bluuebunny/crossref_metadata_2025 huggingface dataset:
Resveratrol's initial transient enhancement of adrenergic contractions of rat tail arterial tissues involves a mechanism limiting its ability to exert a later sustained relaxation
Authors: Stom Sayra M , Peuler Jacob D
Published: November 2023

We have reported that resveratrol can enhance as well as relax adrenergic contractions of arterial tissue rings prepared from both proximal (conductance) and distal (resistance) segments of the long ventral tail artery of the rat (Stom, JSMR 2016; 52:18–35). While the enhancing action only occurred in approximately half of our ring preparations (from proximal and distal segments alike), it always appeared immediately after administration before the later, more sustained relaxing effect (which occurred in all preparations) became apparent. Thus, the enhancing effect seemed to be only transient in duration. However, that does not rule out the possibility of its mechanism lasting long enough to limit the degree to which resveratrol eventually causes relaxation. To determine if that were true, we re‐examined data from our original arterial ring preparations to see if the half in which early transient enhancement occurred showed less eventual relaxation compared to the half in which they did not occur. Indeed, that was the case. This provides a novel explanation for why long‐term administration of resveratrol in vivo is reported to lower arterial blood pressures in some clinical studies but not in others, as highlighted in recent reviews (e.g. Liu, Clin Nutr 2015; 34: 27–34).

Support or Funding Information

MWU Biomedical Masters Program
Non‐adrenergic Vasoconstriction of Human Blood Vessels to the Trace Amine, β‐Phenylethylamine
Authors: Broadley Kenneth John , Mehta Dheeraj
Published: November 2023

Trace amines, such as β‐phenylethylamine (PEA), cause vasoconstriction in animal blood vessels not by indirect sympathomimetic actions but via trace amine‐associated receptors (TAARs). Functional studies were therefore performed on human arteries and veins surplus to coronary artery by‐pass graft (CABG) surgery to establish whether they constrict to PEA and if so whether they are adrenoceptor‐mediated. Internal mammary artery or saphenous vein rings were set up in organ baths containing Kreb's bicarbonate solution at 37±0.5°C gassed with O2:CO2 (95:5) under class 2 containment conditions. 1.5 g resting tension was applied and isometric contraction measured using a computerized Power Lab, Chart 5 data acquisition system (ADInstruments). Cumulative concentration‐response curves (CRC) were obtained for β‐phenylethylamine hydrochloride. Contractions were measured by subtracting baseline tension from plateau responses and expressed as a percentage of the contraction to KCl (60mM) added at the end. Responses were compared by Student's paired t‐test, P≤0.05 indicating significance. PEA showed concentration‐related contractions that were significantly greater at the maximum in the artery (1.53±0.31g, n=9) than the vein (0.55±0.18g, n=10). However, when plotted as % of the KCl maximum contraction, the maximum responses were not significantly different (vein 66.7±9.5%; artery 74.7±4.6% at 3×10−3M), indicating the overall greater contractility of this artery. PEA showed slowly developing contractions in both tissues which reached plateau effect after about 20 min. The reference α‐adrenoceptor agonist, phenylephrine, showed a much more rapid contraction, which was not sustained. In the saphenous vein, PEA (62.8±10.7%KCl) and phenylephrine (61.4±9.7%KCl, n=4) displayed the same maximum responses at 3×10−3M and 3×10−4M respectively, but phenylephrine was more potent. The α1‐adrenoceptor antagonist, prazosin (1μM) dissolved in 1 in 10 DMSO, blocked the responses to phenylephrine in the mammary artery but did not affect the contractions to PEA. Similarly, in the saphenous vein, prazosin (1μM) did not block the contractions to PEA. The DMSO vehicle for prazosin had no effect on PEA in mammary artery or saphenous vein. Thus, the indirect sympathomimetic and trace amine, PEA, has been shown to cause substantial vasoconstriction of human saphenous vein and mammary artery, which would explain its vasopressor actions in humans. This response however was not mediated via α1‐adrenoceptors, but was likely due to stimulation of TAARs.
Understanding the nature of toxicity of Parkinson's disease associated alpha‐synuclein familial mutants H50Q, G51D, and A53E with yeast models
Authors: Ong Emily Nicole , Tembo Maiwase , Mwale Chisomo , Marshall Morgan , Alvarado Charles , Buabeng Michael , Kukulka Natalie , DebBurman Shubhik
Published: November 2023

Parkinson's disease (PD) is associated with the aggregation and misfolding of alpha‐synuclein in midbrain dopaminergic neurons. The gene for alpha‐synuclein has six known mutations that directly cause familial forms of PD. The pathological determinants of three of these mutants (A30P, E46K, and A53T) are well characterized in diverse model systems and they that reveal that each mutant affects cellular toxicity in distinctive ways. The three more recently discovered familial mutants (H50Q, G51D, and A53E) are not extensively studied. We expressed H50Q, G51D, and A53E mutants in budding and fission yeasts model systems and hypothesized that each would generate toxicity by altering their membrane association and aggregation properties, and by disrupting cellular pathways including nitrative stress responses and endocytosis, but each would do so in distinctive ways. First, we found that the H50Q and A53E mutants were toxic to yeast, and bound membranes and aggregated within yeast, while G51D was cytoplasmically diffuse and nontoxic. Secondly, and surprisingly, we found that G51D mutant dominated over H50Q and A53E when these mutants were combined in double/triple mutants. Thirdly, we asked whether the loss of the original amino acid or the gain of the new amino acid in each new familial mutant is responsible for disease. We created four substitution mutations for H50Q, G51D, and A53E in both yeasts models corresponding to the four functional classes of amino acids. We found that H50D was cytoplasmically diffuse and nontoxic, G51A bound membranes and aggregated like WT, G51E was cytoplasmically diffuse and nontoxic like G51D, and A53R was cytoplasmically diffuse and nontoxic, suggesting both the loss of the original amino acid and the gain of the new amino acid are key. Finally, we are currently characterizing these familial mutants in yeast strains altered for endocytosis, nitrative stress, and sumoylation. Collectively, this work adds insight into the pathogenicity of different familial PD mutants of alpha‐synuclein.

