bluuebunny/crossref_metadata_2025 huggingface dataset:
The mechanism of transcription antitermination by RfaH
Authors: Artsimovitch Irina , Belogurov Georgiy A , Sevostyanova Anastasiya
Published: June 2021

RfaH is a bacterial antitermination factor whose function depends on a 12‐nt ops element located in the leader regions of several operons; the ops element both induces RNA polymerase (RNAP) pausing and serves as a recognition site for RfaH. During recruitment, RfaH establishes transient contacts with the ops bases in the non‐template DNA strand exposed on the RNAP surface. After recruitment, RfaH remains bound to RNAP throughout transcription of the entire operon and switches RNAP into a processive state using four mechanisms. First, RfaH competes with σ and prevents σ‐dependent arrest during elongation. Second, RfaH competes with NusG and thus indirectly reduces Rho‐dependent termination. Third, RfaH induces forward translocation of RNAP, thereby inhibiting isomerization into an off‐pathway state that occurs from a pre‐translocated conformation. Fourth, RfaH establishes simultaneous contacts with the β’ clamp and the β lobe, the two elements that maintain the RNAP grip on the nucleic acid chains, thereby preventing the clamp opening that is thought to occur during pausing. While the first three mechanisms are specific to the RfaH‐like proteins, the last mechanism is likely broadly conserved. We propose that structurally diverse antiterminators from bacteria to humans solidify the transcription elongation complex to inhibit conformational transitions that underlie pausing and termination.
Rehybridization of Promoter DNA Targets in Chromatin to Discover Transcription Factors of Oncogenes
Authors: Zhang Huiping , Haegele Joseph , Shin Woo‐Cheol , McGown Linda B
Published: June 2021

Identification of transcription factors (TF) assembled at promoter regions of oncogenes is vital to understand how gene expression is altered in cancer and in tumor microenvironments. Most TF identification methods are based on one‐on‐one DNA/protein interactions in vitro, which may not reflect DNA/protein interactions in vivo and may overlook protein complex information. Moreover, existing methods for chromatin pull down target pre‐identified proteins rather than DNA, which limits the ability to discover new proteins that may bind to specific genomic sequences. We are pursuing a new approach, chromatin rehybridization (CR), which preserves and detects DNA/protein and protein/protein interactions that occur in live cells and targets specific DNA sequences rather than associated proteins.

The G‐quadruplex forming Pu27‐mer located upstream of the c‐MYC oncogene and its associated TFs including cellular nucleic acid binding protein (CNBP) serve as the model system for development of the CR technique. Preliminary results demonstrate that CNBP is pulled down with the targeted Pu27‐mer in MCF‐7 cells using CR. At least one unreported TF associated with c‐MYC was also detected and will be identified using mass spectrometry.

The CR method addresses the need for full proteomic analysis of particular genomic regions. This work is supported by NIH through grant R21 CA134419‐01A1.
Mediator Structure and Interaction with the Basal Transcription Machinery
Authors: Asturias Francisco J , Cai Gang , Imasaki Tsuyoshi , Yamada Kentaro , Cardelli Francesco , Takagi Yuichiro
Published: June 2021

Understanding the structure and conformational dynamics of Mediator is critical for unraveling the essential role the complex plays in regulation of eukaryotic transcription initiation. We used single particle macromolecular electron microscopy (EM) to study the conformational variability of Mediator and that information enabled us to determine a cryo‐EM structure of the complex at ~26Å resolution. Comparing the structures of free and RNA polymerase II (RNAPII)‐associated Mediator made possible a detailed description of Mediator structural modules and the rearrangement they undergo upon interaction with RNAPII, both of which appear to be conserved from yeast to humans. We have also pursued independent EM analysis of the Head module responsible for control of the Mediator‐RNAPII interaction. Conformational analysis of a recombinant Head module revealed distinct conformations and comparison with the structures of sub‐complexes provided information about subunit organization. All of this information, along with results from biochemical and structural analysis of the interaction of the Head module with the TATA‐binding protein (TBP) and RNAPII subunits Rpb4 and Rpb7, suggest a possible mechanism for Mediator regulation of RNAPII interaction with promoter DNA.

