bluuebunny/crossref_metadata_2025 huggingface dataset:
Oxidative stress in diabetic pregnancy alters gene expression and apoptosis leading to congenital heart defects in embryos
Authors: Kumar S.D. , Cheng Y.J. , Lo S.L. , Kang Y.J. , Bay B.H. , Tay S.S.W.
Published: June 2021

Congenital heart defects are frequently present in infants of diabetic mothers. The present study investigated the role of oxidative stress in the developing hearts of embryos from diabetic mothers. SuperArray analysis showed significant alterations of metabolic oxidative stress‐related genes (Gpx3, Fancc, Hbq1, Il22, Nqo1, Sod2 and Tmod1) in the hearts of experimental embryos. Further, the lipid hydroperoxidation level was significantly upregulated in the developing hearts of embryos from diabetic mothers. Western blot confirmed that the protein level of malondialdehyde (MDA) was significantly upregulated in the defective hearts. In addition, MTS assay was performed to assess the viability of H9C2 cell line (cardiomyoblasts) exposed to different glucose concentrations at various time points. Cell viability was significantly decreased in cardiomyoblast cultures treated with high glucose concentrations at all time points studied. Transmission electron microscopy confirmed that apoptotic cell death was upregulated in cardiomyoblasts exposed to high glucose concentrations. It is hypothesized that oxidative stress from maternal diabetes alters the expressions of genes and apoptosis which are involved in the development of the heart, thereby contributing to congenital heart defects.

Supported by grants R‐181‐000‐101‐112 & R‐181‐000‐117‐101 from National University of Singapore.

Grant Funding Source

AAA Postdoctoral Travel Award & AAA Postdoctoral Fellow Poster Presentation Award
Functional TRPC5‐containing channels in human saphenous vein endothelial cells
Authors: Bahnasi Yahya M , O'Regan David J , Porter Karen E , Beech David J
Published: June 2021

Vascular endothelium plays pivotal roles in regulation of vascular tone, blood coagulation, wound healing and cell‐cell adhesion via the expression of vasoactive mediators. Ca2+ plays a central role in the regulation of endothelial cell function. Classical transient receptor potential channels (TRPC1‐7) are widely expressed and some are suggested to play critical roles in regulating endothelial functions. Here, we demonstrate the expression and functionality of TRPC5‐containing channels in human saphenous vein endothelial cells (HSVEC). RT‐PCR analysis suggested that TRPC1, 4, and 5 are expressed in HSVEC. Cells were then studied using fura‐2 real‐time 96‐well plate assays. Challenging of HSVEC with 10 µM histamine or lysophosphatidylcholine (LPC), 1 mM H2O2 or 100 ng/mL vascular endothelial growth factor (VEGF) induced rises in [Ca2+ ]i, which was inhibited by a TRPC5‐specific polyclonal antibody. The inhibition varied from ~80% in the case of LPC, to ~40, 50 and 70% for the histamine, H2O2 and VEGF responses. ATP (100 µM) mediated Ca2+ rise was not affected. Our data support earlier murine studies, and suggest the involvement of TRPC5‐containing channels in mediating Ca2+ signals in human endothelial cells. The channel may be important in inflammatory, oxidative or angiogenic conditions, and is a potential target for therapeutic development.

Work supported by the Wellcome Trust and The Egyptian Ministry of Higher Education.

Grant Funding Source

Wellcome Trust and Egyptian Ministry of Higher Education
Qatari and Indian Asians Type 2 Diabetes in the State of Qatar; IntraCellular Cations and Ultra Structural Study of Platelets
Authors: Afifi Nahla Maher , Ewis Soliman M. , Benslimane Fatiha M. , Bejaoui Yosra Ridha
Published: June 2021

Diabetes mellitus is one of the main causes of cardiovascular diseases in Qatar and platelets play a key role in these events.The aims of this project is to study ultrastructural changes in diabetic platelets, intraplatelets concentration of Ca and Mg and their relation to insulin, and to investigate if there are any racial differences associated with platelets abnormalities. Diabetic groups included 34 Qatari (Q) and 32 Asian Indian (AI). Control groups included 21 (Q) and 13 (AI). The results indicated that intraplatelet Ca was significantly higher in both diabetic (Q & AI) groups compared to corresponding controls (Q; 0.134 ± 0.0728 vs. 0.097 ± 0.034, p< 0.05) and (AI; 0.179 ± 0.0738 vs. 0.117 ± 0.0854, p< 0.01). Comparing to control; a significant decrease in Mg level for the Qatari diabetics (7.4 ± 4.4 vs. 14.4 ± 6.1, p< 0.05) associated with a significant decrease in the insulin level for the same group (26.1 ± 18.2 vs. 21.8 ± 19.6, p< 0.05). The MPV showed significant increase in diabetic groups compared to the controls with (Q; 8.4 ± 1.1 vs. 7.8 ± 0.6, p< 0.05) and (AI; 8.8 ± 0.6 vs. 7.7 ± 0.3, p< 0.05), and a significant increase in (AI) diabetics compared to (Q) diabetics p< 0.05. Abnormal structural characteristics were seen under TEM in both diabetic groups Q & AI with clear evidence of microthrombus in the AI group. This may explain the higher incidence of CV events among the Asian Indians diabetics compared to the other ethnic groups.