Support or Funding Information

E.O. was funded by Parkinson's Disease Foundation
Identification of an Arabidopsis WD‐Repeat Protein that Activates the Deubiquitinase UBP3 and Interacts with Two E3 Ubiquitin Ligases
Authors: Baskerville Andrew T , Donahue Janet , Gillaspy Glenda , Erickson Les
Published: November 2023

Ubiquitination is an essential, dynamic post‐translational modification that regulates a diverse set of cellular processes. Ligation of the small peptide ubiquitin (Ub) onto substrate proteins by E3 Ub ligases can change protein localization, activity or stability. The most common outcome for Ub‐tagged proteins is degradation via the 26S proteasome. Deubiquitinases (DUBs) reverse the effects of ubiquitination by removing Ub from tagged substrates. In animals, Ubiquitin‐specific protease (USP) 12 and USP46 are related DUBs that regulate key proteins in important growth and differentiation signaling pathways. USP12 and USP46 each form trimeric complexes with two WD‐repeat (WDR) proteins, WDR20 and WDR48. The WDR proteins activate the enzymatic abilities of the DUBs while potentially serving as substrate receptors. Numerous animal and fungal DUBs are bound and activated by specific WDR proteins. In Arabidopsis, Ub‐specific proteases 3 (AtUBP3) and 4 (AtUBP4) are related DUBs that have significant sequence similarities to animal USP12/46. Genetic studies have shown AtUBP3/4 are essential for proper pollen development and transmission. It is not yet known if WDR proteins bind and activate DUBs in plants. We have identified At2g37160 as a likely Arabidopsis WDR20 homolog based on amino acid sequence homology and the number and organization of WD motifs. We have shown At2g37160 (AtWDR20) interacts with both AtUBP3 and AtUBP4 in the yeast two‐hybrid system. Biochemical characterization using recombinant proteins shows AtWDR20 increases the DUB activity of AtUBP3 over nine‐fold. A yeast two‐hybrid screen of an Arabidopsis cDNA library using AtWDR20 as bait revealed interactions between AtWDR20 and two different E3 Ub ligases: Myb‐30 Interacting E3 Ub ligase (MIEL1) and CHY Zinc‐finger and Ring Protein 1 (CHYR1). Interactions between a DUB‐associated WDR protein and E3 Ub ligases is intriguing, and implies AtWDR20 may function on both sides of the Ub cycle or is itself a target of ubiquitination. In planta and in vitro analyses will further characterize the interactions between AtWDR20, AtUBP3, and these E3 ligases.
Rotavirus NSP1 as a phosphorylated substrate adaptor of hijacked Cullin‐RING ligases
Authors: Davis Kaitlin A. , Morelli Marco , Patton John T.
Published: November 2023

Rotavirus is a segmented, double‐stranded RNA virus responsible for significant diarrheal disease worldwide, primarily in infants and young children. To circumvent host interferon (IFN) responses, rotavirus NSP1 proteins hijack host Cullin‐RING E3 ubiquitin ligases (CRLs), acting as substrate adaptors to redirect their activity towards proteasomal degradation of IFN signaling proteins. For the NSP1 proteins of many human and porcine virus strains, this activity facilitates the degradation of β‐TrCP, a key regulator of NF‐κB signaling. NF‐κB, a transcription factor required for IFN induction, is activated when inhibitor of NF‐κB (IκB) is phosphorylated on a conserved degron motif by IκB kinase complex (IKK). Phosphorylation leads to recognition by β‐TrCP, which subsequently targets IκB for degradation. NSP1 contains a C‐terminal IκB‐like degron motif that competitively recruits β‐TrCP. Because the IκB degron requires phosphorylation to mediate interactions with β‐TrCP, we sought to address whether the NSP1 degron is also phosphorylated, and whether phosphorylation regulates NSP1 activity. Interestingly, antibodies specific for the phosphorylated IκB degron bound to NSP1, and this recognition was ablated by mutagenesis or phosphatase treatment, suggesting the degron is phosphorylated. Similarly, inhibition of casein kinase II (CKII) activity targeted by siRNAs, small‐molecule inhibitors, or motif mutagenesis disrupted NSP1 degron phosphorylation. Mutagenesis of an N‐terminal RING domain thought to mediate recruitment of CRLs had no effect on NSP1 degron phosphorylation. Our data indicates that binding of β‐TrCP to NSP1 hinges upon degron phosphorylation, and that phosphorylation by CKII occurs independently of binding to hijacked CRLs. The reliance of NSP1 on CKII, rather than IKK, suggests a distinct vulnerability and new potential target for therapeutic intervention.
Redundancy Between Proteasome Chaperones Nas2 and Hsm3 During Proteasome Assembly
Authors: Suppahia Anjana , Chingakham Ranjit Singh , De La Mota Peynado Alina , Roelofs Jeroen
Published: November 2023

