‐Sensitive (LNCaP) and Androgen‐Insensitive (PC‐3) Human Prostate Cancer Cell Lines
Authors: Adedeji Dolapo , Hinton Brittney , Payne Gloria
Published: November 2023

Background

Prostate cancer is one of the diseases worldwide that causes cancer‐related deaths in men. Metformin is an antidiabetic drug that has been in use for over two decades for the treatment of Type II Diabetes mellitus (DM2).

Purpose

The aim of this study was to evaluate the anti‐proliferative property of Metformin Hydrochloride on LNCAP and PC‐3 human prostate cancer cell lines.

Method

In vitro anti‐proliferative assay was used to assess the anti‐carcinogenic activity of metformin hydrochloride on androgen‐sensitive, LNCaP and androgen‐insensitive, PC‐3, human prostate cancer cell lines using MTT (3‐[4, 5‐dimethylyhiazol‐2‐yl]‐2,5‐diphenyltetrazolium bromide) assay. Effect of metformin hydrochloride was observed on each cancer cell lines after 72‐ and 120 – hour exposure at μM (0 μM, 3 μM, 5 μM and 8 μM) and mM (0.1 mM, 3 mM, 5 mM and 8 mM) concentrations.

Results

The average cell death ranging between 20% and 60% was observed when androgen‐dependent, LNCaP, and androgen‐independent, PC‐3, human prostate cancer cell lines were treated with metformin hydrochloride at different concentrations of 3 μM, 5 μM, 8 μM, 0.1 mM, 3 mM, 5 mM, and 8 mM. In PC‐3 cell line, the average cell death percentage was between 21% and 33% at μM concentrations at 72‐ and 120‐hour exposure respectively. The average cell death percentage increases to 53% and 57% after 72‐ and 120 hour exposure respectively at mM concentrations. In LNCaP cell line, the average cell death percentage did not change (25%) at μM concentrations at both 72‐ and 120‐hr exposure. The same trend was observed at mM concentrations. The average cell death percentage did not change at 72‐hour exposure (~47%) and about 49% was observed after 120‐hour exposure of the cell line to metformin hydrochloride.

Conclusion

The result of this study showed that metformin hydrochloride displayed cytotoxicity to some extent regardless of whether the prostate cancer cell line is androgen‐sensitive or androgen‐insensitive after 72 and 120 ‐ hour exposure. A stronger effect was observed on the androgen‐insensitive (PC‐3) than on the androgen‐sensitive (LNCaP) prostate cancer cell lines. Therefore, for elderly males diagnosed with diabetes and prostate cancer, metformin may aid in suppressing the cancer growth by serving as a defense mechanism for DM2; thereby reducing the cancer mortality rates in obese men.

Support or Funding Information

Master's Degree Programs at Historically Black Colleges and Universities (MD‐HBCU).

Grant Award Number: P382G090002
Cytostatic Effects of Pursatilla (Bai Tu Weng) on Mammary Adenocarcinoma Cell Line (MDA‐MB‐231) Along Multiple Signaling Pathways
Authors: Cho Young , Elias Zeyiad
Published: November 2023

Pursatilla (Bai Tu Weng in Traditional Chinese Medicine) from the perennial Ranunculaceae family has been a remedy for various inflammatory symptoms and GI disorders. We studied the cytostatic effects of Pursatilla in human mammary carcinoma cells (MDA‐MB‐238) using the whole plant extract. MAPK (mitogen‐activated protein kinase) and PI3K (phoshoinositide 3‐kinase) pathways were analyzed to examine the cytostatic mechanism of Pursatilla action via immunoblotting assay. Mitogenic stimulation by FBS, measured through phosphorylation on MAPK‐42/44 and PKB, was nearly reversed by Pursatilla in MDA238 cell line. In addition, cell cycle progression, specifically the S‐phase entry in mitosis, is blocked by Pursatilla. However, the cell viability assay (trypan blue) and the cell metabolic activity assay (MTT) indicated that the anti‐mitogenic action of Pursatilla was independent of the cytotoxicity. Consequently, we present the cytostatic and anti‐mitogenic action of Pursatilla via MAPK and PI3K pathways in MDA238 cell line.

