bluuebunny/crossref_metadata_2025 huggingface dataset:
Morphologic evaluation of breast neoplasia in experimental focal hyperthermia
Authors: Calado Ana Isabel , ColaÃ§o Bruno , Oliveira Paula , Portela Ana , Cabrita Antonio Silverio
Published: June 2021

Focal thermotherapy (FT) is a new modality for cancer treatment under study.

The objective of this study was to evaluate the morphological changes of breast tumors induced by 7,12‐dimethylbenz[a]anthrace (DMBA).

At 53 days of age 24 virgin SD female rats were orally given a single dose of DMBA (65 mg/Kg). Twenty‐six weeks later, animals were randomly allocated to two groups: control group (n=12) and treated group (n=12). The treated group was submitted to FT applied on the largest tumor 60 seconds for two days. The animals were ethically euthanized one week after treatment.

Tumors were grouped on: I‐tumors of non‐treated animals; II‐non‐treated tumors of treated animals and III‐tumors injected with ferromagnetic cement heated with electromagnetic energy.

The malignant breast tumors were evaluated by histopathology. Apoptosis, necrosis and hemorrhage did not differ significantly between groups I and II, although the frequency of these parameters was higher on group II. On group III there is a significant increase of necrosis compared to group I, and a higher frequency of necrosis and hemorrhage to group II.

This preliminary study suggests that FT of one breast carcinoma leads to an increase of necrosis in all tumors of one rat experimental model. Although further studies are needed, FT has great potential for future clinical applications in breast cancer therapy.

Grant: FCT‐ PTDC/SAU‐BEB/69497/2006
Evaluation of complementary treatment in DMBA breast cancer model – preliminary results
Authors: Cabrita Antonio Silverio , Verrier Guido
Published: June 2021

Over the last decade, conventional breast cancer therapies have been frequently adjuvated with complementary medicine techniques (CMT). In spite of medical evidence, experimental models are required to validate CMT effect on breast cancer.

This study has two groups of nine Sprague‐Dawley female rats. They were orally given a single dose of DMBA (65 mg/Kg) at one month of age according to the breast cancer protocol. One group (I) was maintained with no other treatment for 24 weeks. The other group (II) was submitted to hands on treatment, three times a week, 15 minutes each, during 24 weeks. All the survival animals were ethically euthanized 24 weeks after carcinogenesis induction.

The malignant breast tumors were evaluated by histopathology. Statistical analysis was carried out using a χ2 and Fisher's tests. The level of significance was set at P< 0,05. There was no statistical difference between the morphology of both groups.

This preliminary study suggests that CMT has no effect on breast carcinoma morphology, although further studies are required to evaluate an action on patient condition.
3D rapid prototyping to model DNA replication and engage students in the scientific process
Authors: Bhattacharyya Basudeb , Marceau Aimee , George Nicholas , Cheema Muhammad , Havlena Zoë , Hua Amy , Huang Susan , Moon Jade , Wang Connie , Nelson David L. , Keck James L.
Published: June 2021

DNA replication is a vital process in all organisms and understanding the fundamental biochemical interactions that drive replication is essential. Single‐stranded DNA‐binding (SSB) proteins form an important component of the replication machinery that facilitates the transfer of RNA primers from the enzyme primase to the replicative polymerase. This activity occurs throughout lagging‐strand DNA replication. The crystal structure of the E. coli χψ subunit of the DNA polymerase III holoenzyme bound to its interaction site on SSB has been determined. The Madison West High School Students Modeling a Research Topic (SMART) Team, in collaboration with the Milwaukee School of Engineering, has modeled this interaction using 3D Rapid Prototyping Technology to gain insights into the physical interactions that drive DNA replication. The SMART team program allows students to experience the scientific process beyond the textbook by investigating the experimental methodology of structural biology and takes students out of the classroom and into the laboratory. Supported by grants from the Howard Hughes Medical Institute and NIH‐NCRR‐SEPA.
Expression of Wnt5a correlates with histopathological features of bladder urothelial carcinoma
Authors: Malgor Ramiro , Brockett Colin , Greco Danielle , House Denise , McCall Kelly
Published: June 2021

Bladder cancer is the second most common malignancy of the genitourinary tract. In the United States, it is the fourth most common cancer in men. Recurrence and progression to an invasive stage are common events urothelial carcinoma (UC).

Wnt5a is a member of Wnt family for which a role in cell migration and invasion has been assigned in cancer.

The goal of this study was to determine the expression of Wnt5a in human urothelium carcinoma (UC) tissue samples and to correlate Wnt5a expression with histological grade and pathological stage of the tumor. We studied 33 tumor samples collected by transurethral resection. Each sample was independently graded and staged and the expression of Wnt5a was determined by immunohistochemistry (IHC) technique. The intensity was scored using a scale of 1+, 2+ or 3+ score. Logistic regression was used for statistical analyses.

The 33 cases were divided as 12 low grade and 21 high grade by the stage 20 pTa, 7 pT1, 5 pT2 and 1 not determined. In all of the tumor specimens, regardless of grade and stage, the expression of Wnt5a was positive in the tumor cells. The IHC signal in high grade tumors was stronger than in the low grade tumors. We found a statistically significant correlation between histopathological grade and the intensity for Wnt5a immunostaining.