Eukaryotic proteasomes consist of a 20S cylindrical core particle (CP) capped with a 19S regulatory particle (RP) on one or both sides. The regulatory particle can be subdivided into base and lid complexes. The base contains a ring of six homologous AAA‐ATPase subunits; Rpt1 to Rpt6. The efficient assembly of the base requires several proteasome‐specific chaperones. Two of these, Nas2 and Hsm3, show redundancy and bind to neighboring Rpt subunits, Rpt5 and Rpt1 respectively. However, Nas2 and Hsm3 are never found on the same sub complex and it is unclear why they are redundant. Here, we show that the reported rescue of Hsm3 deletion strains by over expression of Rpt1 depends on the presence of Nas2. Interestingly, in an Hsm3ΔNas2Δ strain no increased levels of Rpt1 were detected upon overexpression of Rpt1. This suggest that, at least in part, the Nas2 and Hsm3 redundancy depends on the ability of either protein to stabilize Rpt1 in the cell. Consistent with a potential role of Nas2 in Rpt1 stabilization, we observed a direct interaction between Nas2 and Rpt1 in vitro. This binding site mapped to the N‐terminal region of Rpt1. To understand the importance of the domains in Nas2, we studied the effects of over expression of N‐terminal domain or the PDZ domain of Nas2 in hsm3Δnas2Δ strain and observed differences in their ability to rescue the temperature sensitivity of these cells. Biochemical and structural studies have suggested the binding of Nas2 and Hsm3 to proteasome sub complexes is mutually exclusive and suggests a role of Hsm3 in Nas2 expulsion. Consistent with this, we observed the presence of Nas2 on larger assembly intermediates in strains deleted for HSM3. In all, our data suggest Nas2 and Hsm3 show redundancy at the step of Rpt1 incorporation into proteasome complexes and indicate pliability in the assembly pathway.

Support or Funding Information

NIH‐NIMGS (1R15GM112142‐01A1)
Elucidating the Molecular Details of Catalysis for Type III Polyketide Synthases Using Near Natural Substrate/Intermediate Analogs
Authors: Richards Logan R , Stunkard Lee , Benjamin Aaron , Manning Bethany P. , Lohman Jeremy
Published: November 2023

Natural products are a well‐established source of drugs and other commodity chemicals. Polyketide natural products have a high degree of chemical variability and play many roles in biology, making them targets for protein engineering to generate analogs. Polyketide synthases (PKS) exist as three different types, but all require the function of a ketoacyl synthase or ketosynthase (KS). The KS catalyzed reaction is carried out in three steps, transthiolation of an acyl‐chain onto an active site cysteine yielding an acyl‐enzyme intermediate, binding and decarboxylation of a malonyl‐group to yield an enolate, and a Claisen condensation of the enolate onto the acyl‐enzyme intermediate yielding a carbon‐carbon bond. The molecular details of decarboxylation and Claisen condensation are only poorly characterized, yet these details are needed for protein engineering efforts. Type III PKSs exist as an independently functioning KS, which primarily use coenzyme A (CoA) rather than acyl carrier proteins to carry the acyl‐chains and malonyl‐groups. As type III PKS are the simplest system we chose them as our starting point to elucidate the molecular details of KS catalysis. We have expressed and purified wild‐type and mutant forms of germicidin synthase (GCS) and tetrahydroxynaphthlene synthase (THNS) from Streptomyces coelicolor, and chalcone synthase‐2 (CHS‐II) from Arabidopsis thaliana in E. coli. Mutation of the active site cysteine to glutamine results in enzymes that mimic the acyl‐enzyme intermediate state, and can decarboxylate malonyl‐CoA. Interestingly, these mutants are much less stable than the wild‐type enzymes, suggesting that conformational changes occur upon formation of the acyl‐enzyme intermediate. We propose that using near natural substrate and intermediate mimics will allow us to capture the molecular details of catalysis. As such, X‐ray crystallography is underway with these enzymes in complex our synthetic analogs.

Support or Funding Information

We would like to thank the Department of Biochemistry, Purdue University for their sponsorship of the project. We would also like to thank Jeremy Lohman for providing the space needed to conduct our project.
Preliminary Investigation by NMR of the Electron Transfer Complex Between the Vitamin D Metabolizing Cytochrome P450 24A1 and Adrenodoxin
Authors: Estrada D. Fernando , Tu Youbin
Published: November 2023

Background

Deactivation of the bio‐activated form of vitamin D (1,25‐dihydroxy vitamin D3) represents a key metabolic step that helps determine the bioavailability of the active hormone. Deactivation is accomplished by hydroxylation of carbon 23 or carbon 24 of the vitamin D side chain as mediated by the mitochondrial cytochrome P450 enzyme CYP24A1. CYP24A1 belongs to a sub‐set of P450 enzymes that function in the mitochondrial membrane, where interactions with the soluble [2FE‐2S] cluster protein adrenodoxin (Adx) triggers the electron transfer required for P450 catalysis. Adx also serves as the principle redox partner for all other mitochondrial P450 enzymes and their respective reactions, including the activation of vitamin D by CYP27B1. In light of the importance of this electron transfer step in activation and deactivation of vitamin D, there remain basic questions regarding the formation of the Adx‐P450 enzyme complex. Among these are 1) the interface of the protein complex has yet to be mapped out for a vitamin D metabolizing P450, and 2) it is unknown whether Adx binding influences other regions of CYP24A1 that relate to substrate recognition and access.

Objective

In this preliminary study, we have set out to characterize the Adx‐CYP24A1 complex via a combination of spectrophotometric and nuclear magnetic resonance techniques.

Results

Our spectral CYP24A1‐ligand binding data indicate that the affinity between rat CYP24A1 and the vitamin D analog alfacalcidol is strengthened by approximately two‐fold in the presence of adrenodoxin, thereby suggesting a long‐range effect between the putative Adx binding surface of CYP24A1 and the canonical P450 substrate recognition and binding sites. Additionally, we have also optimized the Adx‐CYP24A1 complex for analysis by solution NMR, in which titration with CYP24A1 induces differential line broadening of the spectrum of 15N labeled Adx, with enhanced line broadening occurring in the short α‐helix adjacent to the [2FE‐2S] cluster.