This project was funded by QU&UREP.
Grading system for articular surface degeneration of zygapophysial (Z) joints following induced segmental hypomobility in the rat
Authors: Cramer Gregory D. , Henderson Charles N.R. , Pocius Judith D. , Grieve Thomas J. , Daley Clover
Published: June 2021

Articular surface degeneration (ASD) of the Z joint cephalad articular facets (CAFs) is characterized by roughening, pitting, and remodeling. These changes have been previously found in hypomobile Z joints of rats. Our objective was to refine the method of grading ASD for use in large scale studies. Left and right L4‐S1 CAFs of 6 randomly selected spines (45 CAFs) of control and experimental rats were assessed. The experimental animals had undergone 8 weeks of fixation, using an established, IACUC approved, rat model, in which 3 contiguous segments (L4, L5, L6) were fixed with a specially engineered, surgically implanted, vertebral fixation device. Using a decision tree developed to score overall CAF degeneration, the CAFs were graded under dissection microscopy by 2 observers (blinded to each other and to their own initial scores) to determine: number of regions and size of remodeling; number, location, and depth of pits; and severity and size of roughening. The observers scored the spines on 2 occasions separated by at least 7 days. Weighted kappa showed substantial intra‐observer (k=0.77, SE 0.1) and inter‐observer agreement (first measurement, k=0.72, SE 0.14; second measurement k=0.71, SE 0.14), indicating that the grading system can be used in future studies assessing ASD of CAFs. Support: NIH/NCCAM (Grant #s 1R21 AT00784 and U19 AT002006).
Expression of the PV‐ and BDNF‐immunoreactive neurons in the brain regions associated with emotion and cognition in animal models of autism
Authors: Li Rui‐xi , Wang Wei‐xia , Wang Yue‐hua , Hisao Nishijo , Peng Yu‐wen
Published: June 2021

In this study, immunohistochemical and western blot methods were used to examine the expression of the PV‐ and BDNF‐ immunoreactive neurons in the amygdala, prefrontal cortex, hippocampus and sensory cortex in animal models of autism, which were obtained in offspring of the Wistar rat by means of a single intraperitoneal injection of 600mg/kg sodium valproate (VPA) at the day of 12.5 after pregnancy and by a method of repeated cold stress. The immunohistochemical results showed that PV‐immunoreactive neurons in the amygdala,prefrontal cortex and hippocampus of autism models changed in various degrees in shape and size of the soma of the neurons and in density and length of the process of the neurons; the number of the BDNF‐immunoreactive neurons of the sensory cortex in model group was decreased, and the soma of the neurons were smaller and the processes were shorter in model group at postnatal day of 35 than those of control group; while the morphology of the BDNF‐immunoreactive neurons in model rats tended to be normal at postnatal day of 49. The western blot showed the corresponding results to those by the immunohistochemical methods. The present results suggested that the PV‐containing interneurons might play an important role in autism, and the changed PV interneurons probably weaken their inhibition to the pyramid neurons in the brain circuits that were related to autism; while the BDNF may be closely related with the abnormal development of the brain in autism.
Interaction of Hepatocyte nuclear factor 4alpha (HNF4alpha) with the TATA box binding protein (TBP) contributes to TFIID recruitment and HNF4alpha dependent transcription
Authors: Takahashi Hidehisa , Martin‐Brown Skylar , Washburn Michael P. , Florens Laurence , Conaway Joan W. , Conaway Ronald C.
Published: June 2021

HNF4alpha, a member of nuclear hormone receptor superfamily, is a key transcriptional regulator of many genes that control glucose and lipid metabolism. We have identified HNF4alpha as an interaction partner of the general transcription factor TFIID and of its TATA‐box binding subunit TBP. HNF4alpha binds directly to TBP in vitro and in vivo through HNF4alpha residues 52‐174, which include the DNA binding and hinge domains. We have demonstrated that in vitro, HNF4alpha recruits TFIID or TBP to an immobilized template through direct physical interactions with TBP. An HNF4alpha point mutant that fails to interact with TBP does not recruit TBP or TFIID to promoters in vitro. The same mutation interferes with HNF4alpha‐dependent activation of a reporter gene in vivo. These observations, together with results indicating that HNF4alpha can activate transcription in a highly purified, reconstituted transcription system lacking coregulators, are consistent with the model that the interaction of HNF4alpha with TBP contributes to HNF4alpha‐induced transcription.
Electron microscopy reveals that archaeal box C/D sRNPs are di‐sRNPs
Authors: Bleichert Franziska , Gagnon Keith T , Brown Bernard A , Maxwell Stuart , Unger Vinzenz M , Baserga Susan J
Published: June 2021

Methylation of ribosomal RNA (rRNA) is required for optimal protein synthesis. Multiple 2′‐O‐ribose methylations are carried out by small (nucleolar) box C/D guide ribonucleoproteins (s(no)RNPs), which are ubiquitous in nature from archaea to eukaryotes. Each site of methylation is dictated by base pairing between the specific guide s(no)RNA component of the s(no)RNP and the target rRNA. We have determined the first structure of a reconstituted and catalytically active box C/D sRNP by electron microscopy and single particle analysis. Our results reveal that archaeal box C/D sRNPs form an unexpected di‐sRNP structure which is required for efficient methylation activity, challenging the conventional paradigm of box C/D s(no)RNP architecture.

This work was supported by the NIH and the Boehringer Ingelheim Fonds.
Alternative polyadenylation of poly(A) binding protein nuclear 1 (PABPN1) mRNA
Authors: Hague‐Canto Lisa K. , Tian Bin , Lutz Carol S.
Published: June 2021

Polyadenylation is a 3' end RNA processing event that contributes to the regulation of gene expression by affecting turnover, stability, export, and translation of mRNA. Approximately 53 % of all human genes have multiple polyadenylation sites (PAS). Regulation at the level of alternative polyadenylation has begun to be investigated, but still requires much more elucidation.

Bioinformatics has identified unique alternative polyadenylation of a polyadenylation factor mRNA, poly (A) binding protein nuclear 1 (PABPN1). The mRNAs of this polyadenylation factor contains multiple polyadenylation sites.