The results reveal that the expression of Wnt5a correlates with the histopathological features of the bladder UC; and suggest that Wnt5a could be used as a biomarker for the diagnosis and prognosis of UC.
Reduced side effect liability of novel β‐subunit selective GABAA receptor allosteric modulators
Authors: Yoshimura Ryan F , Tran Minhtam B , Hogenkamp Derk J , Johnstone Timothy B , Gee Kelvin W
Published: June 2021

Current benzodiazepine (BZ) anxiolytics, including diazepam, have well‐known side effects including sedation, memory impairment, abuse‐liability, withdrawal and limited tolerance (Kaplan and DuPont, 2005). We have previously characterized β‐subunit selective GABAA receptor (GABAARs) positive allosteric modulators (PAMs) as anxiolytics with a reduced sedative liability (Gee et al., 2010). Additional testing of the prototypical compound, UCI2‐261, a GABAAR β‐subunit selective anxiolytic, in a number of behavioral models reveal differences between it and typical agonist BZs. Unlike diazepam (2.5 mg/kg i.p.), UCI2‐261 (100 mg/kg, i.p.) does not have a significant amnestic effect in adult, male Sprague‐Dawley rats as measured by the eight‐arm radial arm maze. Furthermore, subchronic (7 days, q.d.) treatment of adult, male CD‐1 mice with UCI2‐261 (10 mg/kg, i.p.) does not induce tolerance to its anxiolytic effect in the elevated plus maze. These results suggest that β‐subunit selective GABAAR PAMs not only have reduced sedative‐liability, but may not share other side effects commonly associated with agonist BZs.
Gene expression in the brain from antidepressants treated mouse using DNA microarray
Authors: Kim Yong , Lee Jun‐Ho , Shin Minkyu , Hong Moochang , Kim Yangseok , Bae Hyunsu
Published: June 2021

The molecular and biological bases for the mechanisms of antidepressants are based on the monoamine depletion hypothesis. However, we do not yet know the full cascade of mechanisms responsible for the therapeutic effect of antidepressants. To identify the genes involved in the therapeutic mechanisms of various antidepressants, we used a cDNA microarray system containing 30000 clones with isolated total RNA from each treated rat brain. In the present study, we demonstrated gene expression changes of treatment with a tricyclic antidepressant (imipramine), a selective serotonin reuptake inhibitor (fluoxetine), monoamine oxidase inhibitor (phenelzine) and psychoactive herbal extracts of PM011. The expression of 35, 20, 37 and 44 genes were decreased and 21, 49, 35 and 27 were over‐expressed by imipramine, fluoxetine, phenelzine and PM011, respectively.

Members of signal transduction, factors related to protein metabolism, cell survival, ligand‐receptor interaction, were some of the genes with altered expression. These data demonstrate that antidepressants interfere with the expression of a large array of genes involved in signaling, survival and protein metabolism.

The results support the hypothesis that the therapeutic effect of these antidepressants is much more complex and not confined to a reuptake inhibition of neurotransmitters and inhibition of MAO

This work was supported by the Korea Science and Engineering Foundation (KOSEF) grant funded by the Korea government(MEST) (No. 2009‐0063466).
The Mitochondrial Protective Effects of Dopamine Neuron Stimulating Peptide‐11
Authors: Bradley Luke H , Kelps Kristen A , Turchan‐Cholewo Jadwiga
Published: June 2021

Dopamine neuron stimulating peptide‐11 (DNSP‐11), an eleven amino acid amidated peptide derived from the glial cell‐line derived neurotrophic factor (GDNF) prosequence, has been shown recently to provide neuroprotective and neurorestorative effects in dopaminergic cell culture and parkinsonian rat model systems. However, unlike GDNF, DNSP‐11 was found to prevent staurosporine‐induced toxicity and cytochrome c release from mitochondria in nutrient‐deprived dopaminergic B65 cells. The objective of this study is to investigate the neuroprotective effects of DNSP‐11 on the mitochondria. We treated dopaminergic cell lines with mitochondrial‐specific toxins and evaluated DNSP‐11’s protective effects by measuring mitochondrial potential, caspase‐3 activity, and TUNEL staining assays. In addition, proteomic profile analyses were performed to determine changes in the membrane, cytosolic and mitochondrial proteins involved in DNSP‐11 protection. These data support a mitochondrial neuroprotective hypothesis and further suggest that DNSP‐11 is a promising candidate for further evaluation as a downstream therapeutic for age‐related neurogenerative diseases, like Parkinson's disease. Support provided by: NIH COBRE Pilot (P20RR20171), NIA (T32 AG000242), NIDA (T32 DA022738), NINDS (NS039787), PhRMA Foundation, and University of Kentucky College of Medicine Start‐up Funds.
The missing piece: Structural identification of the SSB binding site on the Chi/Psi clamp‐loader subassembly
Authors: Marceau Aimee , George Nick , Keck James
Published: June 2021

Bacterial single strand DNA binding proteins (SSBs) are essential for genome integrity and DNA replication. SSB binds to and protects single stranded DNA with its N‐terminal oligosaccharide/oligonucleotide binding (OB) fold whereas the C‐terminus of the protein (SSB‐Ct) is essential for interactions with herterologous genome maintenance proteins. SSB interacts directly with the Chi/Psi subassembly of the Beta‐clamp loader complex, forming a complex that is thought to facilitate processive replication of lagging strand DNA. We have determined a 1.65 Å resolution structure of the E. coli Chi/Psi subassembly bound to a peptide comprising the E. coli SSB‐Ct sequence. We tested the importance of the Chi/Psi SSB‐Ct‐binding site by making alanine substitution variants in Chi that would be predicted to disrupt complex formation and testing Chi/Psi/SSB complex formation in vitro. This approach, together with the structure, identified residues on Chi that are critical for interaction with SSB. We then tested the importance of Chi/Psi/SSB interactions in vivo by incorporating the mutations into E. coli holC (encodes Chi) and following cell growth and DNA replication of the resulting mutants. The phenotypes of these strains show that destabilization of the Chi/Psi/SSB complex has profound effects on E. coli cellular processes.
NAD+ induces C6 glioma cell death by generating oxidative stress and increasing intracellular calcium concentrations
Authors: Zhao Cuiping , Chen Heyu , Xia Weiliang , Li Shengtian , Ying Weihai
Published: June 2021

