bluuebunny/crossref_metadata_2025 huggingface dataset:
Evaluation of the BBZDR/Wor rat: a new animal model for type 2 diabetes
Authors: Negrin Kimberly A. , Tomcho Kayce A. , Birckbichler Paul J.
Published: June 2021

Diabetes is a large concern of the American medical community. A major hallmark of diabetic nephropathy is the decline in the rate of glomerular filtration, thus dramatically affecting the structure and functionality of the kidneys. We hypothesize that changes in biochemical/biomarker expression in the early stages of diabetes will precede morphologic changes, and offer the best approach to identify diabetic patients at risk for development of kidney complications. Our hypothesis was tested by inspection of trichrome‐stained kidney tissue slides for structural and diameter differences, and for severity of damage using the genetically engineered BBZDR/Wor rat, a new animal model that mimics Type 2 diabetes. The diameters of diabetic specimens were statistically larger when compared to age‐matched controls as early as 2 months of age. These results prompted the current study, which involves semi‐quantitative scoring of the severity of kidney damage. This was approached in two ways: visual inspection of the glomerular damage and visual inspection of overall damage. The analysis indicated that diseased specimens have sustained damage by scarring and retraction within the glomerulus, scarring of tubules, increased collagen levels, and an increase in interstitial space. A method to quantitatively analyze the severity of damage is in the early stages of development. The clinical relevance of this study is that the morphological changes may be a future predictor of the pathogenesis of diabetic nephropathy. Support for this research was supplied by the College of Health, Science and Environment and Faculty/Student Research Grant program of Slippery Rock University.
ACTIVITY OF REVERSED DIAMIDINE DB766 AGAINST BLOODSTREAM AND INTRACELLULAR FORMS OF TRYPANOSOMA CRUZI LODGED WITHIN CARDIAC CELLS IN VITRO
Authors: Batista Denise da Gama Jaén , Silva Patrícia Bernardino da , Silva Cristiane França da , Pacheco Michele Gabriele Oliveira , Meuser Andreia Souza , Boykin David W , de Nazaré Correia Soeiro Maria
Published: June 2021

Due to well‐know efficacy of aromatic diamidines and analogues upon a wide range of pathogens including trypanosomatids such as T. cruzi, which still displays a unsatisfactory chemotherapy, our aim was to investigate the biological activity of DB766, a reversed amidine (RA), against bloodstream forms (BT) and amastigotes of T. cruzi (Y strain) in vitro. DB766 displayed an excellent trypanocidal effect towards BT incubated at 37ºC for 24h (IC50 0.05μM). Addition of blood constituents did not impair the drug effect (IC50 0.16μM), measured by incubation at 4ºC/24h. Mammalian cells evaluated by CK‐MB and MTT assays showed the lack of drug toxicity upon uninfected cardiomyocytes (CM) and peritoneal macrophages incubated at doses up to 10.6μM. T. cruzi‐infected CM treated with non‐toxic doses (10.6‐0.04μM) of DB766 resulted in a potent dose and time‐dependent effect (IC50 24h 0.03μM), leading to a considerable reduction in both the percentage of infected cells as well as in the mean number of parasites per infected cells. Fluorescent approaches showed that RA impaired the myofibrils breakdown noticed in T. cruzi‐infected CM not submitted to drug incubation. Our data demonstrate the excellent effect of DB766 against T. cruzi, which merits further in vivo studies currently underway.
Regulation of ubiquitinated protein sorting at multivesicular endosomes
Authors: Piper Robert Charles , Ren Jihui , Pashkova Natasha , Winistorfer Stanley
Published: June 2021

Ubiquitinated membrane proteins are transported into the interior of the lysosome for degradation. This sorting pathway involves incorporation of cargo proteins into endosomal intralumenal vesicles, which accumulate to form a multivesicular endosome/body (MVB). The apparatus that recognizes ubiquitinated membrane cargo proteins in yeast includes Vps27 and Hse1, which are orthologous to Hrs and STAM in animal cells. We find that the Vps27‐Hse1 ubiquitin‐sorting complex associates with a variety of factors that help regulate the fate of ubiquitinated cargo. Recently, we discovered that Ufd3/Doa1 directly binds the SH3 domain of Hse1. Doa1 associates with the AAA ATPase Cdc48, which is involved in processing a number of ubiquitinated substrates. We find that Doa1 possesses 3 distinct ubiquitin binding domains, and a novel SH3 ligand motif required for Hse1 binding. We also found that Doa1 can partially localize to endosomes. Unlike doa1 null mutants, Doa1 mutants lacking the SH3 binding motif for Hse1 have normal levels of ubiquitin and display no growth defects; however, these Doa1 mutants are defective in sorting particular MVB cargo proteins. We also find that the novel Cdc48 cofactor Ubx3 also plays a role at the endosome similar to Doa1 and that ubx3 null mutants are defective in aspects of MVB sorting. These results point to a novel role for a Cdc48‐Doa1‐Ubx3 complex on endosomes that may foster proper processing of ubiquitinated MVB cargo.
Bile Acids Modulate Steroidogenic Gene Expression in H295R Adrenocortical Cells
Authors: Shah Shaili R. , Sewer Marion B.
Published: June 2021