Conclusions

The interaction of Adx with CYP24A1 induces a change in ligand binding, leading to greater affinity for alfacalcidol. Additionally, line broadening of residues in the short α‐helix adjacent to the [2FE‐2S] cluster is suggestive of participation of this region in formation of the complex with CYP24A1. These preliminary studies lay the groundwork for a comprehensive study of the Adx‐CYP24A1 complex under a variety of conditions, including a comparison between the oxidized and reduced states of Adx, and thereby providing insight toward this important step in vitamin D metabolism.

Support or Funding Information

This work was funded in combination by R00GM112862 and SUNY Buffalo startup funds.
Cellular uptake of a cystine‐knot peptide and modulation of its intracellular trafficking
Authors: Gao Xinxin , Stanger Karen , Kaluarachchi Harini , Maurer Till , Ciepla Paulina , Chalouni Cecile , Franke Yvonne , Hannoush Rami N
Published: November 2023

Cyclotides or cyclic cystine‐knot peptides have emerged as a promising class of pharmacological ligands that modulate protein function. Interestingly, very few cyclotides have been shown to enter into cells. Yet, it remains unknown whether backbone cyclization is required for their cellular internalization. In this report, we studied the cellular behavior of EETI‐II, a model acyclic cystine‐knot peptide. Even though synthetic methods have been used to generate EETI‐II, recombinant methods that allow efficient large scale biosynthesis of EETI‐II have been lagging. Here, we describe a novel protocol for recombinant generation of folded EETI‐II in high yields and to near homogeneity. We also uncover that EETI‐II is efficiently uptaken via an active endocytic pathway to early endosomes in mammalian cells, eventually accumulating in late endosomes and lysosomes. Notably, co‐incubation with a cell‐penetrating peptide enhanced the cellular uptake and altered the trafficking of EETI‐II, leading to its evasion of lysosomes. Our results demonstrate the feasibility of modulating the subcellular distribution and intracellular targeting of cystine‐knot peptides, and hence enable future exploration of their utility in drug discovery and delivery.
Lybatide: Naturally‐Occurring Disulfide‐Stapled Helical Peptides from Lycium barbarum
Authors: Tan Wei Liang , Wong Ka H. , Tam James P.
Published: November 2023

Small molecules and proteins represent the two major groups of compounds that are currently approved for human use. In between the molecular size of these two groups are the constrained peptides, a class of compounds that integrate their drug‐like advantages. Constrained peptides share the advantage of proteins for high on‐target specificity and low off‐target adverse side effects. They also have the robustness of small molecules to tolerate thermal, chemical and enzymatic degradation. Currently, naturally‐occurring constrained peptides in plants is an underexplored chemical space for drug discovery. Here we report the discovery and characterization of Lybatides, a constrained peptides with unusual cysteine motif and disulfide connectivity from the root bark of Lycium babarum, the plant that produces the popular functional food and herb, wolfberries. Importantly, an X‐ray crystallographic study revealed the presence of a helix as a dominant feature in the lybatide structure, which is stabilized by four cystine bonds. The presence of a single helix without stabilization by another helix or β‐strand is most unusual and has not been reported. As such, the lybatide structure resembles a naturally‐occurring stapled helical peptide. Lybatides are non‐cytotoxic, up to 100 μM, to the cell lines tested and are stable against thermal, acidic and proteolytic denaturation for up to 2 hours. The presence of the helix in the lybatide scaffold should allow the grafting of helical bioactive peptides for developing peptidyl therapeutics. Overall, this study provides a new scaffold and expands the number of existing constrained peptide scaffolds for drug design.

Support or Funding Information

This research was supported by a Competitive Research Grant from the National Research Foundation in Singapore (NRF‐CRP8‐2011‐05) and an NTU iFood Research Grant (M4081467.080).
Profiling and Authentication of Herbal Products
Authors: Huang Jiayi , Wong Ka Ho , Tam James P
Published: November 2023

Traditional Chinese medicine (TCM) is gaining popularity as an alternative medicine. A central element of TCM is the use of herbal medicine, which could be benefited by a novel authentication method. Astragalus membranaceus, belongs to the Fabaceae family, is a flowering plant native to China. Its root, commonly known as Huang Qi, is a traditional Chinese medicine used for treating diabetes, promoting Qi and boosting immune system. However, there is another medicinal herb, Hedysarum polybotrys, which also belongs to Fabaceae family, sharing the similar Chinese name (Hong Qi) and morphological characteristics with A. membranaceus, making it difficult to differentiate these two species. Here, we report the use of hyper‐constrained, heat‐stable and enzyme‐resistant peptides as a biologic fingerprint for the identification and authentication of herbal medicines. In particular, we use proteomics fingerprints to differentiate 20 samples from each species profiled by MALDI‐TOF MS analysis. Our results showed that A. membranaceus and H. polybotrys are divided into two clusters in a scores plot, suggesting that these two species had distinct proteomic profiles and can be successfully distinguished from each other. Together, the ability to authenticate provides confidence for customers to use herbal medicine and in TCM treatments.