NAD+ plays important roles in various biological processes. We have found that while it is highly protective against oxidative stress‐induced death of primary neuronal cultures and astrocyte cultures, NAD+ can induce glioma and neuroblastoma cell death. In this study we investigated the mechanisms underlying the effects of NAD+ on C6 glioma cells. We found that two antioxidants, Trolox and N‐acetyl cysteine, can markedly decreased the NAD+‐induced glioma cell death, suggesting that oxidative stress mediates the effects of NAD+ on the cell death. Our study has also shown that BAPTA‐AM, a calcium chelator, also largely prevented NAD+‐induced decreases in the cell survival, suggesting a critical role of elevated intracellular calcium concentrations in the cell death. Collectively, our study has provided novel evidence that NAD+ treatment can induce oxidative stress and impaired calcium homeostasis in such tumor cell types as C6 glioma cells, which leads to decreased survival of the tumor cells. (Supported by a Key Basic Research Grant from Shanghai Educational Committee (to WY) and by a Pujiang Scholar Award (to WY)).
Delayed treatment of NAD+ can restore intracellular ATP levels and prevent PC12 cell death induced by oxidative stress
Authors: Chen Heyu , Zhao Cuiping , Shao Jiaxiang , Chen Chen , Xia Weiliang , Ying Weihai
Published: June 2021

Our studies have shown that NAD+ treatment can greatly decreased the infarct formation in a rat model of brain ischemia. However, the mechanisms underlying the effects of NAD+ remain unclear. In this study we tested our hypothesis that NAD+ attenuates oxidative stress‐induced cell death by restoring intracellular ATP levels. Our study applying LDH assay shows that H2O2‐induced PC 12 cell death was completely prevented when 5 mM NAD+ was added one hr after H2O2 treatment. We also applied luciferase‐based ATP assay to show that four hrs after H2O2 treatment the intracellular ATP levels in the cells were profoundly decreased, which were increased by nearly three folds by NAD+ (5 mM) post‐treatment at one hr after H2O2 exposures (p<0.001). In contrast, post‐treatment of the cells with 5 mM pyruvate at one hr after H2O2 exposures did not restore the ATP levels. Due to the critical roles of ATP in cell survival, our studies suggest that the NAD+‐produced profound protection against oxidative stress‐induced cell death is at least partially mediated by its capacity of restoring intracellular ATP levels. (Supported by a key basic research grant from Shanghai Municipal Scientific Committee (to W.Y.), and a National 973 Program Grant #2010CB834306 (to W.Y. and W. X)).
Arrestin Orchestrates Crosstalk Between G Protein‐Coupled Receptors to Modulate the Spatiotemporal Activation of ERK MAPK.
Authors: Xiang Yang Kevin
Published: June 2021

G protein‐coupled receptors (GPCRs) respond to diversified extracellular stimuli to modulate cellular function. Despite extensive studies investigating the regulation of single GPCR signaling cascades, the effects of concomitant GPCR activation on downstream signaling and cellular function remain unclear.

Objective

We aimed to characterize the cellular mechanism by which GPCR crosstalk regulates mitogen‐activated protein kinase (MAPK) activation.

Methods and Results

Adrenergic receptors on cardiac fibroblasts were manipulated to examine the role of arrestin in the spatiotemporal regulation of extracellular signal‐regulated kinase (ERK)1/2 MAPK signaling. We show a general mechanism in which arrestin activation by one GPCR is capable of regulating signaling originating from another GPCR. Activation of Gq coupled‐receptor signaling leads to prolonged ERK1/2 MAPK phosphorylation, nuclear accumulation, and cellular proliferation. Interestingly, coactivation of these receptors with the beta‐adrenergic receptors induced transient ERK signaling localized within the cytosol, which attenuated cell proliferation. Further studies revealed that recruitment of arrestin3 to the beta2‐adrenergic receptor orchestrates the sequestration of Gq‐coupled receptor‐induced ERK to the cytosol through direct binding of ERK to arrestin.

Conclusions

This is the first evidence showing that arrestin3 acts as a coordinator to integrate signals from multiple GPCRs. Our studies not only provide a novel mechanism explaining the integration of mitogenic signaling elicited by different GPCRs, but also underscore the critical role of signaling crosstalk among GPCRs in vivo.
Effect of hedgehog signaling activation on osteoblast differentiation of human mesenchymal stem cells
Authors: Rosa Adalberto Luiz , Oliveira Fabiola Singaretti , Crippa Grasiele Edilaine , de Oliveira Paulo Tambasco , Beloti Marcio Mateus
Published: June 2021

This study aimed at evaluating the effect of hedgehog (Hh) signaling activation by purmorphamine on osteoblast differentiation of human bone marrow mesenchymal stem cells (hBMMSCs). Cultures were grown in presence of 2 μM purmorphamine or vehicle (control) for 7 and 14 days. Hh activation was evaluated by gene expression of Gli1 and osteoblast differentiation by a panel of 84 genes involved in osteogenesis (Osteogenesis PCR Array) using real time PCR. Gli1 expression was compared by Kruskal‐Wallis test and Osteogenesis PCR Array by t‐test followed by Benjamini‐Hochberg multiple test correction. hBMMSCs in presence of purmorphamine displayed a significantly higher expression of Gli1 at 7 and 14 days compared to control. Many genes involved in skeletal development, bone mineral metabolism, cell growth and differentiation, extracellular and cell adhesion molecules, and transcription factors were differentially expressed in purmorphamine treated and control cultures. Noteworthy, genes encoding BMP4 (1.3 and 4.4‐fold), FGF2 (3.3 and 5.8‐fold), IGF1 (3.2 and 6.3‐fold), IGF2 (1.8 and 5.2‐fold), and TGFβ2 (1.4 and 3.4‐fold) were upregulated by Hh activation from day 7 to 14 days respectively. These findings indicate that Hh activation triggers a cascade of intracellular signaling pathways involved in osteoblast differentiation.