Two polyadenylation signals were found for the PABPN1 mRNA. The proximal PAS is predicted from bioinformatics to be the strong polyadenylation site. The distal PAS was suggested to be a weaker site. However, we found the distal PAS contains putative novel auxiliary upstream polyadenylation elements. There are two G‐rich elements found in the upstream region of PABPN1 mRNA that are very highly conserved, suggestive of important function. Mutations of these two G‐rich elements seem to exhibit opposing effects on polyadenylation.

Previous studies from other labs have shown that G‐rich elements located downstream of polyadenylation sites enhance cleavage, and affect polyadenylation signal usage, but remains unexplored when upstream. Research supported by NIH and the Foundation of UMDNJ grants.
Functions of the Uch37 deubiquitinating enzyme in the proteasome and the INO80 chromatin remodeling complex
Authors: Yao Tingting , Ling Song , Jin Jingji , Cai Yong , Swanson Selene K , Washburn Michael P , Florens Laurence , Conaway Ronald C , Cohen Robert E , Conaway Joan W
Published: June 2021

Deubiquitinating enzymes (DUBs) are proteases that can antagonize ubiquitin‐mediated signalling by disassembling ubiquitin‐protein conjugates. How DUBs are regulated in vivo and how their substrate specificities are achieved are largely unknown. The conserved DUB Uch37 is found on proteasomes in organisms ranging from fission yeast to humans. We recently reported that nuclear Uch37 is also associated with the human INO80 chromatin remodeling complex (hINO80). Whereas deubiquitination by Uch37 is activated by proteasomal binding, Uch37 is inactive within hINO80. Interestingly, it can be activated by transient interactions of hINO80 wih the 26S proteasome. Thus, DUB activities can be modulated both positively and negatively via dynamic interactions with partner proteins. We will present evidence that, for Uch37, this regulation plays a role in control of histone H2B ubiquitination. This work was supported in part by National Institutes of Health grants R01 GM37666 (R.E.C.) and R37 GM41628 (R.C.C.). T.Y. is a fellow of the Leukemia and Lymphoma Society.
Purification and Characterization of the Cytosolic TRiC Complex from HeLa Cells
Authors: Knee Kelly Marie , Goulet Daniel Robert , Jameel Shea , King Jonathan A
Published: June 2021

The group II chaperonin TRiC is required for actin and tubulin folding and is thus essential for the reproduction of all eukaryotic cells. We have purified Human TRiC from cervical adenocarcinoma (HeLa) cells. To assess the substrate‐binding properties of Human TriC, we are characterizing its interactions with human γCrystallins. The γCrystallins are a family of β‐sheet eye lens proteins, whose partially folded intermediates have been well‐characterized. The Archaeal group II chaperonin Mm‐Cpn, from M. marapaludis, is a homolog of human TriC, and it has been shown that the complex suppresses aggregation of partially folded Human γCrystallins, and refolds the Crystallins to a native‐like conformation. We are investigating the potential of Human TRiC to suppress aggregation and restore unfolded γCrystallin to a native fold in a similar manner to Mm‐Cpn, using UV/Vis spectroscopy, and size exclusion chromatography. In collaboration with Wah Chiu (Baylor College of Medicine) and Judith Frydman (Stanford University) of the Center for Protein Folding Machinery, we are attempting to visualize the crystallin substrate in the chaperonin/substrate complex by Cryo‐EM.

Supported by an NIH Roadmap Award.
Secondary purification and oligomeric structure of three class II small heat shock proteins from Zea maize
Authors: Nau Christopher T. , Tims Hannah S. , Bouchard Robert A. , Meulia Tea , Pett Virginia B.
Published: June 2021

Small heat shock proteins (sHsps) are chaperones that bind to a substrate in order to reduce undesirable aggregation, holding substrate in a folding intermediate prior to the restoration of the substrate's native conformation by other ATP‐activated chaperones. To this end, sHsps are often expressed in vivo in response to increased temperature or other stress. The goal of the research was to identify a successful secondary purification protocol for three class II small heat shock proteins from Zea maize: 17.0, 17.6, 17.8 (designated according to their size in kilodaltons) and to analyze the oligomeric structure through a combination of native PAGE, glutaraldehyde cross‐linking, transmission electron microscopy (TEM), and fluorescence resonance energy transfer. Anion exchange chromatography under denaturing conditions was found to be effective in purifying the sHsps. TEM revealed two oligomeric sizes of ~12 and ~25 nm, larger than what has been found for other class II sHsps, while chemical cross‐linking suggests that the oligomeric structure of the sHsp is a dodecamer.

This work was supported in part by a grant to The College of Wooster from the Howard Hughes Medical Institute through the Undergraduate Science Education Program.
Characterization and C‐terminally truncational analysis of a 70‐kD molecular chaperone, DnaK, from the industrial bacterium Bacillus licheniformis
Authors: Chi Meng‐Chun , Liang Wan‐Chi , Lin Long‐Liu , Huang Hsien‐Bin
Published: June 2021

Aims

The aim of this study was to characterize the recombinant Bacillus licheniformis DnaK (BlDnaK) and to enhance the understandings of allosteric regulation in this protein by deletion mutations.

Methods and Results

The heat shock protein 70, heat shock protein 40 (DnaJ), and nucleotide exchange factor (GrpE) genes of B. licheniformis were overexpressed in Escherichia coli. The purified BlDnaK had a Vmax of 32.5 nmol Pi/min and a KM of 439 μM. In vivo, the dnaK gene allowed an E. coli dnaK756‐ts mutant to grow at 44oC. Simultaneous addition of DnaJ and GrpE synergistically stimulates the ATPase activity of BlDnaK by 11.7‐fold. A T76W mutant and three C‐terminally truncated BlDnaKs were also constructed to evaluate the role of up to C‐terminal 255 amino acids in allosteric regulation. The specific ATPase activity for BlDnaK, T76W, T76W/δC120, T76W/δC249, and T76W/δC255 was 1.45, 1.06, 2.32, 6.43, and 1.81 nmol/Pi/min/mg, respectively. Measurement of intrinsic tryptophan fluorescence revealed the significant alterations of the microenvironment of aromatic amino acids in the C‐terminally truncated BlDnaKs. The temperature‐dependent signal in the far‐UV region for T76W was consistent with that of BlDnaK, but the C‐terminally truncated mutants showed a higher sensitivity towards temperature‐induced denaturation.