Supporteb by NIH R01 GM67167
TrmB an archael transcriptional regulator
Authors: Thomm Michael , Surma Melanie
Published: June 2021

In Pyrococcus furiosus two binding protein–dependent, substrate–specific ABC transporters could be identified, one of it being a trehalose/maltose (TM)–transporter, which is responsible for the uptake of trehalose and maltose. By binding to the respective operator sites TrmB prevents the further binding of transcription factors and subsequent RNA polymerase recruitment, which leads to an inhibition of transcription. With the aid of in vitro transcription assays using purified components of P. furiosus it could be shown that TrmB–mediated inhibition of transcription on both promoters is released by specific carbohydrates with maltose and trehalose being inducers for the TM operon, and maltodextrins as well as sucrose releasing inhibition on the MD promoter.

TrmBL1 is homologue to TrmB and is involved in the regulation of genes encoding glycolytic and gluconeogenetic enzymes, respectively, where it recognizes a conserved sequence motif called TGM. It inhibits transcription of glycolytic genes with maltose, maltotriose and fructose as inducers (3). On the other hand TrmBL1 activates the expression of gluconeogenetic genes as well. Since the TGM is not present in the promoter region of the TrmBL1 gene, the TGM is not essential for TrmBL1 target recognition (3).

Both TrmB and TrmBL1 share the unique property of recognizing different DNA sequences on different promoters and showing distinctive, promoter–dependent sugar specificity.

In vitro transcription assays, EMSA and DNaseI footprinting experiments proved that TrmB and TrmBL1 have at least two different HTH motifs and recognize their respective target promoters with different helices within their N–terminal DNA binding domains.
Residence Time and in vivo Antibacterial Activity ‐ A Critical Aspect of Lead Compound Optimization
Authors: Lu Hao , England Kathleen , Pan Pan , Slayden Richard , Tonge Peter J.
Published: June 2021

Lead optimization is one of the critical steps in modern drug discovery. However, in many cases the lack of a direct relationship between in vitro activity and in vivo efficacy has significantly hindered the transformation of compounds from “inhibitors” to “drugs”. One possible reason for this failure may arise from the strong dependence of lead optimization on in vitro thermodynamic parameters, such as IC50 and Ki values. However, the length of time that drugs remain bound to their targets may play a more important role in determining in vivo drug activity instead of the thermodynamics of drug‐target interactions. In our study of the enoyl‐ACP reductase from Francisella tularensis, we have found that in vivo efficacy of our designed inhibitors correlates positively with their residence time but not with their in vitro antibacterial activity (Ki and MIC). In addition, a second study that targeted the enoyl‐ACP reductase from Mycobacterium tuberculosis, also demonstrated that inhibitors with longer residence times are more effective at reducing the CFUs in the spleens mice infected with M. tuberculosis. We propose that both the thermodynamics and kinetics of drug‐target complex formation should be determined in drug discovery programs and that residence time is a better predictor of in vivo drug efficacy than in vitro data acquired at equilibrium or at constant drug concentration.
Selective Inhibition of Nitric Oxide Synthase by Suramin
Authors: McNamara Alyssa , Carroll Andrew , Stevens‐Truss Regina
Published: June 2021

Nitric oxide synthase (NOS) is a formidable target for drug design as its reaction product, nitric oxide (·NO), is linked to a number of pathological conditions including Alzheimer's disease. Suramin is a polysulfonated naphthylurea that has been in use clinically for the treatment of trypanosomiasis and onchocerciasis. It has been shown to exhibit a broad range of biological actions in vivo and in vitro, and specifically has been shown to discriminate among calmodulin‐binding sites on proteins. In this study suramin was shown to inhibit both neuronal NOS and inducible NOS, however, it was found to have a greater effect on inducible NOS. Inhibitory constant for the neuronal NOS was found to be 75 μM while for the inducible NOS that value was 2 μM; over a 30 fold selectivity. Dixon analysis suggests that suramin acts as a non‐competitive inhibitor of the neuronal isoform, and as an uncompetitive inhibitor of the inducible isoform. Western analysis suggests that the inhibition is due to suramin's interference with the binding of calmodulin. These results point to a potential new target site on NOS for drug design.
LRRK2 Kinase Activity is Dependent on Dimerization
Authors: Sen Saurabh , West Andrew
Published: June 2021