Grants from FAPESP and CNPq, Brazil.
Hydroxylated polymethoxyflavones induce p53 and Bax dependent apoptosis and cell cycle arrest.
Authors: Qiu Peiju , Guan Huashi , Dong Ping , Li Shiming , Ho Chi‐Tang , Pan Min‐Hsiung , McClements David Julian , Xiao Hang
Published: June 2021

Previously, we reported that hydroxylated polymethoxyflavones (OH‐PMFs, such as 5‐hydroxy‐6,7,8,3′,4′‐pentamethoxyflavone (5HPMF), 5‐hydroxy‐3,6,7,8,3′,4′‐hexamethoxyflavone (5HHMF), and 5‐hydroxy‐6,7,8,4′‐tetramethoxyflavone (5HTMF) potently induced apoptosis and cell cycle arrest in HCT116 colon cancer cells. Herein, using isogenetic variants of HCT116 cells, we investigated the effects of p53 and BAX status on the apoptosis and cell cycle arrest induced by OH‐PMFs. All three OH‐PMFs increased G0/G1 cell population in wt‐ p53 and these effects were abolished in null‐p53 HCT116 cells. In null‐p21 HCT116 cells, none of the three OH‐PMFs induced G0/G1 arrest. These results suggest that OH‐PMFs induce G0/G1 arrest by a p53 and p21 dependent manner. 5HHMF and 5HTMF can significantly induce apoptosis in wt‐ p53 cells, but not in null‐p53 cells. Moreover, 5HHMF and 5HTMF significantly induced apoptosis in BAX(+/−) HCT116 cells, while their pro‐apoptotic effects on BAX(−/−) HCT116 cells were marginal. Western blot analysis showed that 5HHMF and 5HTMF increased the levels of p21, cleaved caspase‐3, cleaved PARP in both wt‐ p53 and BAX(+/−) HCT116 cells, and these effects were much weaker in null‐p53 and BAX(−/−) HCT116 cells. Taken together, our results demonstrated that OH‐PMFs, especially 5HHMF and 5HTMF induce apoptosis and cell cycle arrest by a p53 and BAX dependent manner.
Identification of the SSB binding site of Uracil DNA Glycosylase
Authors: George Nicholas Patrick , Liban Tyler J
Published: June 2021

Uracil DNA glycosylases (UDGs) catalyze the removal of uracil bases that arise in DNA by spontaneous cytosine deamination and dUTP misincorporation by DNA polymerases. UDGs are found in all kingdoms of life and in many cases have been shown to interact functionally with single‐strand DNA‐binding proteins (SSBs). The structural mechanisms that govern UDG/SSB interactions and the in vivo significance of the complex are currently unknown. We present the X‐ray crystal structures of Escherichia coli UDG in complex with the 9 C‐terminal residues of E. coli SSB and of Deinococcus radiodurans UDG in complex with the 12 C‐terminal residues of D. radiodurans SSB. The structures reveal a conserved platform on each UDG for binding SSB. Alanine variants of UDG residues involved in binding the SSB C‐terminal peptides lead to dramatic weakening of the cognate UDG/SSB complexes. These structures and solution studies provide firm evidence for the identification of the SSB interaction platform on UDG. Future genetic studies will examine the importance of UDG/SSB interactions in E. coli cells.
Growth inhibition effects of human breast cancer cell line by ursolic acid
Authors: Gim Gyeong‐Hun , Choi Youn‐Kyung , Shin Yong‐Cheol , Ko Seong‐Gyu
Published: June 2021

Growth inhibition effects of human breast cancer cell line by ursolic acid.

Gyeong‐Hun. Gim, Youn‐Kyung. Choi, Yong‐Cheol. Shin, Seong‐Gyu. Ko

Lab of Clinical Biology and Pharmacogenomics, Department of Basic Science of Oriental Medicine, College of Oriental Medicine, Kyunghee University.

Ursolic acid (3‐hydroxy‐urs‐12‐en‐28‐oic acid) is a pentacyclic triterpenoid derived from leaves, berries, fruits, and flowers of medicinal plants, such as Rosemarinus officinalis. Ursolic acid has been shown to inhibit tumorigenesis, tumor promotion, and suppress angiogenesis. Besides, we found that ursolic acid have been shown to decreased cell proliferation and induce apoptosis in a variety of breast cancer cell line (MCF‐7, MDA‐MB‐231, MDA‐MB‐453, SK‐BR‐3). It support that ursolic acid activates apoptosis of breast cancer cell. So It was detected to protein level of genes associated apoptosis signal by using immunoblotting. Among many proteins, caspase‐3 was increased. Therefore, our results explain that ursolic acid led to activates caspase 3 and poly(ADP‐ribose) polymerase cleavage, result in apoptosis activation. These results of ursolic acid could have predicted the possibility as anticancer therapy drug.
Inhibition of TNF‐α‐induced ROS generation, VCAM‐1 expression and NF‐κB activation by chloroform extract of aged black garlic in human umbilical vein endothelial cells
Authors: LEE EUN NA , Kim Hye Kyung , Kim Myoung June , Kim Hyo Jin , Park Jin Kyeong , Oh Sae‐Ock , Baek Sun‐Yong , Kim Bong‐Seon , Choi Young‐Whan , Yoon Sik
Published: June 2021

The current study investigated if the chloroform extract of aged black garlic (CEABG) could attenuate the activities of adhesion molecules in tumor necrosis factor‐α (TNF‐α)‐stimulated human umbilical vein endothelial cells (HUVECs). The study was performed on HUVECs that were pretreated with 30 μg/ml of CEABG before TNF‐α treatment. When pretreatment of HUVEC with CEABG for 30 min, CEABG suppressed reactive oxygen species (ROS) production induced by TNF‐α. HUVECs treated with CEABG exhibited remarkably suppressed TNF‐α‐induced mRNA expression of VCAM‐1, but little alteration in ICAM‐1 and E‐selectin mRNA expression. CEABG treatment also significantly decreased the TNF‐α‐induced cell surface and total protein expression of VCAM‐1 without affecting ICAM‐1 and E‐selectin expression. Furthermore, CEABG significantly inhibited NF‐κB transcription factor activation in TNF‐α‐stimulated HUVECs. Collectively, our findings suggest that CEABG can be an efficient new anti‐inflammatory agent. It may have a potential therapeutic use for the prevention and treatment of vascular disease such as atherosclerosis.
THE CUTANEOUS PERFORATORS OF THE PERONEAL ARTERY ON ARTERITIC PATIENTS
Authors: MATUSZ PETRU L , MASTACANEANU MIHAELA
Published: June 2021