Bile acids, the end products of cholesterol metabolism, are synthesized in the liver and secreted into the intestines to assist in the absorption of fat‐soluble vitamins and dietary fat. The farnesoid X receptor (FXR) is a member of the nuclear receptor family that is a key modulator of lipid and glucose homeostasis. In addition to being highly expressed in the liver and intestines, FXR is also detected in tissues such as the adrenals and kidneys. Based on FXR expression in the adrenal cortex, we hypothesized that this receptor plays a regulatory role in cortisol production. We stimulated H295R adrenocortical cells with the bile acid chenodeoxycholic acid (CDCA), and assessed the effect on the transcription of CYP17 gene, one of the genes in the steroid hormone biosynthetic pathway. Western blot and lysis demonstrated an increase in CYP17 following CDCA treatment. Using guggulsterone, a plant antagonist of FXR, and CYP17 reporter gene deletion plasmids, we identified the CDCA/FXR‐responsive region of the CYP17 gene. Stimulatory effects of CDCA were also observed on several other steroidogenic genes. Given that CYP17 encodes P450c17, an essential enzyme in the steroidogenic pathway, our studies identify a novel role for bile acids as modulators of cortisol output.
Tetratricopeptide repeats in the anaphase‐promoting complex provide multiple activator binding sites
Authors: Matyskiela Mary Ellen , Morgan David O.
Published: June 2021

The anaphase‐promoting complex (APC) is a 13‐subunit ubiquitin ligase that initiates the final events of mitosis. Substrates are recruited to the APC by an activator protein (Cdc20 or Cdh1). We tested the possibility that activator binding to the APC depends on three related APC subunits (Cdc27, Cdc16, and Cdc23) that contain multiple tetratricopeptide repeats (TPRs): repeated 34 amino acid sequences that often fold into hydrophobic grooves used for protein‐protein interactions. Each subunit contains 9–10 TPRs, which together may form a large array of TPRs on the APC surface. To assess the function of this array, we mutated residues predicted to lie in the TPR grooves of Cdc27, Cdc16, and Cdc23. Mutation of single residues in the grooves of Cdc27 or Cdc23 reduced the ability of the activators Cdh1 and Cdc20 to bind and activate purified APC. Subunit binding and APC integrity were unaffected. The mutations suppressed the lethality of activator overexpression in yeast. We propose that the presence of multiple activator binding sites may explain how the APC can make specific yet flexible interactions with a wide variety of substrates. Supported by NIH GM53270 and an NSF fellowship.
Comparative analysis of the effects of histone deacetylase inhibitors on cell cycle and cell death in MCF‐7 breast cancer cells
Authors: Knutson Andrew Kekupa’a , Taylor Travis , Welsh Jennifer , Roy Somdutta , Tenniswood Martin
Published: June 2021

Histone deacetylase inhibitors (HDIs) are currently being evaluated for their therapeutic potential and have shown considerable promise as a possible adjuvant therapy for many cancer types. Posttranslational modifications of the tumor suppressor protein, p53, stabilize the protein, and affect its downstream target gene activation. This study compared the activity of two HDIs, JW‐1521 and SAHA, on p53+/+ MCF‐7 human breast cancer cells. Crystal violet growth assays of JW‐1521 and SAHA treated cells show differential levels of apoptosis with JW‐1521 having a much earlier and more cytotoxic effect. Flow cytometry confirmed this, showing clear apoptosis in JW‐1521 treated cells (37%) and relatively little apoptosis in SAHA treated cells (4%) after 48 hours. Cell cycle associated genes known to be downstream transcriptional targets of p53, including genes cyclin B1, cdc20, and Kntc2, were less down regulated by JW‐1521 than SAHA. In contrast p53 activated pro‐apoptotic genes such as p21B and Gdf15, were dramatically up regulated by JW‐1521 and only modestly induced by SAHA. These differential levels of gene induction provide molecular evidence of both cell cycle arrest and apoptosis, and suggest that the differential p53 mediated activation of these genes explains the difference in the biological effect of JW‐1521 and SAHA. (Supported by the Coleman Foundation and the Pat Girondi Fellowship in Molecular Medicine)
Cyclin‐dependent kinase 5 mediated phosphorylation of GluProRS induces translational silencing of inflammatory gene expression
Authors: Arif Abul , Fox Paul L.
Published: June 2021

The bifunctional aminoacyl‐tRNA synthetase GluProRS has a regulated noncanonical activity in translational control of multiple gamma‐interferon (IFN)‐inducible mRNAs. GluProRS is a component of the heterotetrameric GAIT (Gamma‐inteferon Activated Inhibitor of Translation) complex that binds the GAIT element in the 3’UTR of ceruloplasmin and VEGF mRNA and blocks their translation. Here we investigate the activation of GluProRS by phosphorylation, and its role in GAIT complex formation and function. In human monocytes, we have identified Ser886 and Ser999 in the non‐catalytic linker domain of GluProRS as the sites of phosphorylation upon induction with IFN. Phosphorylation of the two sites is catalyzed independently by two distinct kinases. Cyclin‐dependent kinase 5 (Cdk5), also known as neuronal CDC2‐like kinase (NCLK), phosphorylates Ser886. We have also elucidated an IFN‐activated kinase cascade in which ERK‐2 activates the Cdk5 activator protein, p35 (NCK5a) that activates Cdk5, the proximal kinase that phosphorylates GluProRS. Cdk5‐mediated phosphorylation induces rapid release of GluProRS from its residence in the tRNA multisynthetase complex and facilitates its interaction with the other GAIT proteins to form the active GAIT complex. Suppression of Cdk5 expression or activity inhibits GluProRS phosphorylation as well as the translational silencing function. The non‐neuronal activity of Cdk5 and its activator proteins, and the identification of GluProRS as a substrate in monocytes clearly indicate a unique extraneuronal function of Cdk5 in translational control of inflammatory gene expression. Research support: P01 HL29582 and AHA.
The phospholipase B homolog, Plb1, and the cAMP•protein kinase A pathway function cooperatively to regulate an osmotic stress‐induced growth control system in fission yeast
Authors: Matsuo Yasuhiro , McInnis Brittney , Marcus Stevan
Published: June 2021