Support or Funding Information

Bastyr University Faculty Seed Grant and Student Research Grant.
A Novel Hydrogen Sulfide Prodrug, BW‐HP‐202, Attenuates Myocardial Ischemia/Reperfusion Injury
Authors: Li Zhen , Organ Chelsea L. , Trivedi Rishi K. , Zheng Yueqing , Yu Bingchen , Wang Binghe , Lefer David J.
Published: November 2023

Background and Hypothesis

Hydrogen sulfide (H2S) protects against acute myocardial ischemia/reperfusion (MI/R) injury and heart failure by ameliorating oxidative stress, improving mitochondrial function, and attenuating apoptosis. One of the major limitations of currently available H2S donors is poor pharmacokinetic profiles that result in very rapid and uncontrolled H2S release. BW‐HP‐202 is a recently developed H2S prodrugs designed for sustained release of H2S upon catalysis in vivo via cellular metabolism. We hypothesized that BW‐HP‐202 would ameliorate myocardial cell death following MI/R in an in vivo murine model.

Methods and Results

Male C57BL/6J mice (10–12 weeks of age) were subjected to 45 minutes of MI followed by 24 hours of R. At the time of reperfusion, animals received BW‐HP‐202 (50 mg/kg, 100 mg/kg) or Vehicle (1% THF) by intracardiac injection. In addition, at 4 hours of R, plasma was collected for troponin‐I measurements. Myocardial infarct size was reduced by 38% (p < 0.05 vs. Vehicle) in mice treated with 50 mg/kg BW‐HP‐202, 45% (p < 0.01 vs. Vehicle) in mice treated with 100 mg/kg BW‐HP‐202. Circulating Troponin‐I levels were also significantly reduced in mice treated with BW‐HP‐202 (41.3 vs. 17.63 vs. 19.86, p < 0.05).

Conclusions

BW‐HP‐202 significantly attenuated MI/R injury in a murine model in a dose‐dependent manner. Experiments are currently underway to further define the pharmacokinetics, mechanisms of action, and safety profile of this H2S prodrug.
Protamine Sulfate Neutralization of Bovine and Porcine Heparins: Developmental Implications.
Authors: Paria Sayan , Atcho Omar , Kouta Ahmed , Abro Schuharazad , Hoppensteadt Debra , Jeske Walter , Kingo Jonas , Fareed Jawed
Published: November 2023

INTRODUCTION

Bovine heparins are currently under consideration for the re‐introduction in the US. Protamine sulfate is commonly used to neutralize heparin. Currently, porcine heparin is used for surgical and interventional purposes. The USP Potency of the currently available bovine heparins is lower than that of porcine heparins. This study investigated the differential neutralization of bovine and porcine heparins by protamine sulfate using various laboratory assays.

MATERIALS & METHODS

8 bovine heparins were obtained from Kin Master, Brazil in powder form with 140–150 U/mg potency. The porcine heparin was obtained from Medefil Inc., U.S.A. in powder form with potency of 190 U/mg. Each of the bovine heparins was supplemented to blood bank plasma at concentrations of 0–10 mg/ml. The effect of protamine sulfate at a fixed concentration of 10 mg/ml was measured using aPTT, thrombin time, amidolytic anti‐Xa and anti‐IIa assays. Saline supplemented heparinized plasmas were used as control. Additionally, the neutralization of the anti‐coagulant effects of a fixed concentration of pooled bovine heparin at 10 mg/ml by protamine sulfate was also studied at a fixed concentration of 10 mg/ml.

RESULTS

All 8 bovine heparins produced a concentration dependent prolongation in aPTT and thrombin time assays. Although porcine heparin exhibited stronger anticoagulant effects in these assays. The protamine neutralization profile of both bovine and porcine heparins was similar. Protamine at concentration of 5 mg/ml completely neutralized the equigravimetric amounts of heparins. However, protamine was not effective in neutralizing the higher concentration of these agents. In the amidolytic anti‐Xa assay both heparins produced concentration dependent inhibitory effects, however porcine heparin was stronger. In this assay, protamine differentially neutralized the bovine heparin in comparison to porcine heparin. At 5 mg/ml almost complete neutralization was noted. In the anti‐IIa aminolytic assay, bovine heparins were weaker than porcine heparin. However, protamine sulfate completely neutralized both bovine and porcine heparins. In the whole blood ACT assay at 10 mg/ml, porcine heparin produced stronger anti‐coagulant effect (380 ± 21sec.) in comparison to bovine heparin pool (304 ± 17 sec.). Protamine sulfate at 10 mg/ml, markedly neutralized the anti‐coagulant effects of both heparins. The relative neutralization was comparable between the bovine and porcine heparins.