The distribution of cutaneous perforators (CP) form the tibioperoneal trunk, posterior tibial artery (PTA) and peroneal artery (PA), is very important for the surgical practice. We analyzed the internal diameter (ID) of the PA CP after injecting plastic mass in the arterial network of the calf followed by vascular wall dissection, on ten legs amputated from arteritic patients (80% male, aged 57–84 years). We have to highlight some particular issues to be considered: the order of arterial blockage is: (1) PTA, (2) anterior tibial artery (3) PA; the CP of the PTA are much fewer in number and suprafascial distribution territory; the CP of the PA had at the level of penetration of the fascia a significantly greater diameter in arteritic patients (mean ID 0.96 ± 0.21 D.S. mm, on an average of 3.4 CP on calf); we have shown the same issue the above lateral malleolar artery; associated path of the sural nerve and lesser saphenous vein with the median superficial sural artery (supplied by the CP of the PA), we highlighted an arterial network consisting of many interconnected parallel vessels. This study shows that the use of various types of fasciocutaneous or osterocutaneous flaps based on PA source, can be extended also to ateritic patients.
Bypass of 8‐oxo‐G by DNA polymerase lambda
Authors: Hambardjieva Elena , Derkunt Burak , Yakubovskaya Elena , Mejia Edison , Diaz Miguel Garcia
Published: June 2021

7,8‐dihydro‐8‐oxoguanine (8‐oxo‐G) is a common oxidative DNA lesion. Synthesis across 8‐oxo‐G leads to frequent (10–75%) misincorporation of dA. The OGG1 DNA glycosylase can remove the oxidized base and initiate base excision repair (BER). However, after an 8‐oxo‐G:dA mispair has been formed, OGG1‐initiated BER would consolidate a transversion mutation. The MUTYH glycosylase can eliminate the misincorporated dA instead, allowing a second round of synthesis opposite 8‐oxo‐G. DNA polymerase lambda (polλ) has been implicated in MUTYH‐initiated repair of 8‐oxo‐G:dA mispairs. Pol λ is unique in its preference for the correct incorporation of dC opposite this lesion. Moreover, the polymerase often polymerizes past the first incorporation, initiating long‐patch BER.

We have solved crystal structures of polλ in complex with DNA containing site‐specific 8‐oxo‐G residues in order to investigate the mechanisms by which the polymerase preferentially incorporates dC opposite 8‐oxo‐G and discriminates against 8‐oxodG:dA pairs. Our data suggest a possible explanation for the discrimination against 8‐oxo‐G:dA pairs. The Glu529 residue of the thumb subdomain is shifted away from its normal conformation in structures containing 8‐oxo‐G:dA mispairs. This suggests an unfavorable interaction of Glu529 with the C8 of 8‐oxoG when the base adopts a syn conformation.

This work was supported by NIH R00 ES015421.
Anti‐allergic effect of Diosgenine in mice
Authors: Choi Ko Woon , Jung Da Hye , Park Hye‐Jin , Pyo Suhkneung
Published: June 2021

Allergies are immediate hypersensitive responses to antigens and IL‐4 is involved in the initiation and development of allergic responses. Diosgenin, a steroidal sapogenin, is extracted from the root of wild yam and has been shown to have a variety of biological activities including anti‐inflammatory activity. The present study was undertaken to examine whether diosgenin has an inhibitory effect on allergic response in mouse splenocytes and mouse model. Our results showed that diosgenin suppressed both T and B cells proliferation induced by mitogens. RT‐PCR analysis also revealed that mRNA levels of both IL‐4 and NFAT1 were in vitro suppressed in the presence of diosgenin. In allergic mouse model, application of diosgenin on atopic dermatitis like skin lesions improved skin condition and inhibited starching behaviors. In addition, diosgenin application suppressed IL‐4 mRNA levels and its secretion. Levels of NFAT1 mRNA were also attenuated by diosgenin in mouse. Taken together, these results suggest that diosgenin has a potent inhibitory role in T cell activation and may be a candidate for therapeutic agent in allergy.
Comparative bioavailability of (−)‐hydroxycitric acid from oral administration of HCA calcium salt and calcium‐potassium double salt in Albino Wistar rats
Authors: Bagchi Debasis , Trimurtulu Golakoti , Krishna Raju A. V. , Sengupta Krishanu , Murthy P.B.S. , Rao T.V. Narasimha
Published: June 2021

(−)‐Hydroxycitric acid (HCA) derived from Garcinia cambogia is a popular nutraceutical product for management of obesity. Various HCA salts are available in the market thus we evaluated the oral bioavailability of calcium‐potassium double salt (HCA‐SX) and calcium salt (HCA‐Ca) in Albino Wistar rats. Rats were divided randomly into 2 groups (n=8). Rats were supplemented orally with a single dose (1000 mg/kg) of each HCA salt. Blood samples were collected before administration followed by 0.5, 1, 1.5, 2, 3 and 5h after administration. Oral administration of HCA‐SX resulted in significantly better bioavailability compared to HCA‐Ca. Peak plasma concentration of HCA‐SX (37.3 μg/mL) is significantly (p = 0.031) higher than HCA‐Ca (12.93 μg/mL). HCA‐SX at 93.93% exhibited better bioavailability (AUC 65.55) compared to HCA‐Ca (AUC 33.80). Statistically significant improvement in peak plasma concentration was also observed in HCA‐SX compared to HCA‐Ca. In summary, the current study provides strong evidence to prove superior oral bioavailability of HCA‐SX compared to HCA‐Ca.
Role of cyclooxygenase (COX)‐1 and COX‐2 in a rabbit model of lipopolysaccharide (LPS)‐induced ocular inflammation
Authors: Waterbury L. David
Published: June 2021

Objective

To evaluate relative contribution of COX‐1 and COX‐2 to the LPS‐induced ocular inflammation in rabbits.