Current therapies for Parkinson's disease (PD) fail to slow the onset or progression of disease. Pathogenic missense mutations in the PARK8 locus encoding the leucine‐rich repeat kinase 2 (LRRK2) gene cause late‐onset disease that mimics the clinical and neurochemical aspects of idiopathic disease. The most common LRRK2 mutations occur in the kinase domain and enhance both kinase activity and toxicity in vitro. LRRK2 represents a clear target for modulation by small molecule kinase inhibitors that may provide neuroprotection in patients that harbor LRRK2 mutations and possibly PD cases without LRRK2 mutations. Through the biochemical characterization of LRRK2 protein, we find that kinase activity is dependent on dimerization whereas further oligomerization results in a loss of activity. We develop a Bimolecular Fluorescence (BiFC) assay based on eGFP fluorescence dependent on protein dimerization. Cell‐based LRRK2 kinase assays should hasten the identification of specific and effective LRRK2 inhibitors.
Analysis of novel low nanomolar thiosemicarbazone inhibitors of cruzain
Authors: Chavarria Gustavo Edgardo , Wiley Jacob A. , Devanna Kishore Kumar Gaddale , Arispe Wara M. , Li Weiyue , Adamson Lauren J. , Jones Lindsay M. , MacDonough Matthew T. , Pinney Kevin G. , Trawick Mary Lynn
Published: June 2021

Chagas’ disease, caused by the protozoan Trypanosoma cruzi, affects millions of people in Latin America where it is a major cause of heart disease. Currently, there is no satisfactory treatment for the chronic stage of the disease and there is an urgent need for new therapeutic agents. Cruzipain, a potent Trypanosoma cruzi protease, and a member of the papain family of cysteine proteases, is a validated molecular target for the parasite. An analysis of a small library of thiosemicarbazones revealed that the 3,3′‐dibromobenzophenone thiosemicarbazone was an excellent enzyme inhibitor (Siles, R. et al., Bioorg. Med. Chem. Lett., 2006,16:4405–4409). Testing of a second generation of substituted monobromo‐benzophenone thiosemicarbazones against cruzain, a recombinant form of the target protease, revealed a number of compounds that were more effective than the dibromobenzophenone thiosemicarbazone lead compound. A group of these thiosemicarbazone derivatives demonstrated low nanomolar IC50 values against cruzain using a fluorometric microplate assay. This study was supported in part by funds from the Baylor University Undergraduate Research and Scholarly Achievement Small Grant Program and the Vice Provost for Research (M.L.T.) and by a Welch Foundation Grant, AA1278 (K.G.P.)
Biochemical and Docking Analysis of Polyisoprenylated Methylated Protein Methyl Esterase with Substrates
Authors: Duverna Randolph , Lamango Nazarius
Published: June 2021

Polyisprenylated proteins (PP) methylation by polyisoprenylated protein methyl transferase (PPMTase) is counteracted by polyisoprenylated methylated protein methyl esterase (PMPMEase), making it the only reversible step of the polyisoprenylation pathway. Since PMPMEase and PPMTase may influence both the structural and functional conformations of PP, a thorough study of these enzymes is essential to our understanding of the physiology of PP. PMPMEase has been reported under various pseudonyms because of its apparent broad substrate metabolism. The current study aimed to reveal its role as the endogenous polyisoprenylation‐specific esterase. A series of N‐, S‐ and O‐substituted substrates were analyzed by Michaelis‐Menten kinetics using porcine liver PMPMEase and in silico determination of binding affinities using Arguslab. Consistent with the biochemical analysis, the S‐ethyl analog yielded an AScore binding energy of −11.32 compared to −13.48, −14.88, −16.15 and −16.81 kcal/mol for S‐prenyl (C‐5), S‐trans‐geranyl (C‐10), S‐trans, trans‐farnesyl (C‐15) and S‐all trans‐geranylgeranyl (C‐20), respectively. As judged by the KM and AScore values, substrates with the natural modifications have the highest affinity for PMPMEase. The results demonstrate that PP are the likely endogenous substrates for PMPMEase.
Isolation and characterization of carboxypeptidase H gene from flounder (Paralichthys olivaceus)
Authors: Kim Young Tae , Seo Yong Bae
Published: June 2021

