bluuebunny/crossref_metadata_2025_split huggingface dataset:
Mutagenesis and Steady State Kinetic Characterization of a Class B Metallo‐β‐lactamase
Authors: Baker Julia P. , Eberhardt Eric 
Published: November 2023

Antibiotic resistance is of growing concern for the medical community worldwide due to the overuse of common antibiotics that has created an opportunity for bacteria to evolve novel mechanisms for resistance to antibiotics. Specifically, bacteria that express enzymes that inactivate β‐lactams prevent the antibiotics from inhibiting cell wall biosynthesis in bacteria.Class B β‐lactamases, metallo‐β‐lactamases, are of particular concern. These metal dependent enzymes are resistant to almost all classes of β‐lactams, including carbapenems, which have the broadest spectrum of activity. One such enzyme is β‐lactamase 2 [Bc(II)] from Bacillus cereus, a mono‐ or di‐zinc dependent metallo‐β‐lactamase. The amide bond hydrolysis of β‐lactams by Bc(II) is likely facilitated by a zinc(II)‐bound water and the amino acid residue aspartate‐90, which is thought to participate in general acid base catalysis. Bicknell and coworkers suggest that the metal acts as a Lewis Acid and binds a hydroxide ion that carries out a direct nucleophilic attack on the β‐lactam carbonyl. The metal is suggested to stabilize the resulting tetrahedral intermediate, allowing for the carboxylate anion of aspartate‐90 to deprotonate the metal‐bound hydroxide and then protonate the amine nitrogen. To more thoroughly understand the role of aspartate‐90, the steady state kinetics of wildtype, aspartate‐90‐asparagine, and aspartate‐90‐alanine mutant are being evaluated.
Topoisomerase II Inhibition by BZP Thiosemicarbazones
Authors: Stephens Madison Taylor , Angulo Yocelyne L. , Eatebarian Roya R. , Medawala Wathsala , Morris William , Deweese Joseph E. , Lisic Edward , Jiang Xiaohua 
Published: November 2023

Topoisomerase II (TopoII) regulates DNA topology during DNA metabolism and is active in replicating cells. Thus, it is the target of several FDA approved anticancer drugs such as Etoposide. Current topoisomerase II inhibiting drugs do not specifically target the alpha form of the enzyme, affecting normal cells and causing serious health problems. There has been evidence that α‐(N)‐heterocyclic thiosemicarbazone (TSC) compounds have demonstrated antiproliferation effects on tumor lines. Our experiment investigates the inhibition of specific BZP thiosemicarbazone compounds on TopoIIα and stimulation of the cleavage complex of this enzyme. Metal‐free as well as Cu(II) and Pd(II) complexes of BZP derivatives of TSCs were examined against the full TopoII relaxation and its ability to create double strand breaks. The Cu(II) complexes showed the highest inhibitory activity against the enzyme and highest stimulation of the cleavage complexes in the absence of ATP. Among the Cu(II) complexes of BZP derivatives, Cu(BZP‐tBTSC)Cl exhibited a significantly high increase in the cleavage complex of TopoIIα compared with Etoposide. In addition, DTT abolished the effect of BZP TSCs on TopoIIα The mechanism of inhibition is not clear, but seems to be different from that of Etoposide.Support or Funding InformationResearch supported by Tennessee Technological University through Undergraduate Research and Creative Activity (URECA) grants.
Structural and Functional Characterization of the Sco Protein from Thermus thermophilus
Authors: Lopez Liezelle , Hunsicker‐Wang Laura 
Published: November 2023

The Sco family of proteins has been implicated in the assembly of cytochrome c oxidase (Complex IV) of the respiratory electron transport chain, but its function is still under debate. TtSco has been shown to bind to Cu (I) and Cu (II) ions. However, it is also structurally similar to thioredoxins and has been shown to be able to reduce the disulfide bond present in the CuA site of cytochrome c oxidase prior to copper insertion. To provide evidence of Sco's role in the assembly of the CuA‐center, single cysteine to serine mutants, TtSco‐C53S and TtCuA‐C149S, were first produced. TtCuA was conjugated to a chromophore that is released upon reaction with TtSco. The two proteins were reacted and monitored via UV‐Visible spectroscopy in a 1:1 ratio at 412 nm to monitor formation of a mixed disulfide intermediate (MDI) between the two. TtSco and TtCuA have been reacted to form the MDI at different stoichiometric ratios and to probe the kinetics of formation of the MDI. Our results indicate much higher production of MDI with Sco in excess. Our second aim is to crystallize the mixed disulfide intermediate to visualize and better understand the interactions between TtSco and TtCuA. Cation exchange chromatography has shown the most success in separating MDI from unreacted starting materials. A successful condition for crystallization of the MDI has been found and consists of ammonium sulfate and polyethylene glycol 8,000. Lastly, the effects of copper binding on the relative stability of apo and holo forms of TtSco have been studied via chemical denaturation. Interestingly, preliminary data suggests that holo‐TtSco is slightly less stable than apo‐TtSco. Further studies aim to investigate the effect of the copper‐ligating His139 on stability of the protein, both with and without metal bound to gain more insight into TtSco's ability to bind metals and the role metal binding plays in its function.Support or Funding InformationWelch Foundation
A New Oridonin Analogue Suppresses Triple‐Negative Breast Cancer Cells and Tumor Growth via Inducing the Expression of Death Receptor 5
Authors: Chen Ceshi , Wu Jing , Zhou Jia 
Published: November 2023