Methods

At hour 0, 27 rabbits received either artificial tears (Refresh Tears®; Allergan, Inc.; Irvine, CA) (3 drops 20 min apart), COX‐1 inhibitors trans‐resveratrol 0.1% (TR) (3 drops 20 min apart) or FR122047 0.1% (FR) (1 drop), COX‐2 inhibitor nimesulide 0.1% (1 drop), or COX‐1 and COX‐2 inhibitor ketorolac 0.45% (Acuvail®; Allergan, Inc.; Irvine, CA) (3 drops 20 min apart). At hour 1, rabbits received intravenous injections of LPS and fluorescein isothiocyanate (FITC)‐dextran. At hour 2, aqueous samples were collected for analysis. Six additional rabbits received only FITC‐dextran 2 hours before sample collection.

Results

LPS increased aqueous prostaglandin E2 (PGE2) and FITC‐dextran levels by 4.2‐ and 137.8‐fold, respectively. Ketorolac 0.45% and COX‐1 inhibitors (TR and FR) significantly reduced PGE2 elevation (P < .01) while COX‐2 inhibitor (nimesulide) was ineffective. Of all inhibitors, only ketorolac 0.45% significantly inhibited FITC‐dextran elevation (P < .01). Inefficacy of TR and FR in reducing FITC‐dextran leakage may be due to their poor penetration into iris–ciliary body.

Conclusion

COX‐1 was the primary isoenzyme responsible for early inflammatory response to LPS. Ketorolac 0.45% suppressed ocular inflammation by inhibiting PGE2 elevation and FITC‐dextran leakage.
Activity of CNTO 530 in a murine model of sickle cell anemia
Authors: Makropoulos Dorie Ann , Achuthanandam Ram , Rafferty Patricia , Brosnan Kerry , Chroscinski Denise , Avery Justin , Miller Andrew , Ogunkua Lawrence , Walker Mindi , Bugelski Peter J
Published: June 2021

CNTO 530 is an erythropoietin receptor agonist MIMETIBODY™ construct. CNTO 530 has been shown to be active in a number of rodent models of acquired anemia and in a model of beta‐thalassemia. Here we report on the activity of CNTO 530 in murine globin KO‐human HbS transgenic (sickle cell) mice. Berkeley mice are knocked out for murine alpha and beta globin and are transgenic for human alpha, beta (sickle) and gamma globin genes. Thus, these mice express exclusively human hemoglobin A (HbAsickle) (or, HbS) and can also express human fetal hemoglobin (HbF). To test the effectiveness of CNTO 530 in this model, mice received a single subcutaneous (s.c.) dose of CNTO 530 (0.3 mg/kg), epoetin‐alpha (0.1 mg/kg) or darbepoetin‐alpha (0.03 mg/kg) (as comparators) at 10,000 U/kg, a dose previously shown to increase hemoglobin in normal mice. Hematology, histology of the spleen, F‐cells and expression of HbF were then evaluated over time. These mice express a severe compensated hypochromic microcytic anemia and display the sickle cell phenotype. Ten days after dosing, CNTO 530 but not epoetin‐alpha or darbepoetin‐alpha caused an increase in reticulocytes, red blood cells, F‐cells, total hemoglobin and HbF. In conclusion, these results demonstrate that a single dose of CNTO 530 can increase expression of HbF in sickle cell mice and suggest that human treatment with CNTO 530 may have beneficial effects in sickle cell anemia patients.
Morphometric evaluation of masticator muscles after experimental hyperthyroidism
Authors: Rebelo Diana , Farinha Rodrigo , Loureiro Marco , Cabrita António Silvério
Published: June 2021

Hyperthyroidism is a clinical condition characterized by excessive production and action of thyroid hormones with various clinical manifestations, one of which being the excessive catabolic reactions in skeletal muscles. This study evaluates the morpho‐histologic effects in masticator muscles of levothyroxine administered by gavage. Ten Wistar male rats were used, randomly distributed in two groups. The control group had no manipulation. The test group had an experimental administration of levothyroxine by gavage 3 times in a week, in a dosage of 250 mg/kg. After two weeks, the animals were sacrificed and fragments from masseter and temporal muscles were collected and analysed in a morphometric program, ImageJ®.

At morphometric evaluation, the masseter muscle from the control group had an average area of muscle fibers (A) of 10704±6835 for a 95% confidence interval (CI), while the test group had an A of 13621±5014 for a CI of 95%. The temporal muscle from control group had an A of 7211±2824 for a CI of 95%, while the test group had an A of 21826±12020 for a CI of 95%.

According to these results, masseter muscle fibers from test group have a slightly large area when compared to control group. The temporal muscle fibers have an enlarged size when compared to the control group, with statistical significance. It suggests both temporal and masseter muscle involvement in this experimental situation.
ACETAMINOPHEN CONSUMPTION ALTERS ACHILLES TENDON COLLAGEN CONTENT IN WISTAR RATS
Authors: Whitt Jamie A , Tedeschi Jamie , McMullan David R , Del Mundo Jonathan D , Broderick Tom L , Carroll Chad C
Published: June 2021

The purpose of this study was to evaluate the influence of acetaminophen (APAP) on collagen content in rat Achilles tendon. Ten‐week‐old male Wistar rats were given saline (SAL, n=8) or APAP (n=10; 200 mg·kg−1) once daily via oral gavage for 8 weeks. Tendon collagen content was determined by detection of the collagen specific amino acid hydroxyproline using HPLC. Following treatment, tendons, mm. gastrocnemius and soleus, fat pad, heart, liver, and femur and tibia bones were harvested. Tendon collagen content was greater (p<0.05) in the rats given APAP (SAL: 510.5±42.3; APAP: 663.4±38.0 μg collagen·mg tendon wet weight). Fat pad weight was greater (p<0.05) in the APAP group (SAL: 11.02±1.87; APAP: 15.83±1.36 g). Both femur and tibia weight [femur: (SAL: 867±19; APAP: 936±23 mg, tibia: (SAL: 644±22, APAP: 718±16 mg)] were greater (p<0.05) in the rats consuming APAP. M. soleus (SAL: 203±11; APAP: 240±9 mg) but not m. gastrocnemius (SAL: 2.73±0.05; APAP: 2.76±0.12 g) weight was greater (p<0.05) in the rats given APAP, whereas heart and liver weight was not different between groups. APAP appears to promote collagen deposition in tendon and has growth promoting affects on bone and skeletal muscle, however, the affect on skeletal muscle appears to be specific to the type I soleus muscle. The specific mechanisms of these effects require further study.