Support or Funding Information

Acknowledgements

This research was supported by a Competitive Grant from the National Research Foundation in Singapore (NRF‐CRP8‐2011‐05) and an NTU iFood Research Grant (M4081467.080)
Bioorthogonal Protein Conjugation: Application for the Development of a Highly Sensitive Bioluminescent Immunoassay for the Detection of Interferon‐γ
Authors: Moutsiopoulou Angeliki , Hunt Eric , Broyles David , Pereira Christie Ataides , Woodward Kristen , Dikici Emre , Kaifer Angel , Daunert Sylvia , Deo Sapna
Published: November 2023

Interferon‐γ (IFN‐γ) is a 144‐amino acid long N‐terminal methionylated recombinant protein and is a potent multifunctional cytokine which is secreted primarily by activated NK cells and T cells. Originally came to attention due to its anti‐viral activities, IFN‐γ can also act as an anti‐proliferative, and immunoregulator, as well as a proinflammatory compound. A continuous production of the IFN‐γ along with other inflammatory cytokines is triggered in order to activate the immune system when the virus such as the HIV infects the immune system. Therefore, IFN‐γ is an important biomarker for monitoring infections.

The hypothesis for this study is that a bioluminescence‐based immunoassay would provide for highly sensitive detection of IFN‐γ due to high signal‐to‐noise ratio obtained with bioluminescence signal. For this purpose, monoclonal anti‐IFN‐γ antibody was conjugated to a mutant of bioluminescent protein, Gaussia luciferase (GLuc) containing a tyrosine at the N‐terminus. The bioconjugation was achieved through orthogonal chemical conjugation at the N‐terminal tyrosine. It has been demonstrated that the Formylbenzene Diazonium Hexafluorophosphate (FBDP) reagent provides bioorthogonal aldehyde functionality and reacts specifically with the ortho position of the phenol group of the tyrosine residue. The conjugation was efficient and retained the functionalities of both the antibody and the luciferase.

A sandwich‐type immunoassay was developed assay and validated for its accuracy and precision. A detection limit of 10−12 M was achieved for the detection of IFN‐γ. We also demonstrated the applicability of the assay for the detection of IFN‐γ in biological fluids such as serum samples.

Support or Funding Information

NIGMS funding (R01GM047915)
Role of albumin on the cellular uptake and selective autophagy of nanodiamonds
Authors: Lawrence Emmanuel Naveen Raj , Chao Jui‐I
Published: November 2023

Albumin is a versatile protein carrier for targeted drug delivery in cancer therapy. Selective autophagy plays a vital role in the targeted recognition and degradation of the cellular components and foreign pathogens in human cells. Nanodiamonds (NDs) are very promising fluorophores used for theranostic applications due to their biocompatible and photostable properties. Recent studies have demonstrated the cellular uptake of nanoparticles, but their interaction with proteins and intracellular trafficking through the selective autophagy pathway are poorly investigated. In our previous study, we found that Ub (ubiquitin) coated NDs bind to autophagy receptors, including SQSTM1 (sequestosome 1), OPTN (optineurin), and CALCOCO2/NDP52 (calcium binding and coiled‐coil domain 2) and are then linked to MAP1LC3/LC3 (microtubule‐associated protein 1 light chain 3) for entry into the selective autophagy pathway. Here, we show the role of albumin on the cellular uptake and intracellular trafficking of ND‐coated with bovine serum albumin (BSA) or human serum albumin (HSA) through the selective autophagy pathway. We observed that EGFR, clathrin and autophagy proteins expression on the influence of ND's endocytosis and uptake ability of lung cancer cells incubated with serum‐free medium. In addition, the existence of serum albumin in culture medium may decrease the uptake ability of NDs. The binding affinity of ND‐BSA and ND‐HSA was analyzed by SDS‐PAGE. The cellular uptake ability and delivery to the lysosomes were decreased using BSA or HSA coated on NDs. Interestingly, NDs coated with albumin bind to the autophagy receptors and delivered to the lysosomes. Taken together, these findings provide a fundamental understanding for the role of albumin on the cellular uptake and selective autophagy pathway of NDs, which may illustrate the cellular interactions of nanoparticle and improve the therapeutic effect of nanomaterials.
Association Between Women with Endometriosis and Human Leukocyte Antigen‐C Genotyping
Authors: Chou Ya‐Ching , Tzeng Chii‐Ruey
Published: November 2023

Study objective

Endometriosis is a frequent benign gynecological disorder that affects 6%–10% of fertile women and causes severe pelvic pain, suffering, infertility, and hysterectomy. The etiology of endometriosis remains unclear and several evidence suggest cell‐mediated and humoral immunity are involved in its pathogenesis. HLA‐C antigen expression is low and only a minority of immunodominant cytotoxic CD8+ T cells response are restrict by HLA‐C. Moreover, HLA‐C has been regarded to act as a ligand for killer immunoglobulin receptors expressed in natural killer cells and inhibit NK cells. Previous studies have showed that HLA‐C affects viral infection and has been implicated in several human diseases. The aim of this study is to investigate the human leukocyte antigen‐C genotype in women with endometriosis.

Method

A total of 52 endometriosis and 47 non‐endometriosis women were diagnosed by laparoscopy and typed for the HLA‐C antigen. The total DNA were extracted from blood and analyzed HLA‐C alleles through sequence based typing methodology.

Results

The prevalence of HLA‐C*0304 in endometriosis group was 18.3% (19/104 alleles), compared with 6.4% in non‐endometriosis group (6/94 alleles). Thus, the prevalence of HLA‐C*0304 in endometriosis women was significantly higher than non‐endometriosis [OR 3.278, 95% CI=1.249 to 8.607, p= 0.0119 (χ2 analysis)].