The phospholipase B homolog, Plb1p, and the cAMP•protein kinase A (PKA) pathway are essential for growth of the fission yeast, Schizosaccharomyces pombe, under high salinity conditions. While plb1Δ, adenylate cyclase (cyr1Δ), and PKA (pka1Δ) mutants are not hypersensitive to sorbitol, we show here that plb1Δ cyr1Δ and plb1Δ pka1Δ double mutants are hypersensitive to both sorbitol and low salt concentrations whereas cyr1Δ pka1Δ mutants are phenotypically similar to the respective single mutants, thus demonstrating that Plb1p and the cAMP•PKA pathway function cooperatively in mediating hypertonic stress response in S. pombe. Hypertonic stress‐induced growth arrest is both asynchronous and reversible in the plb1Δ pka1Δ double mutant as well as the respective single mutants, indicating that Plb1p and the cAMP•PKA pathway are required for cell cycle progression in hypertonic media but not for survival of hypertonic stress. Deletion of cgs1, which encodes a Pka1p regulatory subunit, partially rescues the hypertonic stress‐induced growth defect of plb1Δ cyr1Δ cells, providing further evidence of functional interaction between Plb1 and the cAMP•PKA pathway. Pka1‐GFP fusion proteins are localized in the cytoplasm and concentrated in the nucleus, the latter in a cAMP‐dependent, Cgs1p‐independent fashion, under normal growth conditions and exported from the nucleus in response to prolonged hypertonic stress.
Aurora‐C phosphorylates and regulates Borealin and CENP‐A during mitosis
Authors: Slattery Scott Dunning , Moore Rebecca V , Brinkley Bill R , MacCorkle Rebecca A
Published: June 2021

Aurora‐B and –C kinases are members of the Aurora serine/threonine kinase family of mitotic regulators. Aurora‐B kinase is evolutionarily conserved from yeast to humans and has multiple functions in chromosome condensation, cohesion, biorientation, and in cytokinesis. In contrast, Aurora‐C kinase has only been found in mammals, is upregulated in some tumor cell lines and tissues, and has a unique physiological role in spermiogenesis. Despite these known functions, little is known about the function of Aurora‐C in mitosis. We have explored Aurora‐C function in mitosis, and have found that it interacts with several known substrates of Aurora‐B: the chromosomal passenger complex (CPC) proteins Inner Centromere Protein (INCENP), Survivin, and Borealin. We have also found that Aurora‐C, like Aurora‐B, phosphorylates the centromeric histone Centromere Protein‐A (CENP‐A), and Borealin in vitro. These molecular mechanisms are consistent with our observation that in the absence of Aurora‐B, Aurora‐C is sufficient for proper centromeric localization of the CPC proteins and timely prometaphase progression. Thus, Aurora‐C shares many mitotic substrates and is capable of performing mitotic functions previously attributed to only Aurora‐B. This work was supported by NIH Grant CA 41424 to BRB and The Huffington Foundation Grant to BRB and RAM.
Leukocyte 12‐lipoxygenase: expression, purification and physical characterization
Authors: Funk Max O. , Rapp Johanna , Sharp Allan M.
Published: June 2021

The expression and purification procedure [Richards, K.M. and Marnett, L.J. (1997) Biochemistry 36, 6692–6699] for leukocyte 12‐lipoxygenase was updated for the production of the large quantities of protein necessary for the characterization of its physical properties. A two‐step FPLC ion exchange chromatography procedure was adequate for purification as evaluated by SDS PAGE, specific activity measurement, isoelectric focusing, and electrospray ionization mass spectrometry. The solution properties of the enzyme were examined by variable temperature circular dichroism spectroscopy, differential scanning microcalorimetry, and dynamic light scattering measurements. The redox characteristics of the active site iron atom were determined by EPR spectroscopy following treatment with a variety of fatty acid hydroperoxides. Leukocyte 12‐lipoxygenase was found to be more similar to reticulocyte 15‐lipoxygenase than to soybean lipoxygenase‐1 or human 5‐lipoxygenase.

(Supported by the National Institutes of Health GM 62140)
Role of Purines in the Prevention of Acute Lung Injury (ALI)
Authors: Siddaramappa Umapathy Nagavedi , Gorshkov Boris , Fan Zhenghong , Verin Alexander Dmitriyevich
Published: June 2021

ALI is a major cause of acute respiratory failure that is associated with high morbidity and mortality. Human pulmonary artery EC (HPAEC) expresses both P1 (Adenosine) and P2Y (ATP and ADP) receptors. We have already shown that ATP promotes EC barrier enhancementvia a complex cellsignaling and the involvement of specific trimeric G‐proteins with the association of both Gq and Gi (Circulaion Research, 2005,97,115–24). Recently, we showed the barrier enhancement property of Adenosine in HPAEC cells and specific siRNA studies showed the primary role of A2A in barrier enhancement. Both ATP and Adenosine are involved in the restoration of the microtubule disruption induced by Nocodazole as evidenced by ECIS (Electrical cell‐substrate impedance sensing) and Immunofluorescence. PPase1 inhibitor, Tautomycetin inhibited the ATP and Adenosine induced barrier enhancement in HPAEC cells which demonstrate the possible role of complex PPases pathway in barrier enhancement via ATP and Adenosine. In murine model of ALI, ATP administrated intravenously, attenuated inflammatory response by decreasing accumulation of cells (48%), and proteins (57%), in bronchioalveolar lavage (BAL), and by reducing neutrophil infiltration into alveoli. ATP reduced the LPS induced Evans Blue Dye (EBD) leakage from vascular space into BAL. In the similar murine model of ALI, Adenosine decreased the accumulation of protein (48%) into BAL. These novel findings strongly suggest that purinoreceptors stimulation exert a protective role against ALI, probably via decreasing endothelial junctional permeability in vitro and in vivo. Research Support, NHLBI;HL083327.
Lipid Rafts play a role in human normal follicle stimlating hormone receptor (hFSHR) Signaling
Authors: Libous Jennifer Lauren , Dias James A , Cohen Brian D
Published: June 2021