Conclusions

This study provides evidence that BlDnaK has biochemical and molecular chaperone properties of the Hsp70/DnaK family. Our preliminary results also showed that the ATPase activity of the variants was stimulated differently from wild‐type BlDnaK in presence of BlGrpE.
Identification of substrate binding sites on a small heat shock protein reveals a unique mode of interaction between differentially aggregating substrates
Authors: Jaya Nomalie , Garcia Victor , Vierling Elizabeth
Published: June 2021

Small heat shock proteins (sHSPs) function as molecular chaperones by preventing irreversible protein aggregation. The mechanism of sHSP‐substrate interaction is not clearly understood, due to the difficulty of studying the heterogeneous complex formed between sHSPs and denaturing substrates. Using the photoactivatable cross‐linker, p‐benzoyl‐L‐phenylalanine (Bpa), site‐specifically incorporated into the well‐characterized sHSP from pea, PsHsp18.1, we show for the first time direct interaction between a sHSP and substrate in sHSP‐substrate complexes. Model substrates firefly luciferase (Luc), malate dehydrogenase (MDH) and a putative native substrate fructose bisphosphate aldolase (FBPA) were used in these experiments. Our results show that the N‐terminal arm of PsHsp18.1 makes direct contacts with all three substrates strengthening the relationship between this evolutionary variable region and substrate protection. Interestingly, Luc, a fast aggregating substrate, makes more contacts with the N‐terminal arm and limited contacts in the structurally conserved α‐crystallin region. However, MDH and FBPA, slow aggregating substrates, make direct contacts not only with the N‐terminal domain but also with the α‐crystallin region of the sHSP. These data lead us to propose a model in which the N‐terminal arm makes intial contacts with the hydrophobic regions of the unfolding substrate, but over time those contacts can be altered to include new contacts in the α‐crystallin region. This rearrangement may be substrate dependent and critical to the architecture of the soluble sHSP‐substrate complex. We are using chemical modification and limited proteolysis to gain further insight into the organization of sHSP and susbtrate within the complex.
NUCLEOTIDES BINDING Class A β‐LACTAMASE FROM BACILLUS CEREUS 569/H/9
Authors: Ott Taylor Ann , Cha Peter W , Shin Ben C , Shaw Robert W. , Kim Sung‐Kun
Published: June 2021

The Class A β‐lactamase from the pathogenic bacterium Bacillus cereus 569/H/9 catalyzes hydrolysis of β‐lactam antibiotics (e.g., Penicillin G), that impede the synthesis of bacterial cell wall. Although clavulanic acid is known as an inhibitor of Class A β‐lactamases, we are interested in the utility of aptamer inhibitors of Class A enzymes since they are such highly efficient and specific inhibitors of the Bacillus cereus 5/B/6 Class B enzyme. We have attempted to bind a random pool of nucleotides to the β‐lactamase using SELEX (Systematic Evolution of Ligands by EXponential enrichment) technique. We found that the random pool of nucleotides bind β‐lactamase using the electrophoretic mobility shift assay (EMSA), confirmed by both ethidium bromide staining and coomassie blue staining. After 8 rounds of SELEX, we were able to proceed with inserting the selected nucleotides into a cloning vector in order to identify nucleotide sequences. We also carried out another method for selecting aptamers using the interaction between biotin and streptavidin. After 10 rounds of SELEX, we identified the sequences that bind the β‐lactamase. Using the computer program Mfold, the secondary structures of aptamers that potentially inhibit the β‐lactamase have also been determined.
Screening of peptides binding on total cell extracts of Phytophthora capsici by phage display
Authors: Park Hye‐yeon , Kim Sung‐Kun , Yoon Moon‐Young
Published: June 2021

Phytophthora blight, caused by the oomycete pathogen Phytophthora capsici Leonian, is a completely destructive disease running rampant on the pepper plant, Capsicum annum. This blight is pronounced in regions where the pepper is commonly consumed and grown such as Korea as well as other parts of the world. At large, the main nuisance that has been reported about the Phytophthora disease is its responsibility for the major cultivation losses of the peppers. However, an even graver concern is that the pathogen can survive in the soil devoid of any host plants for many years. Therefore, P. capsici has been an attractive target for the prevention and diagnostics of Phytophthora blight of peppers. Recently, it has been reported that the molecule consists of multiple copies of P. capsici‐binding peptide, which is broadly applicable to the rapid development of plant defense mechanisms against novel plant disease resistance strategy. We screened P. capsici‐binding peptides using a M13 phage display method. In the course of 5 round biopanning, we identified P. capsici protein surface‐binding peptides from an M13 random peptide library, which was originally screened for other novel peptides that bind to P. capsici with a high affinity (picomolar range). As the result of identifying these peptides, they can be used as a basis to develop new potent inhibitors or diagnostic probes with a diverse polyvalence.
An Extended Binding Interface Spanning Two Sites for Interaction with PAI‐1 Alters the Function of Vitronectin in the Extracellular Matrix
Authors: Goswami Sumit , Wickerman Lindsay , Peterson Cynthia
Published: June 2021