Triple‐negative breast cancer (TNBC) remains the leading cause of death among women with breast cancer worldwide. Oridonin is a natural anti‐cancer compound isolated from the traditional Chinese herb Rabdosia rubescens. However, the antitumor efficacy of oridonin for TNBC and other cancers is far away from ideal. In this study, we investigated a series of newly designed oridonin analogues against HCC1806 and HCC1937 TNBC cell lines and identified CYD‐6‐28 that significantly inhibits cancer cell proliferation, induces G2/M phase cell cycle arrests and apoptosis. CYD‐6‐28 induces the expression of p21 and the cleavage of caspase‐8, −3, −7 and PARP, and inhibits the expression of Cyclin D1, FLIP and XIAP. CYD‐6‐28 also inhibits the activation of STAT3 and AKT while inducing the activation of ERK. We demonstrated that CYD‐6‐28 induces apoptosis at least partially through inducing the expression of Death Receptor 5 (DR5). Finally, CYD‐6‐28 significantly suppresses HCC1806 xenograft tumor growth in nude mice at 5 mg/kg without significant toxicity. Taken together, CYD‐6‐28 has the potential to be developed as a promising therapeutic agent to treat TNBC.Support or Funding InformationThis work was supported by the National Natural Science Foundation of China (81120108019, U1132605, and 81325016 to Chen, C), and the grants P30 DA028821 and R01 DA038446 from the National Institutes of Health, and Cancer Prevention Research Institute of Texas (CPRIT) award (to Zhou, J).
The First Example of Allosteric Partial Inhibition of a Monomeric Protease
Authors: Verespy Stephen , Mehta Akul Y. , Afosah Daniel , Al‐Horani Rami A. , Desai Umesh R. 
Published: November 2023

PurposeAllosteric, partial inhibition of monomeric proteases, like thrombin, has not been achieved previously, but may offer a regulatory mechanism for maintaining homeostasis of diseased systems. We therefore sought to identify and study novel agents that induce allosteric, partial inhibition of thrombin.MethodsA library of 36 sulfated coumarins was synthesized and screened against thrombin, factor Xa, and factor XIa; proteases of the coagulation cascade. Eleven were selected to establish their half maximal inhibitory concentration (IC50) and efficacy of inhibition (ΔY). To study binding interactions, the most active compound was chosen to assess binding affinity, Michaelis‐Menten kinetics, competition studies, and recombinant mutant studies. To assess conformational change in and around the active site, fluorescence quenching studies and chemical kinetic studies using the thrombin‐antithrombin system were performed. Assays probing the inhibitors ability to induce partial inhibition of thrombin's endogenous substrates was performed using hydrolysis assays and UPLC‐MS monitoring.ResultsThe IC50s of ten thrombin‐selective compounds ranged from 0.19–58 μM, with ΔY values ranging from 22–73%, indicating that this scaffold induced submaximal activity of thrombin; see Figure. Compound 3g (IC50 = 187 ± 40 nM; ΔY ~47%; 150‐fold selective for thrombin) was found to have a KD of 143 nM. Kinetic studies showed a decrease in the VMAX and no change in the KM, indicating a non‐competitive or allosteric mechanism. 3g was found to compete ideally with heparin for exosite 2 of thrombin, and not compete with hirudin peptide for exosite 1. Thrombin mutant studies suggested a region of binding, but did not identify a unique residue responsible for interaction, indicating the sulfate may engage multiple basic residues. Quenching studies produced Stern‐Volmer relationships showing 3g caused a 49% change in the active site, while BF caused a 71% change; within standard error to their observed ΔY values. Chemical kinetic studies showed that 3g slows the thrombin‐antithrombin complex formation by ~12‐fold, suggesting it induced a conformational change to the active site serine. 3g was found to inhibit thrombin catalyzed fibrin formation with an IC50‐ of 514 nM, but with a residual activity of 94%.ConclusionThis is the first time a phenomenon of allosteric, partial inhibition has been observed with any monomeric protease. Similar to partial agonism of receptors and oligomeric proteins, this work represents the first group of probes found to induce submaximal activity with relatively high potency against thrombin. They will be useful as powerful tools in developing future anticoagulants with higher potencies combined with the possibility of little‐to‐no risk of bleeding events; which may offer a major advantage over clinically used thrombin inhibitors.Support or Funding InformationFinancial support from the NIH in the form of R01 HL090586, HL125639 and P01 HL107152
MAGP2 binding to αvβ3 integrins affects levels of microfibril formation
Authors: Daniels Ryan , Valdivia Michael , Perez Andrew , Miyamoto Alison 
Published: November 2023