CONCLUSIONS

These studies demonstrate that in the whole blood and plasma based clotting assays the protamine neutralization profile of porcine and bovine heparin is similar when compared at equigravimetric concentrations. However, in the anti‐Xa assays, differences are noted between the two heparins. This may be due to the differential compositions of these heparins at the lower molecular weight component.
Distinct MAP kinase activation patterns of Angiotensin and Cannabinoid systems in SHR astrocytes
Authors: Haspula Dhanush , Clark Michelle
Published: November 2023

Objective

To determine the effect of exogenous Angiotensin (Ang) II and ACEA, both alone and in combination, on MAP kinase activation in astrocytes isolated from Spontaneously Hypertensive Rats (SHRs) and Wistar rats.

Background

By activation of astroglial Ang type 1 receptor (AT1R) and Cannabinoid type 1 receptor (CB1R), Ang II and cannabinoids are able to exert potent effects on proliferation and neuroinflammation. An increase in phosphorylation states of MAP kinases such as ERK and p38, has been linked to several functions in astrocytes. Although crosstalk between the two receptors has been reported by us and several other groups, the consequences of this interaction at the level of MAP kinase activation has not been investigated.

Methods

Astrocytes were isolated from the brainstem and cerebellum of Spontaneously Hypertensive Rats (SHR) and normotensive Wistar rats. The cells were treated with 100 nM Ang II or 10 nM ACEA, both alone and in combination, for varying time periods and the extent of phosphorylation was measured using Western blotting.

Results

Ang II treatment resulted in greater activation of MAP kinases in brainstem astrocytes, but not cerebellar astrocytes, of SHRs when compared to Wistar rats. ACEA treatement on the other hand, is associated with a greater activation of MAP kinases in brainstem, but not cerebellar, astrocytes of Wistar rats when compared to SHRs. Co‐treatment of Ang II and ACEA resulted in greater activation of ERK and p38, than Ang II or ACEA treatments alone, in brainstem astrocytes isolated from both SHRs and Wistar rats. In cerebellar astrocytes however, co‐treatment did not result in greater activation of MAP kinases than the treatments alone in both rat models.

Conclusion

Greater MAP kinase activation, by ACEA, in brainstem astrocytes of Wistar rats when compared to SHRs is suggestive of a potential diminished endocannabinoid tone in pathological conditions such as hypertensions. An augmentation of MAP kinase activation by the combination treatment in brainstem astrocytes, but not cerebellar astrocytes, is indicative of a potential dampening of one system by the other in cerebellar astrocytes. Our findings suggest that the crosstalk between AT1R and CB1R may differ depending on the brain area and the underlying pathological condition.

Support or Funding Information

NSU HPD: Grant # 335585
Elemental and Lipophilic Analysis of Kashmir Saffron by ICP‐MS and GC‐MS and its in‐vivo Anti‐inflammatory Potential
Authors: Madkhali Hassan Abdu , Ganaie Majid Ahmad , Jan Basit Latief , Ahmad Ajaz , Raish Mohammad Latief , Ahamad Syed Rizwan
Published: November 2023