The hFSHR is a member of the family of g protein‐coupled receptors that has been shown to signal through both g protein linked and MAP kinase pathways. Little is known about the importance of the membrane microenvironment in normal signaling. To study the role of lipid rafts in hFSHR signaling, cholesterol depletion experiments were carried out with methyl‐â‐cyclodextrin (MBCD) in HEK293 cells expressing hFSHR. Cells were treated with MBCD prior to stimulation with FSH for 0, 15, and 30 minutes. Cells were harvested and activation of MAP kinase enzymes (p38 and p44/42) were measured by western blot. MBCD treatment resulted in constant phosphorylation for p38 and a decrease in phosphorylation for p44/42 in response to FSH. In parallel experiments, cells were treated with MBCD prior to FSH stimulation to measure cAMP accumulation. cAMP production in MBCD treated cells was almost completely abolished, although hormone binding remained normal. One interpretation of the data suggests that the disruption of lipid rafts by depleting cholesterol disturbs the membrane microevironment sufficiently to disturb hFSHR signaling. Currently, a cholesterol add‐back experiment is being carried out to follow up on the cholesterol depletion to see if signaling is restored.
C17orf37 regulates prostate cancer cell migration and invasion by NF‐kB mediated downstream target genes
Authors: Dasgupta Subhamoy , Vishwanatha Jamboor K
Published: June 2021

C17orf37/MGC14832 is located in the hot spot locus of human chromosomal region 17q12‐21 next to ErbB2. Earlier we have characterized C17orf37 protein, and have shown the protein expression significantly higher in prostate cancer cells and tissue specimens compared to normal prostate cells. By gene knock down studies, we have found C17orf37 regulates prostate cancer cell migration and invasion by modulating certain key proteins involved in the process like MMP‐9, uPA and VEGF. Here we hypothesize that C17orf37 acts as a membrane bound signaling molecule and facilitates migration and invasion of prostate cancer cells by NF‐kB mediated downstream target genes. Our results show that knockdown of C17orf37 in DU‐145 prostate cancer cells by increasing concentration of gene specific siRNA significantly reduced the DNA binding activity of NF‐kB in a dose dependent manner. Furthermore, C17orf37 knockdown also decreased the activated phosphorylated Akt to inactive form suggesting C17orf37 must be located upstream of Akt. Imaging DU‐145 and LNCaP prostate cancer cells by total internal reflection fluorescence microscopy we have observed C17orf37 on the membrane of these cells and this observation is supported by the fact that C17orf37 has a potential prenylation motif. By Fluorescence Lifetime Imaging Microscopy based FRET assys we have located the C17orf37 interacts with annexin A2 mostly in the membrane and perinuclear region. Currently we are investigating the functional significance of C17orf37 and annexin A2 interaction and the downstream signaling cascade that regulates migration and invasion in prostate cancer cells. (Supported grants from the National Institutes of Health CA109593 and MD 001633)
Gene Repair in sub‐S phase Cell Populations and Cell Cycle Response to Quadruplex Oligonucleotides
Authors: Warriner Melissa , Schwartz Timothy , Engstrom Julia , Kmiec Eric
Published: June 2021

Targeted nucleotide exchange (TNE) relies on the use of a short synthetic oligonucleotide (ODN) designed to be complementary to a target sequence in the genome except for a centrally located mismatch, which directs the desired base change in the DNA. The repair reaction is believed to be enhanced by mechanisms that promote a more open conformation of the DNA, thus, enabling the target site to be more accessible for the binding of the oligonucleotide. One such method is through the modulation of cell cycle progression to increase the population of cells in S phase, wherein targeting of a single base pair is enhanced. Studies aimed at demonstrating the variations in correction in relation to cell cycle measured repair frequency late in the reaction cycle. Our work aims to discern variations in repair levels in subphases of S as cells enter and progress through this phase, in the presence of the oligonucleotide. In addition, we evaluate gene expression patterns of several cell cycle genes that may help denote the changes in cell cycle distribution as a function of gene repair events.

Additional research has been performed analyzing gene expression patterns in malignant versus non‐malignant esophageal cancer cells in response to treatment with G‐rich oligonucleotides (GRO). It has been shown that these single‐stranded DNA molecules which are comprised primarily of guanosine residues induce retardation in the progression of the cell cycle, and result in a sub‐G1 population of apoptotic cells. Successful inhibition of growth of malignant, but not nonmalignant, cells has been seen. Thus, G‐rich ODNs may be guiding the selective killing of tumor cells. I am currently analyzing the RNA levels of apoptosis genes incubated with GROs to understand how the cells are responding, and which pathway may be responsible for the induction of apoptosis. Funded by Howard Hughes Medical Institute.
Signaling role of NADPH oxidase in Entamoeba histolytica‐induced cell death of HT29 colonic epithelial cells
Authors: kim ju young , Lee Yong Ah , Kim Kyeong Ah , Min Deul‐le , Shin Myeong Heon
Published: June 2021