Plasminogen activator inhibitor‐1 (PAI‐1), a member of the serpin family of proteins, circulates in blood in a complex with vitronectin (VN). In the extracellular matrix, VN exists as an oligomer with altered conformation. Although the binding of PAI‐1 to VN has typically been thought to follow a 1:1 stoichiometry, our lab has gathered substantial evidence to support a 2:1 stoichiometry of PAI‐1 binding to VN. This two‐site binding model involves an extended binding interface and leads to the oligomerization of VN. We thereby propose 1) the formation of this dual binding interface is necessary for the oligomerization of VN; 2) these oligomeric forms of VN associate more with matrix components; and 3) the oligomeric PAI‐1/vitronectin complexes promote cell binding. In this study, we have used mutant forms of PAI‐1 to show that disruption of the second‐site interaction leads to less efficient oligomerization of VN. We have also shown that PAI‐1‐mediated oligomeric forms of VN exhibit increased association with specific matrix components, including Collagen‐IV and Heparan Sulfate proteoglycan (HSPG). Such association promotes cellular binding to the matrix. Thus, we have characterized one of the protease‐independent features of PAI‐1, which affects the assembly and function of matrix‐associated VN.
Overexpression of Cu/Zn‐SOD and/or catalase accelerates benzo(a)pyrene detoxification by up‐regulation of AhR
Authors: Guo Zhongmao , Wang Zefen , Ramesh Aramandla , Zhou Lichun , Yang Hong
Published: June 2021

Overexpression of Cu/Zn‐superoxide dismutase (SOD) and/or catalase has been shown to delay benzo[a]pyrene (BaP)‐accelerated atherosclerosis in hypercholesterolemia mice. The goal of this study is to assess the effect of these antioxidant enzymes on the expression of aryl hydrocarbon receptor (AhR) and its target genes. We observed that the AhR protein level is 18‐20 fold higher in the aorta endothelial cells (MAECs) obtained from mice overexpressing Cu/Zn‐SOD and/or catalase than in those from wild‐type mice. Following BaP exposure, the BaP reactive intermediates accumulated in the transgenic cells are significantly less, while the BaP‐induced cytochrome P450 (CYP) protein levels, and BaP‐elevated glutathione S‐transferase (GST) activity are significantly higher in the transgenic cells, in parallel with elevated CYP1A1, CYP1B1 and GSTp1 mRNA levels, when compared to wild‐type MAECs. Moreover, knockdown of AhR with RNA interference inhibits BaP‐induced CYP and GST expression and suppresses BaP detoxification in MAECs. These findings imply that up‐regulation of AhR is a mechanism for the increased BaP detoxification by overexpressing Cu/Zn‐SOD and/or catalase, which contributes to understanding the inhibitory effect of these antioxidant enzymes on BaP‐accelerated atherosclerosis. NIH grant support: RR003032 and K01HL‐076623 (H.Y.); R01ES014471 (Z.G.).
The crystal structure of CYP3A4 in complex with bromocriptine
Authors: Wester Michael R , He You‐Ai , Gajiwala Ketan S , Parge Hans E , Burke Benjamin , Zientek Michael A , Lee Caroline A
Published: June 2021

CYP 3A4 is a major enzyme in the clearance pathway of many xenobiotics and it has been predicted that the active site of CYP3A4 displays significant structural plasticity in order to accommodate structurally diverse ligands. In an effort to understand the conformational changes in response to ligand binding, we are pursuing co‐crystal structures with CYP3A4 in complex with molecules that sample the diversity of known ligands. The crystal structure of CYP3A4 in complex with bromocriptine has been solved to 2.7 angstroms resolution. Bromocriptine is a CYP3A4 substrate and a dopamine receptor agonist used to treat Parkinson's disease, that falls at the large end of the 3A4 ligand spectrum with a molecular weight of 655 daltons. In the crystal structure, bromocriptine is bound in the active site of CYP3A4 in an orientation that places the proline ring 3.8 angstroms from the heme iron in an orientation consistent with the known site of metabolism. Surprisingly, only minimal conformational changes to the 3A4 protein backbone are required for binding of bromocriptine. The RMSD of Ca atoms in the bromocriptine co‐crystal structure from those in the apo 3A4 structure 1TQN.pdb is 0.89 angstroms. Bromocriptine is oriented in the active site through hydrogen bonds with SER119 and ARG212. Only minor side‐chain rotamer changes of ARG 212 are required to accommodate these binding interactions. These results suggest that further crystal structures that probe the diversity of CYP3A4 ligands will be necessary to characterize the conformational flexibility of this enzyme.
Bioactive Nanostructures for Regeneration
Authors: Stupp Sam
Published: June 2021

Bioactive nanostructures molecularly crafted to signal cells or deliver cargo to specific cells have great potential to regenerate tissues and cure disease. The chemistry of such nanostructures should allow them to interact specifically with cell receptors or intracellular structures. Ideally, they should also disintegrate into nutrients within an appropriate time frame. The organization of these nanostructures at larger length scales comparable to cells and large colonies of cells will also be critical for their function. Our laboratory has developed an extensive family of amphiphilic molecules that self‐assemble into supramolecular nanofibers with the capacity to display a large diversity of signals to cells1‐5. Using both polymers and small molecules we have also developed macroscopic constructs that can organize or confine cells in three dimensions. This lecture will illustrate the use of these systems to regenerate axons in the central nervous system for spinal cord injuries, to promote angiogenesis in cardiovascular therapies, induce bone formation, and mediate the differentiation of stem cells.
A novel approach for investigating glycan involvement in embryonic cellular interactions. III. whole embryo periodate studies
Authors: Ghazarian Haike , Oppenheimer Steven B.
Published: June 2021