Saffron has been widely used worldwide and its health benefits have been recognized since ancient times. Recently, there has been renaissance in research to establish its beneficial properties and biological action mechanisms. Thus the aim of our study was to analyze the lipophilic and elemental content of Kashmir saffron and also study its effect in in‐vivo systemic inflammation. Lipophilic and elemental analysis was done by GC‐MS and ICP‐MS respectively and in‐vivo systemic inflammation was produced by administration of D‐GalN/LPS to Wistar rats. Lipophilic analysis was done by sequence of various steps viz extraction, methoxymation and derivatization by GC‐MS. This is a quick technique to identify and quantify lipophilic metabolites like fatty acids, fatty alcohols, sterols, sugars, higher alkanes, alkenes and flavonoids. Sugars like D‐Ribose, Xylitol, Ribopyranose and D‐fructose were present in higher amount as compared to fatty acids or higher alkanes. The elemental composition of saffron was analyzed by ICP‐MS and it is interesting to find out that Na, Ca, K, Mg, Sb and Al concentrations are high but within limits. Heavy metals like As, Ag, Ba, Cd, Cr, Mn, Mo, Se, U, V, Ni and Co are present in very low concentration. The administration of D‐GalN/LPS to rats produced an acute systemic injury manifested by a significant increase TNF‐α, compared to controls (p<0.05). However, pretreatment with saffron extract at 250 and 500 mg/kg doses ameliorated this injury as evident by a reduction TNF‐α in a dose‐dependent manner. In the current study we establish the complete lipophilic and elemental analysis of Kashmir saffron. The method used is very rapid, simple and reliable. Furthermore, our in‐vivo results suggest that saffron may be a promising option as either an alternative or an adjunctive therapy in disorders involving systemic inflammation.
Sestrin2 Inhibited the Activation of Hepatic Stellate cells and Liver Fibrosis via Blocking Transforming Growth Factor‐β Signaling
Authors: Yang Ji Hye , Ki Sung Hwan
Published: November 2023

Hepatic fibrosis results from chronic liver injury and inflammatory responses. Sestrin2 (Sesn2), an evolutionally conserved antioxidant proteins, prevents acute hepatitis and metabolic liver diseases. However, the role of Sesn2 in the pathogenesis of hepatic fibrosis remains obscure. In the current study, we first investigated whether Sesn2 protects fibrogenesis in cultured hepatic stellate cells (HSC) or in chronic carbon tetrachloride (CCl4)‐treated or bile duct ligation (BDL)‐performed mice. Activated primary HSC up‐regulates the levels of Sesn2 protein and mRNA. Transforming growth factor‐β (TGF‐β) also increased Sesn2 expression in cultured HSC, which was due to increased transcription. TGF‐β signaling occurs primarily by activation of Smad proteins and overexpressed Smad3 increased Sesn2 luciferase activity. In silico analysis of the 5′ upstream region of Sesn2 gene identified a putative Smad binding element (SBE) sequence. Deletion of the putative SBE demonstrated in the human Sesn2 promoter was critical for the TGF‐β‐mediated response. Moreover, ectopic expression of Sesn2 reduces epithelial‐mesenchymal transition (EMT) markers, which is accompanied with a marked decrease in SBE luciferase activity and Smad phosporylation. A recombinant adenovirus Sens2 (Ad‐Sesn2) administration presents less severe hepatic injury as supported by decreases in CCl4‐or BDL‐induced ALT/AST levels. Furthermore, Ad‐Sesn2 reduced liver injury and collagen accumulation. Finally, expression of Sesn2 is down‐regulated in the livers from patients with liver cirrhosis or CCl4‐or BDL‐induced hepatic fibrosis mice. Taken together, our findings suggest that Sesn2 protects HSC activation and hepatic fibrosis via antagonizing TGF‐β signaling.

Support or Funding Information

This research was supported by the National Research Foundation of Korea (NRF) funded by the Ministry of science, ICT & Future Planning (No. R13‐2008‐010‐00000‐0)
A SUMO‐ubiquitin mediated proteasome pathway in repair of DNA damage induced by topoisomerase II inhibitors
Authors: Sun Yilun , Nitiss John
Published: November 2023