Conclusion

The result may be useful to implicate the role of HLA‐C antigens and NK cells in the pathogenesis of endometriosis.
Effect of Outer Hair Cell‐Specific STAT3 Deletion in the Noise‐induced Cellular Stress Response
Authors: Dziennis Suzan , Wilson Teresa L , Foster Sarah L , Nuttall Alfred L
Published: November 2023

Background

Signal transducer and activator of transcription 3 (STAT3) is part of Janus kinase/signal transducer and activator of transcription (JAK/STAT) signaling pathway. STAT3 is activated by phosphorylation and translocates to the nucleus in response to inflammatory cytokines to induce a transcriptional response. Non‐canonical actions of STAT3 are becoming elucidated such as those effected in the cytoplasm and mitochondria. Loud sound exposure induces several stress responses including increased reactive oxygen species (ROS) which contribute to noise‐induced cochlear tissue damage. We previously reported the general JAK2/STAT3 inhibitor JSI‐124 reduced levels of ROS after noise exposure suggesting STAT3 contributes to noise‐induced cellular damage.

Objective

Here, we generated outer hair cell (OHC)‐specific STAT3 knock‐out mice to better understand the function of STAT3 in OHCs in response to cellular stress pathways induced after loud sound exposure.

Methods and Results

Floxed STAT3 mice were crossed with prestin‐CreERT2 mice for several generations to produce STAT3fl/fl × prestin‐CreERT2+/− (STAT3fl/fl) mice. Tamoxifen treatment resulted in STAT3 deletion specifically in OHCs (STAT3OHCΔ/Δ). The OHCs of 8 week old STAT3OHCΔ/Δ mice appeared morphologically normal and a full complement of OHCs was present. Ex vivo cochlea from STAT3fl/fl and STAT3OHCΔ/Δ mice were treated with IL‐6, antimycin A or corresponding vehicle followed by addition of the ROS detector CellROX. Nuclei were stained with Hoechst. IL‐6 or antimycin A treatment resulted in contrasting effects on nuclear morphology and ROS based on the loss of OHC‐specific STAT3, demonstrating the diverse contribution of STAT3 in the noise‐induced cellular stress response.

Conclusions

STAT3 either prevents or contributes to detrimental cellular stress pathways in OHCs. This may be due to multiple and diverse activities of STAT3 including both genomic actions as a transcription factor and non‐genomic actions such as interaction with the mitochondrial electron transport chain.

Support or Funding Information

Supported by grants 5R01DC000105 (ALN) and P30DC005983.
CD95‐Ligand from Myeloid Cells Contributes to Abdominal Aortic Aneurysm Formation
Authors: Wiong Wanfen , Meisinger Trevor , Fitzgerald Matthew , Batra Rishi , Baxter B. Timothy
Published: November 2023

Abdominal aortic aneurysm (AAA) is one of a number of diseases associated with a prominent inflammatory cell infiltration, matrix protein degradation, and smooth muscle cell apoptosis. This chronic infiltrate is predominantly composed of macrophages and T lymphocytes. Active T cells produce a variety of cytokines, including CD95‐ligand (CD95L). Elevated expression of CD95L in the aortic tissue was observed in patients with AAA, suggesting that CD95L may play a role in the pathogenic mechanism of AAA. The purpose of this study was to investigate the role of CD95L in AAA formation. By studying a murine model of aneurysm, we found that both mRNA and protein levels of CD95L were increased in aneurysm tissue compared with normal aortic tissue. We further tested the response of mice lacking expression of CD95L to aneurysm formation. These mice were resistant to aneurysm formation. Our results show that CD95L deficiency attenuates macrophage and T cell infiltration into the aortic tissue. To study the role of CD95L in the myelogeous cells in AAA formation, we created chimeric mice and showed that myelogeous cell‐derived CD95L plays a dominant role in disease progression. These studies demonstrated that inflammatory cell infiltration during AAA formation is dependent on myelogeous cell‐derived CD95L.

Support or Funding Information

R01HL130623 (To W. X) and R01HL062400 (To B.T.B)
Carbon Monoxide as a Mediator of Ca2+ Signaling for Insulin Secretion in Pancreatic Islets
Authors: Rahman Faiz Ur , Kim Uh‐Hyun
Published: November 2023

Glucagon‐like peptide‐1 (GLP‐1) increases intracellular Ca2+ concentrations ([Ca2+]i), resulting in insulin secretion from pancreatic β‐cells through the sequential production of Ca2+ mobilizing second messengers: nicotinic acid adenine dinucleotide phosphate (NAADP) and cyclic ADP‐ribose (cADPR). Ca2+ signals and gasotransmitter formation are closely related in maintaining cellular signaling function, including insulin secretion from pancreatic β‐cells. However, the interrelationship between Ca2+ signals and gas formation is poorly understood. Previously, we demonstrated that NAADP activates the neuronal type of nitric oxide (NO) synthase (nNOS) during the stimulation of insulin secretion in β‐cells. NO activates guanylyl cyclase (GC) to produce cyclic GMP (cGMP), which in turn induces cADPR formation. We investigated the influence of GLP‐1/NAADP on nNOS activity. NAADP regulates nNOS enzyme activity by increasing CaMKII phosphorylation levels, which was abolished by pre‐treatment with a CaMKII inhibitor (AIP). We then studied the effects of NO and carbon monoxide (CO) on cGMP formation and Ca2+ signals by using a NO donor (SNP) and a CO donor (CORM‐3). Exogenous NO and CO amplify cGMP formation, Ca2+ signal strength, and insulin secretion in mouse islets. However, this signal is impeded when exposed to combined treatment with NO and CO. Furthermore, CO potentiates cGMP formation in a dose‐dependent manner in the range of 10–100 μM, whereas higher doses of CORM‐3 inhibited cGMP formation, suggesting that high CO concentrations block GC activity. Furthermore, our data with regards to ZnPP, a HO inhibitor, and heme oxygenase (HO)‐2 knockdown revealed that NO‐induced cADPR formation and insulin secretion is dependent on HO. HO‐2 knockdown or ZnPP blocked NO‐induced cGMP formation and insulin secretion in pancreatic islets, whereas exogenous CO repaired the blockade. These results demonstrate that CO, a downstream regulator of NO, plays a role in bridging the gap between the Ca2+ signaling second messengers.
C‐Terminal 15kDa Fragment from β‐Actin Causes Apoptotic Cell Death
Authors: Ito Nanako , Tone Shigenobu , Tanaka Masato
Published: November 2023