MWU College of Health Sciences and MWU Office of Research and Sponsored Programs
Integrating Novel Protein Engineering and Nanotechnology for Therapeutic Angiogenesis
Authors: Roy Rituparna Sinha , Soni Shivani , Harfouche Rania , Vasudevan Pooja R , Paraskar Abhimanyu , Sengupta Shiladitya
Published: June 2021

Therapeutic angiogenesis is an emerging concept that can potentially be harnessed for management of ischemic pathologies. Hepatocyte growth factor/scatter factor (HGF/SF) promotes angiogenesis but the native protein has limited therapeutic potential due to its complex multidomain structure with glycosylation sites. To address this translational challenge, a novel spliced HGF/SF variant, 1K1 encoding the N‐terminal domain and first kringle (K1) domain was engineered with reverse mutations (K132E:R134E) introduced in the K1 domain. To address the clinical requirement of sustained delivery, 1K1 was formulated in a polymeric nanoparticle of 60–140 nm size and with the total loading efficiency 54.27±7.12%. 1K1‐nanoparticle (NP) induced significantly greater angiogenesis both in vitro and in vivo than free 1K1 (P<0.05). 1K1‐NP induced angiogenesis were blocked by PHA665752, LY294002 and PD98059 inhibitors in a concentration‐dependent manner (P<0.05), suggesting that 1K1‐NP induces angiogenesis through MET receptor and downstream MAPK and PI3K pathways. Integrating novel protein engineering strategy and nanotechnology offer stimulating revascularization at the site of ischemic pathology.
The Role Of Autophagy In Angiogenesis In Aortic Endothelial Cells
Authors: Du Jianhai , Teng Ru‐Jeng , Guan Tongju , Xu Hao , Prichard Kirkwood A , Shi Yang
Published: June 2021

Angiogenesis plays critical roles in the pathogenesis of ischemic heart diseases and peripheral vascular diseases. The objective of this study is to examine the role of autophagy in angiogenesis. Using cultured bovine aortic endothelial cells (BAEC), autophagy was induced either by nutrient‐deprivation or by overexpression of ATG5. Both treatments significantly increased autophagosome formation and increased expression of light chain3 II (LC3II). Interestingly, both nutrient‐deprivation and ATG5 overexpression increased EC tube formation, migration as well as wound‐healing process. To determine whether autphagy is required process for angiogenesis, EC were treated with a specific auotphagy inhibitor 3‐methyladenine (3‐MA) or Beclin‐1 siRNA to suppress autophagy activation. Interestingly, 3‐MA or Beclin‐1 siRNA greatly decreased EC tube formation, migration and wound‐healing under nutrient‐deprived conditions, and blocked VEGF‐induced tube formation and migration. Autophagy inhibition did not affect the expression of VEGF, PDGF and integrin αV. Furthermore, autophagy inhibition decreased AKT activation and ROS production, two important contributors to angiogenesis. In conclusion, we report for the first time that autophagy appears to be essential for angiogenesis and that targeting autophagy provides an important new route in treating cardiovascular diseases.
Acute Caloric Restriction is Cardioprotective in Adult Rats
Authors: Snorek Michal , Neckar Jan , Wilhelm Jiri , Skoumalova Alice , Hodyc Daniel , Sedivy Vojtech , Tousek Frantisek , Kolar Frantisek , Herget Jan
Published: June 2021

Reduced coronary perfusion in acute coronary syndrome causes myocardial ischemia. Also the effect of reactive oxygen species (ROS) induced ischemia‐reperfusion injury contributes to myocardial lesion. ROS generation in hypoxia is modulated by the redox state. Fasting before exposure to ischemia should increase ketogenesis that consumes reduced coenzymes and consequently reduces free radical damage.

We investigated whether the 3‐day fasting affects production of ROS, incidence of ventricular arrhythmias during ischemia/reperfusion and myocardial infarction size.

We analyzed production of the lipid peroxidation end products (LFP) in the heart tissue. Open‐chest rats were subjected to 20‐min LAD coronary artery occlusion followed by reperfusion. A single‐lead ECG was recorded and arrhythmias were assessed. The infarction size in excised hearts was determined by triphenyltetrazolium chloride staining and it was normalized to area at risk (IS/AR).

Three day fasting reduced LFP production in heart tissue. During early reperfusion the number of premature ventricular complexes was reduced in fasting rats compared to controls, as well as ventricular tachycardia duration. In fasting rats the IS/AR reached 48.5 ± 3.3 % while in controls it was 74.3 ± 2.2 %, p<0.005.

Acute caloric restriction limits free radical damage in heart, reperfusion ventricular arrhythmias and myocardial infarction size in rats.

Supported by GAUK 60009/2009 and MSMT 1M 0510.
Interferon‐gamma Induced Adipose Inflammation Linked to the Impairment of Vascular Function in Type 2 diabetic mice
Authors: Zhang Hanrui , Zhang Cuihua
Published: June 2021