DNA topoisomerases play a critical role in nuclear processes such as replication and transcription by managing the topology of DNA duplexes. Topoisomerase II (Top2) is an established target for anti‐cancer agents such as doxorubicin and etoposide, which act by trapping Top2 on DNA. This leads to accumulation of Top2‐DNA covalent complexes (Top2ccs) and Top2‐induced DNA double‐strand breaks (DSBs). The mechanisms causing tumor‐specific cell killing by Top2 inhibitors may arise from DNA repair defects in cancer cells. Therefore, targeting pathways for repair of Top2‐mediated damage is a potential strategy to enhance the action of Top2 inhibitors. We hypothesize that the primary step in repair of Top2‐mediated DSBs is removal of trapped Top2 from DNA, and that the ubiquitin‐proteasome system is required for this removal. To explore the proteolytic pathway for processing Top2cc, we developed a sensitive method to detect ubiquitylation and other post‐translational modifications (PTMs) on Top2cc by modifying the ICE assay, a biochemical approach for detecting protein‐DNA crosslink. Using a yeast system, we showed that the proteasome plays a key role in repair of Top2 damage by degrading Top2cc. We found that deletion of genes encoding ubiquitin ligase Slx5/Slx8 led to decreased ubiquitylation of Top2cc. Depletion of Siz1, a SUMO ligase, was found to reduce not only SUMOylation of Top2cc but also ubiquitylation of the enzyme. These findings taken together suggest a signaling axis wherein Siz1‐mediated SUMOylation primes Top2cc for Slx5/Slx8‐mediated ubiquitylation and the consequent proteasomal degradation. Finally, we demonstrated that Rnf4, the human ortholog of Slx5/Slx8, plays an important role in repairing Top2cc by modulating its ubiquitylation and proteolysis in human cells. Our work is a key step in elucidating how DNA‐protein adducts are processed in eukaryotic cells, and may lead to the identification of novel anti‐cancer drug targets.
Selective Stimulation of Inhibitory Motorneurons in the Mouse Colon Following Light Activation of Channel Rhodopsin‐2 (ChR2) Ex Vivo
Authors: Perez‐Medina Alberto , Manfredsson Fredric , Galligan James
Published: November 2023

Inhibitory motor neurons in the enteric nervous system (ENS) are responsible for producing relaxations of gastrointestinal (GI) smooth muscle. Neurogenic relaxations are required to produce the pressure gradients needed to propel GI content along the gut. Nitric oxide (NO) and ATP (or related purine) are the predominant neurotransmitters released by inhibitory motorneurons in the ENS. Immunohistochemical studies using antibodies against nitric oxide synthase (NOS) and the vesicular nucleotide transporter (VNUT), protein markers for nitrergic and purinergic inhibitory motorneurons respectively, showed that there are NOS+ neurons, VNUT+ neurons and NOS/VNUT+ neurons in the myenteric plexus of the mouse colon. To determine if NO and ATP were released by the same or separate neurons, we injected an adeno‐associated virus (AAV9) containing a construct with genes encoding ChR2 and yellow fluorescent protein (YFP) bordered by loxP sites into the proximal colon of NOS‐cre expressing mice. 4–6 weeks later, the colon was harvested for electrophysiological measurements of circular muscle inhibitory junction potentials (IJPs, the electrophysiological correlate of muscle relaxation) evoked by blue light (470 nm) and transmural electrical stimulation. IJPs were recorded using intracellular microelectrodes. Light stimulation (5–20 mW/mm2) evoked biphasic IJPs in tissues from all mice successfully transduced by AAV9 (as measured by detection of the YFP reporter), but not in non‐transduced tissues (lacking YFP). Electrical stimulation evoked IJPs in tissues from all mice. The amplitude and duration of the light‐ and electrically‐evoked IJPs increased with the number of stimuli (1–7 stimuli). The fast, early phase of light‐ and electrically‐evoked IJPs was blocked by MRS2179 (10 μM, a P2Y1 receptor antagonist) while the slow, late phase of the IJP was blocked by nitro‐L‐arginine (100 μM, NOS inhibitor). IJPs were blocked completely by combined application of MRS2179 and nitro‐L‐arginine. These data provide direct evidence that inhibitory motorneurons in the mouse colon co‐release NO and ATP (or related purine) to cause relaxation of colon circular smooth muscle. Selective expression of ChR2 in subtypes of neurons will enable more precise identification of synaptic mechanisms and nerve pathways in the ENS.

Support or Funding Information

Supported by R01DK094932
Neuroprotective Potential of Transient Receptor Potential Melastatin‐2channel Inhibitor Flufenamic Acid in Experimental Model of Focal Cerebral Ischemia
Authors: Medhi Bikash , Harikrishnareddy Dibbanti
Published: November 2023

Introduction

Cerebral Ischemia remains third leading cause of death and disability worldwide. Although progress has been made in supportive care, efforts to guard the brain from ischemic neuronal death have failed. Recently transient receptor potential melastatin‐2 (TRPM‐2) ion channel have been identified as major culprit that initiates neuronal death in cerebral stroke. These channels raises the intracellular Ca2+ levels and oxidative stress there by initiating mitochondrial membrane disruption, endoplasmic reticulum stress, glutamate excitotoxicity which initiates neuronal death. We therefore investigated the neuroprotective effect of TRPM‐2 channel inhibitor flufenamic acid (FA) for the treatment of Cerebral Ischemia.