Entamoeba histolytica is an enteric tissue‐invasive protozoan parasite that causes amoebic colitis and occasionally liver abscess in humans. During tissue invasion, E. histolytica is exposed to elevated amounts of exogenous reactive oxygen species (ROS). ROS cause severe damage to biological macromolecules, leading to metabolic malfunctions. NADPH oxidase is one of the major sources of ROS in vasculature; it consists of a catalytic subunit, p22phox, p47phox, p67phox, and the small GTP binding protein Rac1. In this study, we investigated the roles of ROS and of NADPH oxidase in the E. histolytica‐induced cell death of HT29 cells. NADPH oxidase components were highly expressed in HT29 cells. When HT 29 cells were co‐incubated with E. histolytica, PI and tryphan blue stained cell portion markedly increased compared with results for cells incubated with medium alone. In addition, a large amount of intracellular ROS was detected after exposure to viable trophozoites. The treatment with diphenyleneiodonium chloride (DPI), an unspecific inhibitor of NADPH oxidase and rac1 siRNA strongly inhibited the E. histolytica‐induced ROS generation. Moreover, the E. histolytica‐induced cell death was effectively blocked by pretreatment of cells with DPI and rac1 siRNA. These results strongly suggest that NADPH oxidase‐generated ROS is essential for the E. histolytica‐induced HT29 cell death.
Tolerance of a choanoflagellate to environmental stressors
Authors: Misiak Kristen , Dunlap Kristen J , Rodriguez Sara S , Julian David
Published: June 2021

Choanoflagellates are the protozoan antecedents of multicellular animals, but little is known about their response to environmental change. Recently, the arctic choanoflagellate Diaphanoeca grandis was found in a supersulfidic, anoxic fjord in Norway, suggesting that at least some choanoflagellates are highly stress tolerant. We investigated this by testing the 24 h survival of D. grandis exposed to variations in pH (7.3–8.7), temperature (4, 10, 15, 20, 25, 30°C), Po2 (normoxia vs. anoxia), salinity (13, 19, 26, 32, 48, 64‰), H2O2, (0.01, 0.1 and 1 mM) and H2S (0.03, 0.3, 1%). Cell death was assessed by morphology and propidium iodide labeling. Compared to control conditions (normoxia, 10°C, 32 ppt salinity, pH 8.0), cell death was significantly increased only at the highest pH (8.7, p = 0.023), temperatures of 20°C and above (p < 0.0001), the lowest and highest salinity (13 and 64 ppt; p < 0.0001), H2O2 of 0.1 mM and above (p<0.0001), and H2S of 0.1% and above (p=0.0002). Furthermore, 65% of D. grandis were alive after 24 h of anoxia (p= 0.0023). These results indicate that D. grandis is surprisingly tolerant of typical environmental stressors. Whether this is characteristic of all choanoflagellates is still unknown.
Pro‐Apoptotic Bnip3 Functions as a Mitochondrial Sensor of Oxidative Stress in Myocardial Ischemia/Reperfusion
Authors: Quinsay Melissa , Kubli Dieter , Gustafsson Asa B
Published: June 2021

Bnip3 is a member of the BH3‐only subfamily of pro‐apoptotic Bcl‐2 proteins and is associated with mitochondrial dysfunction and cell death. We have investigated potential mechanism(s) by which Bnip3 activity is regulated. We found that Bnip3 forms a DTT‐sensitive complex in isolated myocytes treated with H2O2 and in hearts subjected to ischemia/reperfusion (I/R). Overexpression of Bnip3 in HL‐1 myocytes revealed that most of Bnip3 existed in the 48‐kDa complex which correlated with increased cell death. Bnip3 contains a single Cys at position 64 and mutation of the Cys or deletion of the N‐term (aa 1–64) caused a reduction in the cell death activity of Bnip3. Co‐immunoprecipitation studies and separation of purified Bnip3 on SDS‐PAGE showed the presence of the same DTT sensitive complex, suggesting that the complex is a Bnip3 homodimer. Mutation of a His residue in the C‐terminal transmembrane domain resulted in complete loss of homodimerization and cell death activity of Bnip3 compared to wild type. A consequence of I/R is the production of ROS and oxidation of proteins, which promotes formation of Cys disulfide bonds between proteins. Thus, these studies suggest that Bnip3 functions as a redox sensor in cells where increased oxidative stress induces homodimerization of Bnip3 via N‐terminal Cys residue and the C‐terminal TM domain. This works was supported by grants from TRDRP (14KT‐0109) and NIH (HL087023).
DUSP1 is controlled by p53 during the cellular response to oxidative stress
Authors: Liu Yuxin , Wang Jianli , Yin Yuxin
Published: June 2021

p53 controls the cellular response to genotoxic stress through multiple mechanisms. We report here that p53 regulates DUSP1, a dual‐specific threonine and tyrosine phosphatase with stringent substrate specificity for MAP kinase. DUSP1 is a potent inhibitor of MAK kinase activity through dephosphorylation of MAPK. In a colon cancer cell line containing inducible ectopic p53, DUSP1 protein level is significantly increased upon activation of p53, leading to cell death in response to nutritional stress. In MEF cells, DUSP1 protein abundance is greatly increased following oxidative stress in a p53‐ dependent manner, also when apoptosis is triggered. We show that p53 induces the activity of a human DUSP1 regulatory region. Furthermore, p53 can physically interact with the DUSP1 regulatory region in vivo and p53 binds to a 10‐bp perfect palindromic site in this DUSP1 regulatory region. We demonstrate that overexpression of DUSP1 or inhibition of MAK kinase activity significantly increases cellular susceptibility to oxidative damage. Those findings indicate that p53 is a transcriptional regulator of DUSP1 in stress responses. Our results reveal a mechanism whereby p53 selectively regulates target genes, and suggest a way in which subgroups of those target genes might be controlled independently.
Carbachol Regulation of AKT in LNCaP Prostate Cancer Cells
Authors: Fletcher Luke , Schmitt John Michael
Published: June 2021