Carboxypeptidase H (CPH) is a putative post‐translational processiong enzyme, which removes basic amino acids from the intermediates protein biosynthesis. CPH gene was isolated from the flounder (Paralichthys olivaceus) brain cDNA library and consisted of 1590 base pairs encoding 455 amino acids residues. The nucleotide sequence of CPH gene was analyzed with that of other species including zebrafish and mouse turned out to be well conserved during evolution. The expression of the CPH mRNA was confirmed by Northern blot analysis. A recombinant protein of approximately 50 kDa, which corresponds to CPH, was functionally expressed in E.coli. The biochemical studies on the expressed protein were performed and the results obtained from this study will provide a wider base of knowledge on the primary structure of the flounder CPH at the molecular level.
Proanthocyanidins from cranberry (Vaccinium macrocarpon) inhibit matrix metalloproteinase expression in prostate cancer cells by affecting multiple cell signaling pathways
Authors: Deziel Bob Andrew , Patel Kunal , Neto Catherine , Gottschall‐Pass Katherine , Hurta Robert
Published: June 2021

This study examined the effects of a proanthocyanidin‐enriched fraction (PAC) from cranberry (Vaccinium macrocarpon) on matrix metalloproteinase (MMP) activity in DU145 human prostate cancer cells in vitro. MMP‐2/−9 activity was inhibited by PAC in a dose/time dependent manner. PAC (25 ug/mL) decreased cellular viability 30% post 6 h.of treatment and had no significant effect on cellular viability post 3 h. Treatment of DU145 cells with PAC for 3 h. resulted in increased expression of p‐p38, pERK‐1, pERK‐2 protein levels. Increased JNK1 and decreased JNK2 protein expression occurred in response to PAC with corresponding alterations in p‐JNK1 and p‐JNK2. PAC treatment decreased protein expression levels of AKT, p‐AKT, P‐I‐3 kinase p85/p110 proteins. Treatment of DU145 cells with PAC resulted in inhibition of translocation of NF‐kB as suggested by decreased protein expression of p65 in the nuclear fraction. No apparent change in IkB alpha protein levels were noted. Decreased protein levels of p‐IkB alpha were observed. PAC fraction has the ability to inhibit MMP‐2/−9 activity at a concentration which is not cytotoxic to these cells suggesting that PACs inhibit MMP activity levels in DU145 cells in a targeted manner independent of cellular cytotoxicity effects. (N.C.I.C.‐ Canadian Cancer Society, P.E.I. Health Research Program, The Cranberry Institute (Wisconsin Board) funded)
Cleavage of HJV is independent of its GPI‐anchor
Authors: Maxson Julia Elizabeth , Zhang An‐Sheng , Enns Caroline
Published: June 2021

Mutations in hemojuvelin (HJV) lead to the iron overload disorder juvenile hemochromatosis. HJV is a GPI‐linked protein that increases expression of the iron regulatory peptide hormone, hepcidin. Hepcidin negatively regulates iron uptake from the intestine. GPI‐linked HJV potentiates bone morphogenetic protein (BMP) signaling to upregulate hepcidin expression. HJV can also be cleaved by the proteases furin and matriptase‐2, releasing it from its GPI‐anchor and allowing it to be secreted from cells. In contrast to GPI‐linked HJV, secreted HJV negatively regulates BMP signaling and decreases hepcidin expression. To investigate the role of the GPI‐anchor in cleavage and secretion of HJV, we created chimeric proteins with the ectodomain of HJV and the transmembrane and cytoplasmic domains of either low‐density lipoprotein receptor (LDLR) or furin in lieu of the GPI‐anchor. These chimeric proteins appear to traffic similarly to GPI‐HJV, and are cleaved by furin and secreted from cells. Truncated forms of HJV that have neither a transmembrane or GPI‐anchor, are also cleaved by furin and are secreted from cells, but appear to have different kinetics of post‐translational processing than membrane anchored HJV. These studies indicate that the GPI‐anchor is not required for cleavage and secretion of HJV. This work is funded by NIH‐DK080765.
Overexpression of recombinant human procathepsin B in E. coli
Authors: Wen Lisa , Chmielowski Jennifer , McConnell Matthew , Hunter Jessica , Keefer Ryan , McConnell Rose
Published: June 2021

Cathepsin B is a lysosomal cysteine proteinase that is implicated in the processes of tumor invasion, metastasis, and Alzheimer's disease. Development of specific inhibitors of cathepsin B could lead to the discovery of therapeutic agents for treatment of many types of carcinomas, as well as Alzheimer's disease. The objective of this research is to produce functional recombinant cathepsin B enzyme which will be used in screening of new cathepsin B inhibitors. Human procathepsin B cDNA has been successfully amplified by PCR and sub‐cloned into pET15b expression vector. Overexpression of the procathepsin B protein has been accomplished by IPTG induction. The ability to produce large quantities of procathepsin B enzyme will permit a drug discovery program for development of newer effective inhibitors of cathepsin B.