Periodate oxidation has been widely used to degrade the carbohydrate components of molecules to help determine the role of sugars in molecular function. In this study, 24.5‐26 hr early gastrula Lytechinus pictus sea urchin embryos were incubated with specific ultra‐low concentrations of sodium (meta) periodate that degrades some carbohydrates by selectively oxidizing C‐C bonds between vicinal diols. Incubations were carried out in 15 degrees C artificial seawater in 96 well microplates for 24‐25 hrs. After the incubation period, embryos were examined for viability, fixed in 5% formaldehyde, and the morphological characteristics of 5664 embryos were quantitatively assessed as embryos possessing complete attached archenterons, incomplete unattached archenterons, no invagination, exogastrulation, or embryos that were dead. At 1.2x10−6 ‐3.9x10−5 mg periodate/ml 15‐30% of the embryos were swimming and possessed incomplete unattached archenterons, while the controls without periodate displayed 2.3% incomplete archenterons. At higher concentrations of periodate (7.8x10−5‐1.0x10−2 mg periodate/ml), embryo death occurred. Using a two‐sample equal variances t‐test, the numbers of unattached archenterons in the 1.2x10−6‐3.9 x10−5 mg periodate/ml treated embryos were significantly different from those in the controls without periodate at a p value of less than 0.001. The use of periodate in probing whole, living embryos is novel and could become widely used to help identify the role of glycans in interacting systems (Supported by NIH NIGMS SCORE (S0648680), MARC, RISE, the Joseph Drown Foundation, and the Sidney Stern Memorial Trust).
Activity of immobilized lectin stored at different temperatures
Authors: Zem G. C. , Khurrum M. , Allen J. , Petrosyan S. , Dayan M. , Kawashima R. , Goldstein L. , Bagerdjian A. , Uber L. , Nguyen M. , Borna L. , Adams L. , Fukasawa A. , Simon C. , Karout R. , Yaghoubi A. , Mesrkhani A. , Talebi M. , Recinos S. , Khojastehfar P , Varvaryan B. , Khademi T. , Hashemieh M. , Shaklian A. , Termendjhian A. , Kaur Rajal M. , Nersesyan A. , Adorjan G. , Manukyan M. , Dadayan R. , Andrews T. , Gabrielyan Z. , Oppenheimer S. B.
Published: June 2021

Here, the mannose‐rich yeast binding activity of immobilized Con A was tested when the beads were stored for up to 60 days in a freezer (‐20 C), a refrigerator (4C), and at room temperature (24C), compared with controls stored in a refrigerator under the manufacturer's specifications. In about a total of 9,000 replicate experiments, beads stored at the 3 temperatures studied, maintained their ability to effectively bind yeast (Saccharomyces cerevisiae) over the 60 day period. Linear trend lines for the 3 storage temperature graphs confirmed that the room temperature condition was slightly better than the refrigerator condition, that was slightly better than the freezer condition. The difference between the trend lines was at their widest divergence about 12%. While the beads bound yeast effectively after being stored at the temperatures tested, the experiments were conducted at 24C. It is likely that if the actual experiments were done at different temperatures, the kinetics of yeast binding to the beads would be affected by temperature. This issue was not examined in this study but should be investigated, as many experiments in a wide variety of systems might require being conducted at different temperatures. This is a model system for studying the efficacy of potential glycan‐based anti‐infection drugs (Supported by NIH NIGMS SCORE (S0648680), MARC, RISE, the Joseph Drown Foundation and the Sidney Stern Memorial Trust).
Plasmodium falciparum adhesive protein that mediates sequestration of parasite‐infected red blood in human placenta and cause pregnancy specific malaria
Authors: Goel Suchi , Goyal Atul , Kumar Manoj , Achur Rajeshwara N , Li Guangfu , Gowda D. Channe
Published: June 2021

Malaria caused by Plasmodium falciparum results in millions of deaths every year. In endemic areas, although immunity to malaria develops during childhood years, women become susceptible to malaria during pregnancy. This is due to the sequestration of P. falciparum infected erythrocytes (IEs) in the placenta by binding to host chondroitin 4‐sulfate (C4S). A parasite protein called var2CSA, a member of antigenically diverse family of 60 parasite proteins expressed on the erythrocyte membrane is involved in IE adherence to C4S. Var2‐CSA consists of several Duffy binding like (DBL) domains and how exactly they bind C4S remain unclear. We expressed various DBL domains on yeast cell surface and analyzed cell adherence to C4S. None of the DBL domains could support binding of yeast to C4S, suggesting that individual DBL domains cannot support IRBC binding. It is possible that a conformational structure involving two or more domains is involved in IRBC binding. Alternatively, var2CSA alone may not be sufficient for IE binding, instead a multiprotein complex is involved. To test this hypothesis, we performed affinity binding of parasite ligand from the lysates of 35S‐labeled IEs using C4S‐conjugated beads. In addition to var2CSA, several other proteins could specifically bind to C4S‐beads. Together these data suggest that a complex of var2CSA and other proteins is involved in IE binding. Funded by AI45086 from NIAID, NIH.
Activation of Metalloproteinase in Breast Epithelial Cells by Direct and Secondhand Cigarette Smoke.
Authors: Mukherjee Shyamali , Das Salil K
Published: June 2021