Hormones and agonists that enhance cell survival through binding specific G Protein‐Coupled Receptors (GPCRs) are of particular interest in cancer cell survival. LNCaP cells have been shown to express muscarinic cholinergic receptors that are responsive to the agonist, carbachol. In addition, LNCaP cells specifically express the M3‐subtype of GPCR's that may couple carbachol to Gq, increases in intracellular calcium, and activation of the intracelluar Ca2+/calmodulin‐dependent protein kinases (CaM Ks). The CaM Kinase family of proteins includes CaM KII, CaM Kinase Kinase (CaM KK) and its substrates CaM KIV, CaM KI and the protein kinase AKT. AKT is an anti‐apoptotic enzyme that phosphorylates BAD and inhibits caspase activation. Our goals were to examine the mechanism of carbachol activation of AKT in LNCaP prostate cancer cells and evaluate whether CaM Ks may be mediating carbachol's activation of AKT and cell survival in LNCaP cells. The results suggest that AKT phosphorylation was increased in response to a five‐minute stimulation with 10μM carbachol in LNCaP cells which was blocked by the CaM K family inhibitor, KN‐93 suggesting the involvement of the CaM Kinase proteins in the pathway. In contrast, there was no inhibition by the MEK inhibitor, U0126 suggesting that AKT is activated independently of MEK and ERK. Interestingly, the CaM KK inhibitor, STO‐609 potently inhibited cabachol's activation of AKT. Furthermore, our results indicate that carbachol treatment of LNCaP cells promoted cell survival through CaM Ks. Carbachol's survival effects also appear to be operating through the M3‐subtype of GPCRs, CaM KK, and its phosphorylation of AKT.
Dynamic Intracellular Glycosylation: O‐GlcNAc, is a Key Modulator of Glutamine‐Mediated Cellular Protection and Heat Shock Protein 72 Induction
Authors: Haserodt Sarah Kristen , Pett Virginia , Zachara Natasha E
Published: June 2021

The enzyme O‐GlcNAc transferase (OGT) dynamically modifies over 500 intracellular proteins by adding O‐linked β‐N‐acetylglucosamine (O‐GlcNAc) to threonine and serine residues, regulating protein function in a manner analogous to protein phosphorylation. In response to numerous stressful stimuli, O‐GlcNAc attachment to proteins increases. Augmenting O‐GlcNAc modification prior to cellular injury improves cellular survival, suggesting that this is a pro‐survival response of cells. The mechanism by which O‐GlcNAc increases cellular survival is unknown. However, increased O‐GlcNAc correlates with the enhanced expression of heat shock protein 72 (HSP 72); in contrast, decreased O‐GlcNAc is associated with the reduced expression of HSP72. Interestingly, Glutamine is known to modulate both O‐GlcNAc and HSP72 levels, and supplementation with glutamine improves survival rates in both in vivo and in vitro models. In this study we have used Cre‐Lox technology to delete OGT and to determine if glutamine could modulate either cellular survival or the expression of HSP72. Our data show that in the OGT knockout, glutamine is not protective and can not alter HSP72 expression levels. Moreover, HSP72 induction is ablated in the OGT knockout. We are currently investigating the molecular mechanism by which O‐GlcNAc mediates the expression of HSP72 at the transcriptional level.
VEGF‐B is an apoptosis inhibitor via suppression of BH3‐only protein gene
Authors: Li Xuri
Published: June 2021

Despite its early discovery and high sequence homology to the other VEGF family members, the function of VEGF‐B thus far elusive.

We have recently found a novel function of VEGF‐B as a potent apoptosis inhibitor. VEGF‐B treatment significantly rescues neurons from apoptosis in both the retina and brain. Mechanistically, we reveal that VEGF‐B inhibits apoptosis via suppressing the expression of the BH3‐only protein and other apoptotic/cell death‐related genes. Remarkably, the anti‐apoptotic/survival effect of VEGF‐B is not associated with undesired angiogenesis. VEGF‐B thus appears to be the first member of the VEGF family that has a potent anti‐apoptotic effect, while lacking a general/universal angiogenic activity.
Neutralization of the transcription factor nuclear factor erythroid derived‐2 (NF‐E2) causes robust increases in the anti‐apoptotic protein Mcl‐1 in human neutrophils
Authors: Johnson Paul Richard , Kausar Hina , Wu Rui , Rane Madhavi J
Published: June 2021

Human neutrophils (PMNs) are short‐lived cells that undergo constitutive apoptosis in vivo. When challenged by pathogens, cytokines, or ROS, PMNs survive longer. One hallmark of prolonged PMN lifespan is up‐regulation of the anti‐apoptotic protein, Mcl‐1.

We previously showed that GM‐CSF delays PMN apoptosis via the Akt pathway. Proteomic analysis of neutrophil Akt immunoprecipitates identified NF‐E2 p45 as an Akt binding protein. To explore the possible role of NF‐E2 in regulating PMN apoptosis, neutrophils collected from healthy human donors were subjected to NF‐E2 or isotope control (IC) antibodies or control buffer (Krebs) for 1 hr at 37°C. LPS, TNFα, or control buffer was then added to cells and incubated overnight at 37°C.

PMNs pre‐incubated with NF‐E2 antibody showed an increase in NF‐κB activation and Mcl‐1 protein versus IC or control buffer. LPS overnight incubation caused a slight increase in Mcl‐1 protein, whereas TNFα showed no effect. Also, we demonstrated that Akt phosphorylates NF‐E2 on Ser170 and that additional Akt sites exist. Akt‐mediated NF‐E2 phosphorylation mediates its nuclear localization and DNA‐binding activity.