Materials and Methods

Adult male Wistar rats weighing 250±20g were divided into six different groups viz sham operated group, vehicle treated group, four different doses of flufenamic acid (FA‐5mg/kg, FA‐10mg/kg, FA‐20mg/kg, and FA‐40mg/kg groups). Focal Cerebral Ischemia was induced by middle cerebral artery occlusion (MCAO) model. Neurobehavioral assessmentwere monitored by Longa et al., five point scale with 10 grading scores, infarct size was calculated by using of 2,3,5‐triphenyltetrazolium chloride (TTC) staining, oxidative stress parameters were estimated by biochemical parameters, and brain water content was measured by wet‐dry method.

Results

FA showed neuroprotection in a dose dependent manner. FA 20mg significantly diminished neurological deficits and reduced infarct size in rats. Further, an early calcium dependent rise in levels of MDA was also found to be significantly reduced in ischemic brain regions following FA treatment. With higher doses of FA brain edema was also significantly reduced.

Conclusion

The present study thus provide neuroprotective profile of FA in focal Cerebral Ischemia with its selective effect onTRPM‐2 channel.

Support or Funding Information

Post Graduate Institute of Medical Education and Research
Subcellular localization and relevant mechanisms of human cancer‐related micropeptides
Authors: Deng Jing , Xu Wenli , Jie Yusheng , Chong Yutian
Published: November 2023

Rapid advances in high‐quality sequencing and bioinformatics have invalidated the argument that noncoding RNAs (ncRNAs) are junk transcripts that do not encode proteins. Increasing evidence suggests that small open reading frames (sORFs) in ncRNAs can encode micropeptides and polypeptides within 100 amino acids in length. Several micropeptides have been characterized and proven to have various functions in human physiology and pathology, particularly in cancer. The present review mainly highlights the latest studies on ncRNA‐encoded micropeptides in different cancers and categorizes them based on their subcellular localization, thereby providing a theoretical basis for micropeptide applications in the early diagnosis and prognosis of cancer and as therapeutic targets. However, considering the inherent characteristics of micropeptides and the limitations of the assay technology methods, more detailed information is warranted.
Acute, pro‐contractile effects of prorenin on rat mesenteric arteries
Authors: Rognant Salomé , Baldwin Samuel N. , Pritchard Harry A. T. , Greenstein Adam , Calloe Kirstine , Aalkjaer Christian , Jepps Thomas A.
Published: November 2023

Prorenin and the prorenin receptor ((P)RR) are important, yet controversial, members of the renin–angiotensin–aldosterone system. The ((P)RR) is expressed throughout the body, including the vasculature, however, the direct effect of prorenin on arterial contractility is yet to be determined. Within rat mesenteric arteries, immunostaining and proximity ligation assays were used to determine the interacting partners of (P)RR in freshly isolated vascular smooth muscle cells (VSMCs). Wire myography examined the functional effect of prorenin. Simultaneous changes in [Ca2+]i and force were recorded in arteries loaded with Fura‐2AM. Spontaneously transient outward currents were recorded via perforated whole‐cell patch‐clamp configuration in freshly isolated VSMCs. We found that the (P)RR is located within a distance of less than 40 nm from the V‐ATPase, caveolin‐1, ryanodine receptors, and large conductance Ca2+‐activated K+ channels (BKCa) in VSMCs. [Ca2+]i imaging and isometric tension recordings indicate that 1 nM prorenin enhanced α1‐adrenoreceptor‐mediated contraction, associated with an increased number of Ca2+ waves, independent of voltage‐gated Ca2+ channels activation. Incubation of VSMCs with 1 nM prorenin decreased the amplitude and frequency of spontaneously transient outward currents and attenuated BKCa‐mediated relaxation. Inhibition of the V‐ATPase with 100 nM bafilomycin prevented prorenin‐mediated inhibition of BKCa‐derived relaxation. Renin (1 nM) had no effect on BKCa‐mediated relaxation. In conclusion, prorenin enhances arterial contractility by inhibition of BKCa and increasing intracellular Ca2+ release. It is likely that this effect is mediated through a local shift in pH upon activation of the (P)RR and stimulation of the V‐ATPase.