Degradation of extracellular matrix by metalloproteinases (MMPs) plays an important role in tumor development and its progression. Epidemiological studies have linked cigarette smoking to breast cancer. However, the exact mechanism has not been explored. Due to difficulties in conducting such studies in humans, we have developed a cell culture smoke exposure system using the principle of the air/liquid exposure technique. Breast epitheial cells (MCF 10, MCF 12A and MCF 12F) were grown in DMEM/F‐12 media and exposed to both direct and secondhand smoke generated from 1R3F cigarettes in a THRI MS‐SS smoke exposure systems. A sham control group was also used. For the dose response study breast cells were exposed to 0.03, 0.06, 0.12 and 0.18 puff/ml and incubated for 24 h. For time response study, cells were exposed to 0.18 puff/ml and then incubated for 6, 12, 24 and 36 h. Our results indicated that both direct and secondhand smoke exposure caused an increase in cell proliferation at low doses and cellular apoptosis at higher doses. Cells were isolated after 7 days, and Western blot analysis was performed to determine whether smoke exposure causes any change in the MMP2 and MMP9 protein levels. Both direct and secondhand smoke exposure caused a significant increase in the MMP protein levels in all three breast cell lines. (Supported by FAMRI Clinical Innovator Award # 062415).
Targeting the Cysteine‐rich domain in HIV‐1 Tat by 15‐deoxy‐delta(12,14)‐prostaglandin J2
Authors: Narayan Vivek , Kalantari Parisa , Henderson Andrew J , Prabhu K. Sandeep
Published: June 2021

Anti‐retroviral therapies for AIDS have typically targeted HIV proteins integrase, protease, and reverse transcriptase. Transactivator of transcription (Tat), an early HIV protein that increases the efficiency of proviral transcription and replication, has not yet been targeted successfully. Tat has a cysteine‐rich region within its transactivation domain consisting of seven cysteines (Cys). Endogenous cyclopentenone prostaglandins (CyPGs), such as 15d‐PGJ2, are involved in regulating cellular growth, differentiation, and homeostasis. CyPGs have been shown to possess anti‐viral activity by an unknown mechanism. 15d‐PGJ2¬ reacts with Cys selectively via the reactiveá,â ‐unsaturated ketone moiety. Our studies indicate that 15d‐PGJ2 causes a dose‐dependent inhibition of Tat‐dependent HIV transcription and replication, which is independent of the NFêB pathway. Using a biotinylated derivative of 15‐d‐PGJ2, we demonstrate that the Cys‐thiols in Tat are targeted in a covalent Michael addition reaction. Such a 15d‐PGJ2 modified Tat lost its ability to transactivate the HIV long‐terminal repeat (LTR) in human macrophages. Treating HIV‐infected human macrophages with 15d‐PGJ2 also inhibited viral replication. These data show that Tat acts as a molecular target of CyPG and suggests a novel therapeutic approach to complement current anti‐retroviral strategies for HIV/AIDS.
Ca2+ regulation of the Na+/Ca2+ exchanger
Authors: chaptal vincent , mercado‐besserer Gabriel , ottolia michela , nicoll deborah , philipson kenneth , abramson jeff
Published: June 2021

Ca2+ is an essential ion involved in a multitude of processes essential for life. In the heart, Ca2+ influx into the cytosol leads to muscle contraction. In order for the heart to function properly, the same amount of Ca2+ entering the cytosol must be removed, to allow the heart to relax and refill with blood. Defects in Ca2+ cycling result in smaller and slower calcium transients that are characteristic of heart failure. The Na+/Ca2+ exchanger (NCX1.1) serves as the major mechanism for Ca2+ extrusion from the myocyte, utilizing the Na+ electrochemical gradient to counter‐transport three Na+ into the cell while extruding one Ca2+. In addition, NCX1.1 is also regulated by these two ions, although these mechanisms remain incompletely understood.

Structurally, NCX1.1 has nine predicted trans membrane helices and a large cytoplasmic loop of approximately 500 residues. This loop contains two Ca2+ Binding Domains (CBD1 & CBD2) that regulate NCX1.1 activity. We solved the structures of CBD1 and CBD2 using X‐ray crystallography and performed a comprehensive mutational and electrophysiological analysis that shed new light on their crucial role in the regulation of NCX1.1 activity.

To learn how regulation occurs, we will explore the interactions of these regulatory Ca2+ binding domains with each other, as well as with other parts of NCX1.1.

This work has been funded by AHA and HFSP.
Structure of voltage sensitive calcium channels in mechanosensitive osteocytes
Authors: Majid Amber Sobia , Thompson William R. , Czymmek Kirk J. , Duncan Randall L. , Farach‐Carson Mary C.
Published: June 2021

Regulation of skeletal remodeling is a major focus of attention in translational osteoporosis research. A recent association study including 200 single‐nucleotide polymorphisms on human chromosome 3p21 revealed CACNA2D2, (one of four α2d subunits of the voltage sensitive calcium channel (VSCC) complex), as a novel susceptibility marker for bone mineral density (BMD) variation. VSCCs are a complex of polypeptides that influence osteoblast mechanosensitivity and consist of a pore forming α1 subunit, an intracellular ? subunit, a dimer of α2 and d subunits, and a ? subunit in some tissues. This study characterized the structure of the VSCC complex in an osteocyte‐like cell line, MLO‐Y4, using RT‐PCR, Western blot, and immunostaining. We demonstrated that the T‐type Cav3.2 (α1H) subunit is the predominant α1 subunit expressed in osteocytic cells in vitro and in cortical bone. Additionally, MLO‐Y4 cells express α2d1, ?7, and ?1‐4 subunits. These findings represent a shift in the expression of VSCC from long lasting (L‐type) in osteoblasts to transient (T‐type) channels in osteocytes. An association of the α2δ subunit with T‐type channels in osteocytes could stabilize the functional channel and provide a mechanism for interaction with the extracellular environment. This key protein complex may provide a crucial function for mechanosensitive osteocytes in bone, having implications in skeletal remodeling and BMD.
Filling the Gap: A streamlined approach for monitoring expression and purification of membrane proteins with a periplasmic C‐terminus via GFP fluorescence
Authors: Hsieh Jennifer M , Mercado Gabriel C , Bui Ha‐Quyen , Abramson Jeff
Published: June 2021