We hypothesized that IFNγ induces inflammatory cell infiltration and chemoattractant gene expression in mesenteric adipose tissue (MAT) and impairs vascular function of mesenteric arteries (MA) of type 2 diabetic mice. Acetylcholine (ACh)‐induced vasodilation was impaired in Leprdb vs. m Leprdb, but sodium nitroprusside (SNP)‐induced vasodilation was comparable. Both anti‐IFNγ and anti‐MCP‐1 improved endothelial function in Leprdb, while IFNγ induced impaired vascular function in m Leprdb. Mesenteric bed weight and MAT adipocyte size were greatly elevated (P<0.05) in Leprdb vs. m Leprdb. Immunohistochemical staining results revealed that CD3‐positve T‐lymphocytes, Mac‐3 positive macrophages infiltration into the adventitia of small mesenteric arteries was significantly increased in Leprdb and IFNγ‐treated m Leprdb, suggesting the possible crosstalk between adipose inflammation and vascular dysfunction. Western blotting results showed that anti‐IFNγ decreased MAT IFNγ and MCP‐1 protein expression in Leprdb,while IFNγ and MCP‐1 expression was elevated in IFNγ‐treated m Leprdb. Anti‐MCP‐1 treatment only decreased MCP‐1 expression in Leprdb without affecting IFNγ levels, indicating that IFNγ may act as an initiator of MAT inflammation. These results suggest a role for IFNγ in the regulation of visceral adipose inflammatory response and vascular dysfunction in type 2 diabetes.
Increased NO‐dependent dilation of rat arterioles in diet‐induced obesity
Authors: Howitt Lauren , Grayson T Hilton , Morris Margaret J , Murphy Timothy V
Published: June 2021

Obesity is a risk factor for hypertension. The large‐conductance Ca2+‐activated potassium channel (BKCa) plays a central role in the regulation of vascular tone. The aim of this study was to examine the effect of diet‐induced obesity on endothelium‐ and BKCa‐dependent dilation of rat cremaster muscle arterioles. Male Sprague‐Dawley rats (213 ± 1g) were fed a high fat or control diet for 16 weeks. Isolated, pressurized (70 mmHg) arterioles were studied. Control rats weighed 570 ± 7 g compared with obese rats 768 ± 13 g (n = 35 – 37, P < 0.05). Obesity had no effect on acetylcholine (ACh; 0.001 – 3 μM)‐induced dilation. ACh‐induced dilation of arterioles from control rats was abolished by a combination of the SKCa, IKCa, and BKCa blockers apamin, TRAM‐34 and iberiotoxin (IBTX, 0.1 μM) respectively, with no apparent role for NO. In arterioles from obese rats however, IBTX had no effect on responses to ACh while the NO/GC inhibitors L‐NAME (100 μM)/ODQ (10 μM) partially inhibited ACh‐induced dilation. Removal of the endothelium constricted arterioles from obese, but not control rats. Expression of eNOS was not altered by obesity, but expression of high molecular‐weight complexes of caveolin‐1 and ‐2 was increased. In summary, diet‐induced obesity abolished the contribution of BKCa to ACh‐mediated endothelium‐dependent dilation of rat cremaster muscle arterioles, while inducing an NO‐component.

Support: NHMRC 455243.
Impaired cardiac functional reserve in type 2 diabetic db/db mice is associated with metabolic, but not structural, remodeling
Authors: Daniels Anneleen , van Bilsen Marc , Janssen Ben , Brouns Agnieszka , Cleutjens Jack , Roemen Theo , Schaart Gert , van der Velden Jolanda , van der Vusse Ger , van Nieuwenhoven Frans
Published: June 2021

To identify the nature of myocardial alterations associated with diabetes, cardiac function, structural remodeling, and gene expression in hearts of type 2 diabetic db/db mice were studied.

Db/db mice showed typical characteristics of type 2 diabetes and preserved cardiac function up to 18 wks of age under non‐challenging conditions. However, β‐adrenergic stimulation with dobutamine revealed impaired contractile and relaxation reserve capacity. Tissue analyses showed no signs of structural remodeling. Neither β1‐adrenoceptor (β1AR) expression nor phosphorylation state of contractile protein targets of β1AR – protein kinase A (PKA) signaling were altered. In contrast, the substantially increased expression of uncoupling protein‐3 (UCP3) and angiopoietin‐like‐4 (Angptl4), along with decreased phosphorylation of AMP‐activated protein‐kinase (AMPK) in the heart of diabetic animals, are indicative of altered mitochondrial efficiency, decline in plasma triacylglycerol consumption and changes in intermediary substrate metabolism, respectively, which together may compromise ATP synthesis, required for maintenance of cardiac function at increased work load.

We conclude that impaired cardiac functional reserve capacity during maximal β‐adrenergic stimulation, is an early sign of cardiac dysfunction in db/db mice which is primarily associated with unfavorable changes in cardiac metabolism.
Low dose chronically infused hemoglobin induces pulmonary hypertension or excerbates hypoxia‐induced pulmonary hypertension
Authors: Irwin David , Foreman Ben , Connor Ian , Lisk Christina , Buehler Paul
Published: June 2021

Observations from examining past medical records from patients with sickle cell anemia or thalassemia suggest these diseases may be a risk factor for the development of severe pulmonary hypertension (PH). Recently it has been proposed that the chronic exposure to cell free hemoglobin is a major contributor to the development of PH in these patients. Therefore, we hypothesized that chronic infusion (3 and 6 wk) of cell free hemoglobin in rats would either induced or exacerbate PH in rats exposed to room air or hypoxia.

Methods

Male, age matched (8–10 wk) Sprague dawley rats were treated with chronically infused free hemoglobin (500 ug/hr) or left untreated and either exposed to chronic hypoxia (18,000 ft; PB ~ 379 mm Hg;) or remained in normoxic conditions (5,280 ft; PB ~ 620 mm Hg). After wards, cardiac output (CO), mean pulmonary artery (Pa) and systemic pressures (MAP) and hemotocrit (Hct) were recorded, and RV/LV+S ratio was determined and lungs were examed as indication of the severity of PH.

Results

Overall, hemoglobin (Hb) infusion increased Pa pressures and lowered Hct compared to untreated cohorts. In the normoxic Hb infused animals MAP and Pa pressures increased between 3 and 6 weeks. Compared to hypoxic control rats, Pa pressures were greater at 3 and 6 wks, whereas MAP was greater only at 3 wk in Hb infused animals.

Conclusion

Chronic infusion of CFH can induce or exacerbate hypoxic‐induced PH. However, whether CFH exerts this effect by oxidative damage, inflammation or NO scavenging is unknown.