Supported by National Cancer Institute at NIH (Grant No. 3R15CA086933‐04 and 3R15CA086933‐04A2S1)
Genome‐wide screens in yeast reveal that diverse cellular pathways influence the toxicity of various neurodegenerative disease‐associated misfolded proteins
Authors: Cameron Dale Matthew , Savare Jean , Higgins Matthew , Galarza Vicente Sancenon , Sia Angela , Collins Sean , Shales Michael , Krogan Nevan , Farese Robert , Muchowski Paul , Weissman Jonathan
Published: June 2021

A common pathological feature of many fatal neurodegenerative diseases is the formation of neuronal protein aggregates. Despite differences in the composition and localization of the aggregates, the near universality of neuronal protein deposition and neurodegeneration in these diseases suggests the existence of a common pathogenic mechanism related to the aggregation process. To identify cellular pathways that modulate toxicity associated with expression of different disease‐associated proteins, we systematically screened the yeast genome, comprising non‐essential gene deletions and strains in which the levels of essential genes are depleted. We identified a number of genes that enhance or suppress toxicity associated with expression of these proteins. However, rather than illuminating a common mechanism of toxicity, our results suggest that distinct pathways influence the toxicity of each protein. This work was supported by the Larry L. Hillblom Foundation and the National Institutes of Health.
Single molecular observation of the apolipoprotein B and its folding intermediates in reassembled low density lipoprotein
Authors: Chang Chia‐Ching , Chu Hsueh‐Liang , Cheng Tsai‐Mu
Published: June 2021

Low density lipoprotein‐cholesterol (LDL‐C) is a clinical significant marker of cardiovascular disease risk. Each particle of LDL contains only one molecule of apolipoprotein B (apoB). In human, the liver secretes full‐length apoB (apoB‐100) which also serves as a ligand for receptor‐mediated uptake of LDL by a variety of cell types. The deficiency of the LDL assembly may play a vital role in many diseases progression. Therefore, it is desired to reveal the folding/assembly process of ApoB. However, it is challenge to monitor its folding/assembly intermediates due to its high insolubility in an aqueous solution. In this study, we had purified LDL from human plasma by sequential ultracentrifugation and the apoB‐100 had been isolated from LDL by ice cheer method. ApoB was dissolved in the denature buffer which contained detergents. The apoB‐100 protein was refolded by the way of an over‐critical refolding process. The folding intermediates of apoB‐100 and its truncated mutants could be observed by immunofluorescence microscopy analysis at the single molecular level. Our data indicate that the N terminus of apoB is important for LDL assembly and stabilization. This study can help the understating the mechanism of lipoproteins folding. Meanwhile, the transportation of LDL in the living body can be monitored, too.
Order in Disorder: Aggrecan CS Region Predicts a New Class of Protein Structure
Authors: Hardingham Timothy Edward , Jowitt Thomas , Murdoch Alan , Day Joanna , Berry Richard , Baldock Clair
Published: June 2021

Aggrecan is a glycosylated multi‐domain protein. Secondary structure analysis of the extended CS sequence predicts no known folded motifs. It is assumed to be unstructured and its elongation caused by high glycosylation. We expressed a 31KDa CS‐peptide sequence from human aggrecan in E Coli and compared its unglycosylated structure with human aggrecan globular G3 domain.

The CS‐peptide and globular G3 domain were analyzed by MALLS/SEC, SAXS, sedimentation velocity and CD (Table 1). The CS‐peptide was hydrodynamically much larger than G3 and its sedimentation coefficient 1.67S was much less than predicted (2.1S) for a random‐coil. This suggested that the CS‐peptide was very extended, and its sedimentation rate increased and CD spectrum changed in 6 M urea suggesting a significant collapse of secondary structure. Modelled as a segmented flexible chain, (Kratky/Porod and Holtzer) gave a segment length 3.3 nm, with 13 repeats and the calculated mass per unit length of 831 gmol‐1 nm‐1 was also much higher than an extended polypeptide chain (308 gmol‐1 nm‐1).