Crystal structures of membrane proteins are notoriously difficult to obtain. These proteins reside in phospholipid bilayers making them difficult to express, purify, stabilize and structurally resolve. Previously, inner membrane proteins (IMP) have been recombinantly expressed and monitored using a C‐terminal green fluorescent protein (GFP) fusion in Escherichia coli. This method proved extremely useful, but is only effective for IMP with C‐termini localizing to the cytoplasm (CI), where the GFP folds correctly. To fill the gap for IMP with C‐termini localizing to the periplasm (CO), we developed the expression vectors: pWarf(‐) and pWarf(+). While pWarf(‐) fuses GFP to the C‐terminus of the IMP, pWarf(+) fuses the single transmembrane helix protein Glycophorin A and subsequent GFP to the IMP. The pWarf(+) vector effectively converts CO IMP to CI IMP. Eleven E. coli IMP from three families were selected for their predicted CO topology. High level whole cell fluorescence was observed only in the pWarf(+) system. Using the pWarf(+) vector, overexpression was rapidly screened, detergents were selected by fluorescent size exclusion chromatography and stability was monitored. Large scale purification is in progress for crystallization trials. By developing the pWarf vector system, a comprehensive, streamlined approach to monitoring overexpression, purification and stabilization has been achieved for IMP. Funding from AHA.
HDAC inhibitor, TSA, downregulates Myc transcription in human cervical cancer cell lines
Authors: Li Hui , Li Xiaoling , Liu Xuefeng , Wu Xinxing
Published: June 2021

Histone deacetylase (HDAC) inhibitors have shown promise in clinical cancer therapy and to induce p21 expression in a p53‐independent manner via increased acetylation of the chromatin at the Sp1 sites in the p21 promoter region. Our previous study has shown that Trichostatin A (TSA), a HDAC inhibitor, induces p21 through downregulation of Myc expression and release of Myc bound from the p21 promoter in HeLa cells. However, the mechanism of how TSA downregulates Myc remains unclear. In this study, we treated two human cervical cancer cell lines, HeLa and SiHa, with TSA, confirming that TSA decreased Myc protein with Western blot. The decreased level of Myc might be due to a decreased stability of Myc protein or downregulated transcription of Myc in TSA treated cells. Our results from both regular RT‐PCR and real time RT‐PCR demonstrated a significant decrease of Myc mRNA in TSA treated cells compared to that in untreated cells. Thus, TSA treatment downregulates Myc expression at the transcriptional level in human cervical cancer cell lines. The results provide the possibility that TSA may alter chromatins at the Myc promoter via direct or indirect modifications. Chromatin changes at the Myc promoter in TSA treated cells are being investigated.

This work was supported by grant from the National Natural Science Foundation of China (No. 30500465, No. 30810103052) .
Regulator of G protein Signaling 6 (RGS6) is Upregulated in Cancer cells by Doxorubicin and Modulates Doxorubicin‐induced DNA damage signaling
Authors: Huang Jie , Chatterjee Tapan K , Mayuzumi Daisuke , Fisher Rory A
Published: June 2021

RGS proteins were discovered as negative regulators of heterotrimeric G protein signaling. The precise biological role of all but a few RGS proteins is unknown, as is the need for such a large structurally diverse protein family if RGS proteins function only as G protein GAPs. Here, we provide new evidence suggesting a role of RGS6 in regulating DNA damage responses in cancer cells. Doxorubicin (Dox) treatment of cancer cell lines led to dramatic increases in RGS6 protein levels, which were undetectable in unstimulated cells. Dox induction of RGS6 protein was accompanied by significant increase in RGS6 gene transcription as assessed using an RGS6 gene promoter reporter. Further, expression of RGS6 in MCF7 cells promoted phosphorylation of p53 in response to a low dose of Dox, which alone had no effects. This activity of RGS6 requires its DEP domain but is independent of its interaction with G proteins. RGS6 expression in several cancer cell lines promoted cell cycle arrest and growth suppression. ATM, an upstream activator of p53, may be involved in RGS6‐induced p53 phosphorylation, as expression of RGS6 promotes ATM activation in response to a submaximal dose of Dox. Our data identify RGS6 as a target of Dox and suggest that RGS6 may play a novel role as a modulator of chemosensitivity in cancer cells. Importantly, these actions of RGS6 are independent of its interaction with G proteins.

NIH (GM075033) and AHA (0750057Z).
Single and multiple courses of antenatal steroids regulate connexin 36 abundance in the ovine fetal brain
Authors: Sadowska Grazyna B. , Stonestreet Barbara S.
Published: June 2021

Connexin 36 (Cx 36) forms gap junctions (GJ). GJ facilitate intercellular communication and are important in neuronal differentiation during development. Cx 36 is the main GJ between neurons, increases during rodent development and is up regulated by dexamethasone (DEX) in pancreatic islets. The effects of steroids on GJ proteins have not been examined in fetal brain. We examined the effects of single and multiple courses of DEX on Cx 36 abundance in the cerebral cortex (CC), cerebellum (CL) and cervical spinal cord (SP), and differences in Cx 36 protein abundance among fetal brain regions. Ovine fetuses at 70% of gestation were examined after a single course of four 6 mg DEX or placebo (PL) injections were given every 12 h for 48 h to the ewes, or the same DEX or PL given course once a wk for 5 wks. Regional brain tissue was frozen after the last DEX or PL dose. Cx 36 protein expression was determined by Western immunoblot. A single course of DEX increased (P < 0.05) Cx 36 protein abundance compared with PL in the CC and SP, and multiple courses increased Cx 36 in SP, but not in CC or CL. Cx 36 was higher in SP than CL. Maternal DEX treatment regulates Cx 36 proteins in vivo in the fetal CC and SP, but not in CL. Different DEX regimens regulate Cx 36 protein abundance in specific brain regions. The greater abundance of Cx 36 protein in SP than CL may suggest the presence of more functional channels in SP in the premature fetal brain.