Matrix metalloproteases (MMPs) are secreted proteins that degrade extracellular matrix (ECM) proteins and are thought to play a major role in cancer cell motility, invasion, and angiogenesis. Activation of αvβ3 integrins has been shown to increase expression or activation of MMPs. Microfibril‐associated glycoprotein 2 (MAGP2) is a small secreted protein that binds to microfibrils in the ECM, and overexpression of MAGP2 in human papillary serous ovarian carcinomas correlates to poor prognosis. MAGP2 has an RGD integrin binding motif in the amino‐terminal half (NT). This motif was shown to be necessary to stimulate cell motility in an an αvβ3 integrin dependent manner. The carboxy‐terminal half of MAGP2 contains a matrix binding domain that is necessary and sufficient for MAGP2 to associate with microfibrils. Direct transfection of a construct expressing the NT‐MAGP2 shows a decrease in extracellular fibers via immunocytochemistry (ICC). This construct can bind to αvβ3 integrins but not microfibrils, suggesting that this effect is mediated by the MAGP2 RGD integrin binding motif. Therefore, we hypothesize that NT‐MAGP2 binding to αvβ3 integrin promotes the activity of MMPs to degrade the ECM in order for the cell to be motile during metastasis. To test the requirement for the RGD motif two mutant constructs were generated in the RGD integrin binding motif of the NT‐MAGP2 construct with a conservative (RGE) and non‐conservative (RGA) mutation to assess levels of cell‐associated MAGP2 and microfibril levels via ICC. As expected, the wild type NT‐MAGP2 construct had the most cell association and greatest loss of microfibrils, followed by the RGE mutant, and lastly the RGA mutant. These preliminary results suggest that there is an inverse relationship between amount of cell associated MAGP2 and amount of microfibrils present. Current studies are testing whether the NT‐MAGP2 treatment increases MMP activity via zymography. These studies could provide insights for possible anti‐MAGP2 treatments of ovarian cancer.Support or Funding InformationHoward Hughes Medical School Two‐Year Undergraduate Research Scholars Programs (RD), California Institute of Regenerative Medicine Bridges to Stem Cell Research Program (MV, AP), Department of Biological Science, CSUF (AM).
Small fungal molecule metabolites targeting mosquito midgut FREP1 block malaria transmission
Authors: Li Jun 
Published: November 2023