Mass (Da)
Mass (Da) exptl
S20, W
f/f0
Rh (nm)
Rg (nm)

G3
25700
26800 ± 700
2.38 ± 0.14
1.31
2.57 ± 0.17
N/D

CS‐peptide
30827
28670 ± 1787
1.67 ± 0.11
2.01
3.99 ± 0.28
4.71

The CS‐peptide of aggrecan thus has an elongated and stiffened structure even when unglycosylated and it identifies a new class of semi‐ordered structure within disordered sequences. As 50% of all expressed protein sequences in the genome are predicted to be disordered these semi‐ordered sequences may be more commonly found than is currently recognized.
Ectopic expression of eukaryotic initiation factor 2B epsilon (eIF2Bε) in rat skeletal muscle rescues the sepsis‐induced reduction in guanine nucleotide exchange activity and protein synthesis
Authors: Tuckow Alexander Paul , Vary Thomas C , Kimball Scot R , Jefferson Leonard S
Published: June 2021

eIF2B is a guanine nucleotide exchange factor (GEF) whose activity is both tightly regulated and rate controlling with regard to global protein synthesis rates. Skeletal muscle eIF2B activity and catalytic epsilon subunit (eIF2Bε) expression have been implicated as potential contributors to the altered rates of protein synthesis in a number of physiological and experimental models. The objective of the current study was to directly examine the effects of exogenously expressed eIF2Bε in vivo on GEF activity and protein synthetic rates in rat skeletal muscle. A plasmid encoding FLAG‐eIF2Bε was transfected into the tibialis anterior (TA) of one leg while the contralateral TA received a control plasmid. Ectopic expression of eIF2Bε resulted in increased GEF activity in TA homogenates of healthy rats demonstrating that the expressed protein was catalytically active. In an effort to restore a deficit in eIF2B activity, we utilized an established model of chronic sepsis in which skeletal muscle eIF2B activity is known to be impaired. Ectopic expression of eIF2Bε in the TA of septic rats rescued the deficit in GEF activity and muscle protein synthesis caused by sepsis. The results demonstrate that ectopic expression of eIF2Bε may be sufficient to rescue the sepsis‐induced reduction in skeletal muscle protein synthesis. (Supported by grants DK15658, GM 39277 and the PA Department of Health).
In vitro Inhibition of Sindbis Virus Replication by Glycyrrhizin
Authors: Guevara Luz E.
Published: June 2021

Alphaviruses, a genus from the Togaviridae family, are responsible for a wide spectrum of human and animal diseases, ranging from silent asymptomatic infections to fever to encephalitis. Alphaviruses are transmitted via mosquito bites and have a high incidence of disease in Africa, Asia, Australia and Eastern Europe. Previous research had shown that certain medicinal plant compounds present antiviral activity and can be used to treat infections. The purpose of this study was to determine if glycyrrhizin, a natural compound found in the roots of the Glychyrrhiza glabra plant, found in Europe, was able to inhibit the infection of Sindbis virus in Vero cells. Vero cells, treated with glycyrrhizin, were infected with Sindbis virus. Viral media from these experimental cells and from control cells were then used to determine the viral titer by plaque assay. Cells were also examined microscopically for morphological differences. The results indicated that there were fewer viral particles released in the experimental plates, with the glycyrrhizin treatment, compared with the control plates. Based on the results, glycyrrhizin is able to inhibit Sindbis virus infection, and can be further investigated for treatment of alphavirus infections. This research is supported by MARC Program – NIH (LG).
Effects of hibernation on fibrinolysis and platelet clot stability in 13‐lined ground squirrels
Authors: Kaczmarek Gina Marie , Talhouarne Gaelle , Kohlnhofer Bridget , Cooper Scott
Published: June 2021