Collectively, our results suggest that Akt regulates NF‐E2 activity and that NF‐E2 plays a role in constitutive apoptosis of human neutrophils. Neutralization of NF‐E2 causes a robust increase in PMN survival as determined by levels of the anti‐apoptotic protein, Mcl‐1.

This work supported by AHA (G030700) Scientist Development Grant (MJR).
Human bronchial/tracheal epithelial cells (BEC) are more sensitive than small airway epithelial cells (SAEC) to sulfur mustard‐induced apoptosis apparently due to a Fas (death receptor) response amplification loop
Authors: Ray R. , Keyser B. , Andres D. , Hauck S. , Benton B. , Carpin C. , Daher A. , Simbulan‐Rosenthal C. , Rosenthal D.
Published: June 2021

Sulfur mustard (SM; bis‐(2‐chloroethyl) sulfide) causes skin blisters, airway injury and ocular damage. Apoptosis is one of its mechanisms of action. Here, we examined SM‐induced apoptosis pathways in vitro in cultured BEC and SAEC. At 16 h after cells were exposed to increasing SM concentrations (0, 50, 100, 300μM), apoptosis pathways were characterized by (a) measuring hydrolysis of fluorogenic substrates of caspases‐3, ‐8 and ‐9, and (b) studying the effects of caspase type‐specific peptide inhibitors on each caspase activity. SM activated caspase‐8 and caspase‐3 in both cell types, but caspase‐9 only in BEC. Immunoblot analyses revealed proteolytic processing of the proenzymes to the active forms of caspases‐8 and ‐3. Moreover, caspases were activated at a lower SM concentration in BEC than in SAEC. Each caspase type‐specific inhibitor tested was most effective for that particular caspase, but also inhibited other caspases. These results lead to the following conclusions. BEC are more sensitive to SM‐induced apoptosis than SAEC. This is due to a mechanism of Fas response (caspase‐8 activation) stimulation via an amplification loop involving caspases‐8, ‐9, ‐3 and ‐6 in BEC, but not in SAEC. These findings may be useful in developing therapeutics against SM injury. Research supported by JSTO‐CBD Award # M0005_04_RC_C. The views represented are those of the authors and are not necessarily endorsed by the US Army.
A Compensatory Mechanism for Acute Hyperglycemia Induced Apoptosis
Authors: Wisdom Erika , George Jancy , Thekkumkara Thomas
Published: June 2021

Studies have shown that prolonged exposure of hyperglycemia may injure cells and contribute in the development of renal diseases in diabetes. In a recent study, we have demonstrated that acute (10–20 min) exposure of human proximal tubule epithelial cells (hPTECs) to 25 mM high glucose (HG) induces a time‐dependent dual effect, involving an early proliferation and a late apoptosis. Acute exposure of HG initiated intrinsic apoptotic pathway activated by reactive oxygen species (ROS). Furthermore, we observed increased protein expression of p53 that was inhibited by ROS inhibitor NAC. Under similar conditions we observed changes in Bcl‐2 and Bax proteins expression. Chronic HG exposure characteristic of diabetes induces a high Bax to Bcl‐2 ratio indicative of cellular apoptosis. However, acute HG exposure reversed the Bax to Bcl‐2 ratio indicating a possible physiological regulatory and recovery phase in which the hPTECs attempt to overcome damage caused by the reactive oxygen species produced by HG exposure. Consistent with these observations, we identified time‐dependent increase in caspase‐3 protein expression. Our study demonstrates for the first time that a single HG exposure for 10–20 min alone is sufficient to elicit cellular apoptosis inducing epithelial cell injury. However hPTECs may initiate intrinsic physiological machinery towards a recovery phase. Supported by NIH Grant DK072140
Anthrax lethal toxin triggers the formation of a membrane‐associated inflammasome complex in murine macrophages
Authors: Nour Adel M , Yeung Yee‐Guide , Santambrogio Laura , Stanley E. Richard , Brojatsch Jürgen
Published: June 2021

Multiple microbial toxins, including the anthrax lethal toxin (LT), trigger an inflammatory response by activating caspase‐1. The caspase‐1 activator Nalp1b protein controls the susceptibility of murine macrophages to LT‐mediated cell death. In this study, we generated antibodies against the murine inflammatory proteins Asc and Nalp1b to localize and identify the LT‐triggered inflammasome complex. Using subcellular fractionation, we found that caspase‐1 and Nalp1b in the triton X‐100 soluble post‐nuclear particulate fraction of murine macrophages. Furthermore, we used immunostaining to show that both proteins co‐localized to the trafficking motor protein dynamin‐2. Unlike caspase‐1 and Nalp1b, Asc and the caspase‐1 substrate interleukin (IL)‐18 were cytosolic. Using size exclusion chromatography (SEC), we discovered that procaspase‐1 preassembles as a 200 kDa complex in untreated macrophages, which shifts to an 800 kDa complex upon LT treatment. Then, we immunopurified caspase‐1 complexes from SEC fractions and identified the caspase‐1‐associated proteins using mass spectroscopy. Consistent with formation of the inflammasome complex, the 800 Kda complex contained proteins involved in inflammation including Nalp1b, enolase, caspase‐1, and caspase‐4. The identified proteins could serve as potential therapeutic targets for management of a variety of microbial and inflammatory diseases.
Kinetic Characterization of Transcriptional Activator‐Coactivator Interactions
Authors: Wands Amberlyn M , Lum Jenifer K , Mapp Anna K
Published: June 2021