Malaria transmission depends on sexual stage Plasmodium parasites successfully invading Anopheline mosquito midguts following a blood meal. However, the molecular mechanisms of Plasmodium invasion of mosquito midguts have not been fully elucidated. Previously, we showed that genetic polymorphisms in the fibrinogen‐related protein 1 (FREP1) gene are significantly associated with P. falciparum infection in An. gambiae, and FREP1 is important for P. berghei infection of mosquitoes. Here we identify that the FREP1 protein is secreted from the mosquito midgut epithelium and integrated as tetramers into the peritrophic matrix, a chitinous matrix formed inside the midgut lumen after a bloodmeal feeding. Moreover, we show that the FREP1 can directly bind Plasmodia sexual stage gametocytes and ookinetes. Notably, ablating FREP1 expression or targeting FREP1 with antibodies significantly decreases P. falciparum infection in mosquito midguts. Our data support that the mosquito‐expressed FREP1 mediates mosquito midgut invasion by multiple species of Plasmodium parasites via anchoring ookinetes to the peritrophic matrix and enabling parasites to penetrate the peritrophic matrix and the epithelium. Thus, targeting FREP1 can limit malaria transmission. To test the hypothesis that small molecules that disrupt this interaction will prevent parasites from infecting mosquitoes, we developed an ELISA‐based method to screen a fungal extract library. We obtained a candidate fungal extract of Aspergillus niger that inhibited the interaction between FREP1 and P. falciparum infected cells by about 92%. The inhibition specificity was confirmed by immunofluorescence assays. Notably, feeding mosquitoes with the candidate fungal extract significantly inhibited P. falciparum infection in the midgut without cytotoxicity or inhibition of the development of P. falciparum gametocytes or ookinetes. A bioactive natural product that prevents FREP1 from binding to gametocytes or ookinetes was isolated and identified as P‐orlandin. Importantly, the nontoxic orlandin significantly reduced P. falciparum infection intensity in mosquitoes. Therefore, disruption of the interaction between FREP1 and parasites effectively reduces Plasmodium infection in mosquitoes. Targeting FREP1 with small molecules is thus an effective novel approach to block malaria transmission.Support or Funding InformationOklahoma Center for Advancement of Science & Technology (HR13‐055), National Institute of Health (1R21AI11517801A1), and National Science Foundation Career Award (1453287)
Towards Understanding Cell Cycle Regulation in the Pathogenic Cryptococcus neoformans and Saccharomyces cerevisiae
Authors: Kawamoto Susumu , Virtudazo Eric , Ohkusu Misako , Suganami Akiko , Tamura Yutaka 
Published: November 2023

We have reported that the cell cycle behavior of the pathogenic yeast Cryptococcus neoformans (C. neoformans) is different from the cell cycle control exhibited by the model yeast Saccharomyces cerevisiae (S. cerevisiae), and also have reported the molecular characterization and physiological roles of the two main eukaryotic cell cycle genes, C. neoformans cyclin dependent kinase 1 (CnCdk1) and cyclin homologues. Only a single Cdk1‐related G1 and G1/S cyclin homologue was found in the genome sequence of C. neoformans and was designated CnCln1. Surprisingly, CnCln1 was not only able to complement the function of the G1 cyclins of S. cerevisiae, such as ScCln3, but also the G1/S cyclins of S. cerevisiae, such as ScCln1 and ScCln2. Our in silico analysis demonstrated that the CnCln1/ScCdk1 complex was more stable than any of S. cerevisiae cyclins (ScCln1, ScCln2, ScCln3) and ScCdk1 complexes. These results are consistent with in vitro analysis that has revealed the flexible functional capacity of CnCln1 as a Cdk1‐related G1 and G1/S cyclin of S. cerevisiae.On the other hand, Cln1 and Cln2, G1/S cyclins of S. cerevisiae, oscillate during the cell cycle, rising in late G1 and falling in early S phase. We have been tried to elucidate the structure basis of the functional distinction between Cln1 and Cln2. We performed in silico simulations: construction and evaluation of three dimensional structures of Cln1‐Cdc28 and Cln2‐Cdc28 complexes. Our in silico simulations suggested that the interaction of Cln1 and Cln2 with Cdc28 were in the two distinct situations, designated as flip and flop conformation, at the extra amino acid region in the cyclin box of Cln1 and Cln2. We speculated the trigger of this flip‐flop conversion of the extra amino acid region in the cyclin box of Cln1‐Cdc28 and Cln2‐Cdc28 might be regulated by the ubiquitination of the sequences rich in Pro (P), Glu (E), Ser (S) and Thr (T), so‐called PEST motifs, in Cln1 and Cln2. Furthermore, we presumed that the functional superiority between Cln1 and Cln2 in the G1/S phase of S. cerevisiae might be controlled by flip‐flop conversion and ubiquitin‐proteasome pathway.Support or Funding InformationThis work was partially supported by Grants‐in‐Aid for Scientific Research from the Ministry of Education, Science, Sports, and Culture of Japan and Cooperative Research Program of Medical Mycology Research Center, Chiba University.
Tellimagrandin I Decreases Cyclin B1 Protein Levels through SP1 in MDA‐MB‐231 Cells
Authors: White Brandon , Luis Tiani , Rodriguez y Baena Alessandra , Wang Laura , Shah Savita 
Published: November 2023