Truncated 15kDa‐β‐actin fragment was originally detected in apoptotic cancer cells treated with etoposide and its over‐expression of the fragment induced apoptotic morphological changes (Mashima, T. et al. Oncogene 18, 2423–2430, 1999). Our final goal is to reveal how the 15kDa‐β‐actin fragment induces the apoptotic morphological changes. To distinguish the transfected cells from non‐transfected ones, we have developed a new expression vector in which two coding sequences are able to be simultaneously expressed in a mammalian cell. This vector enabled to express a fluorescent protein with the 15kDa‐β‐actin in an identical cell. Using these co‐expression plasmids, we have investigated the effects of the 15kDa‐β‐actin on the apoptotic processes of HEK293, COS‐7 HeLa S3, and Jurkat cells.

The over‐expression of 15kDa‐β‐actin fragment was seemed to cause the loss of adhesive properties of COS‐7 cells onto culture dish, since the GFP‐labeled cells were observed to detach from dishes by losing the adhesion ability. The numbers of detached cells from HEK293, COS‐7 and HeLa S3 transfected with the 15kDa‐β‐actin cDNA were determined by counting chamber. The numbers of detached cells were significantly increased by transfection of 15kDa‐β‐actin cDNA, but not by any mock transfections. To examine the relationship between the detachment and cell death, the detached cells were stained with Hoechst33342 and NucView488 caspase‐3 vital staining. The almost detached cells showed caspase‐3 activation and nuclear condensation in HEK293, COS‐7 and HeLa S3 cells. The cell death is not seemed to result from anoikis, since the transfection of the 15kDa‐β‐actin cDNA gave the similar effects on Jurkat cells. We also confirmed that the DNA fragmentations in HEK293, COS‐7, HeLa S3 and Jurkat cells by TUNEL assay.

Cytoskeletal β‐actin has three targets of caspase‐3 motif but β‐actin has been so far considered to be a poor target of caspase‐3. We showed that the 15kDa‐β‐actin C‐terminal fragment actually activated caspase‐3. These results suggest that the cleaved molecule of cytoskeletal β‐actin is not an innocent bystander but an apoptogeneic inducer. Furthermore, we found that an actin binding protein, profilin suppressed the cell death by either 15kDa‐β‐actin or etoposide, VP‐16. The cleavage of cytoskeletal β‐actin would play a key role in the process of apoptotic cell death.
Paspalum scrobiculatum polyphenol rich extracts inhibit human cervical cancer cell proliferation and induce apoptosis in vitro
Authors: Ramasamy Sakthivel , Anupam K , Ratnavathi C.V , Karunagaran D , Chandra T.S
Published: November 2023

Natural products and their derivatives have historically been a major source of new pharmaceuticals. Many dietary derivatives act as an effective anticancer agents such as curcumin, resveratrol and tea polyphenols which selectively inhibits the growth of malignant cells by inducing apoptosis. Cereals are valuable source of bioactive compounds which are found to be very effective in preventing and treating several diseases. Paspalum scrobiculatum is a type of small seeded grain (Kodo Millet) mainly consumed by India and also in Indonesia, Philippines, Thailand, Vietnam and in Africa. It has found its use in traditional medicines against inflammatory diseases, diabetes and several other diseases. In the present study, polyphenol‐rich extract (PEKM) of kodo millet was evaluated for its ability to inhibit the growth of human cervical cancer line (HeLa), at concentrations ranging from 100–500μg/ml. With increasing concentration of PEKM, a decrease in cell proliferation was observed in HeLa cell lines. The concentrations needed for 50% growth inhibition (IC50) were found to be 212μg/ml and 178μg/ml for Kodo Millet Market (KMM) and Kodo Millet Vamban (KMV), respectively. The growth inhibition by PEKM was accompanied by membrane blebbing, appearance of apoptotic bodies, loss of mitochondrial membrane potential, increased production of reactive oxygen species and DNA damage, typically seen during apoptosis. In addition, it activated caspase‐9, decreased the expression of Bcl‐2 and induced poly ADP‐ribosome polymerase (PARP) cleavage. The phytochemical profiling of PEKM by HPLC revealed the presence of phenolic acids like quercetin and cinnamic acid in it. These results suggest that PEKM possesses the ability to inhibit cell growth in a dose dependent manner and could stimulate apoptosis in cancer cells.

Support or Funding Information

Indian Council of Agricultural Research (ICAR) and Indian Institute of Millets Research (IIMR), Govt. of India, New Delhi ‐110001
Role of Primary Cilia in Cell Migration
Authors: Ganesh Jyothsna , Miskevich Frank
Published: November 2023

Cellular migration is a common activity for many different types of cells. If cellular migration is disrupted, cells may be unable to repair damaged tissue (wound repair), migrate appropriately as in cancer, or correctly signal their cellular state. In several situations, cilia have been implicated in helping to determine the direction of cellular migration. As primary cilia express a number of distinct receptors for particular hormones, these organelles have the required machinery to influence cellular behavior. We are using ARPE‐19 cells, a human retinal pigment epithelial cell line capable of forming cilia in culture, as an assay for cellular motility. Using a 3D model of cellular migration (matrigel invasion chambers) and a 2D scratch assay for cellular motility, we are testing various reagents for their ability to alter RPE cell motility. Preliminary results suggest that a number of agents which alter ciliary function, particularly protein kinase A modulators, are able to inhibit the migratory ability of ARPE cells. These results suggest that protein kinase A may be a key link between ciliary signaling and the direction of cellular migration.