Transcriptional activators play an important role in the process of gene expression, and a more detailed understanding of how they interact with the transcriptional machinery and recruit it to a promoter is needed for the development of effective therapeutic replacements. Traditionally these key interactions have been described in terms of binding affinities. However, activator‐transcriptional machinery complexes are not necessarily at equilibrium in the cellular environment, and their kinetic characterization should provide greater insight into the factors which contribute to their differential levels of transcriptional output. To this end, we have used stopped flow fluorescence polarization techniques to generate kinetic profiles of the interactions between the transcriptional activation domains (TADs) of the natural activators Gal4, Gcn4, and VP16 with a component of the yeast Mediator complex, Med15. These TADs possess equilibrium dissociation constants in the low micromolar range for the amino‐terminal portion of Med15, and this affinity can be further enhanced up to a factor of 10 when the TADs are presented in a DNA‐bound form that more closely mimics natural activators. Initial kinetic studies reveal estimated koff rates of greater than 200 s−1 for the isolated TADs against Med 15, and currently rates for the DNA‐bound activators are being investigated. Future work will entail the characterization of additional TAD/coactivator pairs, as well as increasing the complexity of the system through the use of larger coactivator complexes. This research is funded through the National Institutes of Health (NIH) and the Pharmaceutical Sciences Training Program (PSTP).
Facilitating lead optimization with Receptor Image from Fragment Footprinting (RIFF)
Authors: Dwyer Donard S.
Published: June 2021

Lead optimization in drug discovery programs is quite challenging especially when the structure of the drug target is not known. To overcome this limitation, we are developing a computational method called Receptor Image from Fragment Footprinting or RIFF. The goal is to devise a general method for creating realistic models of binding sites starting with only the structure of established ligands or active compounds from screening. The drug or fragment is soaked with a layer of solvent composed of individual amino acids, which is then dispersed (evaporates) from the ligand in molecular dynamics (MD) simulations as a function of time and temperature. The tightest‐binding amino acids from the MD simulations define potential binding modes and can be used to construct model binding sites. RIFF was applied to ligands that interact with HIV reverse transcriptase (HIV‐RT) to validate the method. RIFF correctly identified 6/7 amino acids in the binding site of HIV‐RT (composition matching) located in close proximity to their positions in the actual binding site determined by x‐ray crystallography (topology matching). This method will complement pharmacophore‐based approaches and may speed drug discovery.
Thermal stabilizing mutants of cocaine esterase
Authors: Narasimhan Diwahar , Ko Mei‐chuan , Nance Mark , Macdonald Joanne , Landry Don , Woods James , Zhan Chang‐Guo , Sunahara Roger
Published: June 2021

Despite advances in pharmacotherapeutics that target the dopamine transporter the development of therapeutics that combat cocaine abuse and overdose have been less fruitful. More classical approaches to therapeutic development against cocaine abuse and overdose have inherent challenges in that competitive and allosteric inhibitors of cocaine binding to the transporter exhibit similar behavioral effects of cocaine: inhibition of dopamine uptake. We have recently reported the use of cocaine esterase as a protective therapy against cocaine‐induce lethality. The acceleration of enzyme‐mediated digestion of systemic cocaine by exogenously added cocaine esterase represents a significant paradigm shift in cocaine abuse therapy. Here we report the design and generation of significantly more stable enzyme preparations using computational approaches. We provided evidence from both in vitro and in vivo studies that the modified enzyme displays a prolonged half‐life (up to 30‐fold) and improved thermostability than the wild‐type enzyme. Moreover we have obtained x‐ray crystallographic evidence that provide a structural rationale for the improved enzyme stability. The improved enzyme stability will have a profound influence on the potential of this agent to challenge cocaine‐induced lethality and cocaine addiction in humans.
Strengthening assessment in an interdisciplinary biochemistry major through the implementation of electronic portfolios
Authors: Temple Glena Gilbert
Published: June 2021

The use of portfolios has been documented to have a positive impact on students, faculty and programs. The portfolio provides a roadmap of the skills and program outcomes for students as they progress through the curriculum. Biochemistry majors at undergraduate institutions are often coordinated through multiple departments, causing unique challenges for assessment. The portfolio can serve as the central device for program‐level assessment, bridging various disciplines. This presentation focuses on: 1) the benefits and limitations of electronic portfolio systems in program‐level assessment in the biochemistry major; 2) steps required for implementation and 3) survey results of student and faculty perceptions of the use of portfolios.
Single Cell q‐PCR Analysis of The Molecular Regulation of T Cell Commitment
Authors: Al‐Kuhlani Mufadhal M , Carroll Tanya , Garcia‐Ojeda Marcos E
Published: June 2021

Hematopoietic stem cells (HSC) differentiate into all mature blood cells, including common lymphoid progenitors (CLPs) that can give rise to NK, dendritic, B and T cells. The final fate of CLP depends on the signals and growth factors they receive during development. Notch‐1 is a highly conserved transmembrane receptor that instructs CLP to differentiate into T cells while blocking B cell development. However, the molecular mechanism that regulates this process is not completely understood. Notch‐1 induction of T cell development has been shown to be dose‐dependent, suggesting regulation by a threshold mechanism. In order to elucidate the molecular mechanism exerted on Notch‐1 signaling we use single‐cell Real Time PCR to establish the gene expression level of the regulators of Notch‐1 Deltex‐1, Mint, Nrarp and Numb. Lymphocytes development will be monitored when these regulators are expressed at their normal level and then compared to their development when these regulators over‐expressed, through retroviral infection, or down regulated through the use of siRNA. Understanding the threshold level of the expression of these genes that regulate T cell development will allow us to predict the interaction mechanism of those genes on the CLP cells and the decisions made to determine the fate of these cells.

Source of Support comes from Start‐up Fund from University of California‐Merced