Consumption of walnuts has been shown to inhibit MDA‐MB‐231 breast cancer xenograft growth in nude mice. However, identification of compounds and mechanism of action have not been identified. We recently reported that a walnut extract treatment of the triple‐negative breast cancer cell type, MDA‐MB‐231 cells, could a) arrest the cells in G2/M and decreased cyclin B1 levels, b) decrease mitochondrial membrane potential and release cytochrome c from the mitochondria, and c) increase phosphotidyl serine flipping in the cell membrane, and that these events were associated with cell growth inhibition and apoptotic death. Over‐expression of Cyclin B1 has been shown in breast cancers that lack estrogen receptor, progesterone receptor, and HER2 (i.e. the triple‐negative breast cancer) and is a potential emerging biomarker as well as therapeutic target. In an attempt to conduct detailed mechanistic studies with active compounds from walnut extracts, we were able to identify numerous ellagitannins which induced cell death in breast cancer cells. Of the ellagitannins isolated, tellimagrandin I (TMI) showed the strongest cell death. We have gone on further to characterize the mechanism of action of TMI. We have performed time and dose response analysis of TMI on MDA‐MB‐231 cells for cell death and show that TMI has an IC50 of 60 μM. TMI is also able to reduce Cyclin B1 mRNA and protein levels similar to a walnut extract. We see that mRNA levels are reduced relatively quickly (within 1 hour) while protein levels begin to be reduced at a much later time suggesting that the effects are transcriptional. We have identified other cell cycle specific mRNAs that are also reduced by TMI. A careful examination of all mRNAs that are reduced show that they all have SP1 binding sites. Interestingly, we show that SP1 mRNA levels are also reduced upon TMI treatment in a similar fashion as Cyclin B1. Taken together our results suggest that TMI is able to reduce Cyclin B1 through inhibition of SP1 in MDA‐MB‐231 cells.Support or Funding InformationThis work was supported by a grant from the California Walnut Commission to BW.
Curcumin Inhibits Development and Cell Adhesion in Dictyostelium discoideum: Implications for YakA Signaling and GST Enzyme Function
Authors: Garige Mamatha , Walters Eric 
Published: November 2023

The molecular basis for nutraceutical properties of the polyphenol curcumin (Curcuma longa, Turmeric) is complex, affecting multiple factors that regulate cell signaling and homeostasis. Here, we report the effect of curcumin on cellular and developmental mechanisms in the eukaryotic model, Dictyostelium discoideum. Dictyostelium proliferation was inhibited in the presence of curcumin, which also suppressed the prestarvation marker, discoidin I, members of the yakA‐mediated developmental signaling pathway, and expression of the extracellular matrix/cell adhesion proteins (DdCAD and csA). This resulted in delayed chemotaxis, adhesion, and development of the organism. In contrast to the inhibitory effects on developmental genes, curcumin induced gstA gene expression, overall GST activity, and generated production of reactive oxygen species. These studies expand our knowledge of developmental and biochemical signaling influenced by curcumin, and lends greater consideration of GST enzyme function in eukaryotic cell signaling, development, and differentiation.Support or Funding InformationDepartment of Biochemistry and Molecular Biology, Howard University College of Medicine, Washington D.C.
Alcohol and HIV Protease Inhibitor‐induced inflammasome activation and liver lipotoxicity
Authors: Hinton Michael 
Published: November 2023

The development of highly‐active‐antiretroviral therapy(HAART) has allowed management of HIV and extended the lives of those infected. Alcohol abuse, which is very common in HIV‐1 infected patients, is one of the most important co‐morbid risk factors for liver injury and has been associated with the occurrence of serious metabolic syndrome and subsequent discontinuation of HAART in HIV patients. The importance of the NLRP3 inflammasome in liver diseases has been recognized. Kupffer cells( KC) are the major resident macrophages and play a critical role in regulating hepatic inflammatory response. We have shown that HIV protease inhibitors (PI) increase the production of mature IL‐1β production in macrophages suggesting the activation of NLRP3. The aim of this study is to examine the effect of alcohol and HIV PIs on NLRP3 activation in KCs and characterize the impact of infllammasome activation in KCs on alcohol and HIV PI‐induced hepatic lipotoxicity.MethodsPrimary mouse KCs will be isolated and treated with individual HIV PIs with or without alcohol. The mRNA levels of inflammasome component genes including NLRP3, ASC, and pro‐caspase will be measured by real‐time RT‐PCR. The cell culture medium will be collected for ELISA analysis of IL‐1β and IL‐18.ResultsWe find both alcohol and HIV PI‐induced activation of NLRP3 in KCs. The expression of mature IL‐1β and IL‐18 will be increased by alcohol and HIV PIs. The active form of caspase‐1 will be increased.ConclusionsActivation of the KC infllammasome is a key cellular mechanism underlying alcohol and HIV PI‐induced hepatotoxicity.