Support or Funding Information

University of Michigan Graduate Student Research Assistantship; University Research and Creative Activities grant
Prolyl isomerase Pin1 regulates the stability, transcriptional activity and oncogenic potential of Brd4
Authors: Hu Xiangming
Published: November 2023

The multi‐functional Brd4 belongs to the two tandem bromodomains and an extra terminal (ET) domain‐containing family proteins (BETs), which have emerged as important epigenetic modulators for gene transcription and cancer development. Brd4 has been implicated in the development of hematological malignancies, including mixed‐lineage leukemia (MLL), multiple myeloma, and adult T cell leukemia. While accumulating evidence indicates that Brd4 has a pivotal role in the tumorigenesis by activating the genes responsible for cell proliferation and survival, how Brd4 itself is regulated in cancer remains elusive.

In this study, we report that the stability and functions of Brd4 are positively regulated by prolyl‐isomerase Pin1. Pin1 directly binds to phosphorylated Brd4 and enhances Brd4's stability by inhibiting its ubiquitination. Pin1 also catalyzes the isomerization of Brd4 and induces its conformational change, which promotes its interaction with CDK9 and increases Brd4's transcriptional activity. Substitution of Brd4 with Pin1 binding‐defective mutant reduces Brd4's stability, attenuates Brd4‐mediated gene expression by impairing its interaction with CDK9, and suppresses cancer cell proliferation, migration and invasion, and tumor formation.

Our results identify Brd4 as a new target of Pin1 and suggest that interfering with their interaction could be a potential therapeutic approach for cancer treatment.

Support or Funding Information

UIUC (to L.F.C.) and NIH grants DK085158 and CA179511 (to L.F.C.)
Characterization of human SH2 domain‐containing protein 4A (SH2D4A) in HeLa cells
Authors: Tamborlin Leticia , Pereira Karina Danielle , Meneguello Leticia , de Proença Andre Ricardo Gomes , Luchessi Augusto Ducati
Published: November 2023

The SH2 domain‐containing protein 4A (SH2D4A) has a single Src homology region 2 domain (SH2 domain) and a proline‐rich stretch on its C‐terminal. In humans, SH2D4A gene is located on chromosome 8p close to other genes associated with cancer. This region is deleted in patients with hepatocellular carcinoma (HCC) with poor outcomes, suggesting that depletion of SH2D4A could be involved with tumor progression. Besides, SH2D4A inhibits the STAT3 transcription factor activity mediated by interleukin 6 (IL‐6) signaling pathway, which interacts physically with STAT3, inhibiting the transcription of genes involved with cell proliferation, survival, and tumor development. Five transcripts corresponding to SH2D4A gene are described (variants 1, 2, 3, X1 and X2). The transcript variants 1 and 2 are predicted to encode the same isoform (isoform A), which has 454 amino acids and is considered canonical. The transcript variant 1 presents an upstream Open Reading Frame (uORF). Thus, this study aimed to characterize SH2D4A gene and protein in a model of tumor cells (HeLa cells), looking for new evidences to evaluate the involvement of this protein in development and tumor progression. First of all, we performed amplification of the cDNA by RT‐PCR and subsequent cloning. Thereby, it was possible to identify the transcript variants 1, 2 and X1 of SH2D4A, which was confirmed by sequencing of the samples. Analyzes of the sequenced samples revealed some mutations in SH2D4A gene, among them, the most significant was observed in the start codon, which results in the use of an alternative start codon to production of the protein. In addition, the presence of the uORF in SH2D4A variant 1 downregulates the protein production from transcript variant 1 in transfected cells. In fact, a depletion of the uORF increased the protein content compared to original variant 1. Our results suggest these mutations in SH2D4A gene and the presence of the uORF in transcript variant 1 may contribute to the abnormal characteristics observed in this type of cells.

Support or Funding Information

São Paulo Research Foundation (FAPESP)
LPIN1 promotes epithelial cell transformation and mammary tumourigenesis via enhancing insulin receptor substrate 1 stability
Authors: Choi Hong Seok
Published: November 2023

LPIN1, a phosphatidic acid phosphatase enzyme, dramatically influence tumor progression, however, their mechanism of action is still poorly understood. Here, we demonstrate that LPIN1 enhances the tumour‐promoting function of IRS1 by controlling IRS1 stability. LPIN1 interacts with IRS1 in an IGF‐1‐dependent signalling pathway and inhibits its serine phosphorylation, thereby eliminating ubiquitin‐dependent degradation of IRS1 via proteasomal and lysosomal pathways. Consequently, LPIN1 overexpression increases IRS1 abundance and enhances IRS1's ability to induce epithelial cell proliferation and mammary tumourigenesis. By contrast, depletion or inhibition of LPIN1 in breast cancer cells leads to a decreased IRS1 level, which subsequently inhibits the RAF1‐mediated signalling pathway as well as AP‐1 transcriptional activity. In the syngeneic 4T1 breast cancer model, LPIN1 overexpression increased tumour development. Consistent with these observations, LPIN1 levels were positively correlated with IRS1 expression in human breast cancer. Thus, our results indicate that LPIN1 may be a promising drug target for anticancer therapy.

Support or Funding Information

This research was supported by a grant of the Korea Health Technology R&D Project through the Korea Health Industry Development Institute, funded by the Ministry of Health & Welfare, Republic of Korea (grant number: HI14C2004).