During hibernation, a ground squirrel (Spermophilus tridecemlineatus) has decreased blood flow, which could lead to the formation of stasis blood clots. Ground squirrels have been shown to suppress blood clotting in order to survive hibernation. However, the stability of blood clots and the degradation process through fibrinolysis have not been determined. Preliminary results indicate no significant difference in plasminogen levels during hibernation. To see if fibrinolytic pathways were being activated during hibernation, plasmin activity was measured, and found to be 4‐fold higher in pooled hibernating versus pooled non‐hibernating plasma. These results suggest that fibrinolysis may be activated during hibernation, suggesting that some clots are still forming in spite of the suppression of hemostasis by the ground squirrels. A clot retraction assay was used to compare the stability of clots, and human platelet clots retracted in 45 minutes on average, compared with 25 minutes for non‐hibernating ground squirrels. The stability of blood clots in hibernating and non‐hibernating squirrels will be compared with the clot retraction assay when hibernating ground squirrel blood becomes available in the winter. A decreased stability of platelet clots may also protect ground squirrels from the formation of clots during hibernation. This research was funded by an NIH R15 grant.
Characterization of fluorescent probe partitioning in giant unilamellar vesicles of “lipid raft” mixtures using confocal fluorescence microscopy
Authors: Sharom Frances J. , Juhasz Janos , Davis James H.
Published: June 2021

Direct visualization of raft‐like liquid ordered (lo) domains requires fluorescence probes with known partitioning preference for a specific lipid phase. Therefore, a detailed understanding of the behaviour of commonly used fluorescent probes in defined lipid bilayer systems is needed before they can be used to draw conclusions about the physical state of the membrane. Using giant unilamellar vesicles composed of a ternary “lipid raft” mixture (DOPC/DPPC/cholesterol) for which the phase behaviour is known, we examined nine commonly used fluorescent probes using confocal fluorescence microscopy. The partitioning preference of each probe was assigned using either a well‐characterized ld phase marker, or by quantitation of the domain area fraction. Fluorescent molecules were examined individually, in pairs, and in threes; most of them partitioned into the ld phase, while two probes preferred the lo phase or gel phase. Interestingly, the partitioning of DiIC18 was influenced by Bodipy‐PC, and Lissamine rhodamine B‐DPPE affected the partitioning preference of other fluorescence probes when used with them. We compare and contrast the different fluorescence probes in terms of their partitioning preference, ability to detect phase separations, photostability, and any induced change in lipid miscibility transition temperature.

Supported by the Natural Sciences and Engineering Research Council of Canada
Comprehensive metabolic analysis for understanding of obesity and diabetes mechanisms
Authors: Newgard Christopher B. , Stedman Sarah W.
Published: June 2021

We seek to apply comprehensive metabolic analysis tools (sometimes called “metabolomics”) for understanding of mechanisms underlying chronic human diseases such as diabetes, obesity, and cardiovascular disease. Current approaches include analysis of metabolic flux by 13C NMR‐based mass isotopomer analysis (in collaboration with Drs. Shawn Burgess and A. Dean Sherry and associates, Dallas, TX) and metabolic profiling of important groups of metabolic intermediates by both “targeted” and “unbiased” mass spectrometry (in collaboration with Drs. James Bain, Robert Stevens, Olga Ilkayeva, Brett Wenner, Michael Muehlbauer, Mark Butler, and David Millington at Duke). These tools have been used to investigate the metabolic mechanisms underlying islet β‐cell dysfunction and development of peripheral insulin resistance in animals and humans. Studies in islet β‐cells have demonstrated a critical role of a pyruvate/isocitrate cycling pathway in generation of signals for insulin secretion, and impairment of this cycle associated with loss of β‐cell function. We have also recently identified perturbations of branched chain amino acid (BCAA) catabolism in obese, insulin resistant humans compared to normally insulin sensitive lean controls and have translated these findings to rodent models to demonstrate a contribution of BCAA to development of insulin resistance that is independent of body weight. Finally, in collaboration with Dr. Alan Attie at the University of Wisconsin, we have integrated transcriptomic and metabolomic analysis to identify new pathways that control hepatic gluconeogenesis and PEPCK expression. These examples will serve to illustrate the potential of comprehensive metabolic profiling methods for providing insights into diabetes and obesity mechanisms.
Endoplasmic Reticulum Stress Mediates Palmitic Acid‐induced Insulin Resistance in Skeletal Muscle Cells
Authors: Liu Pingsheng , Peng Gong , Li Linhai
Published: June 2021

Pathological elevation of plasma fatty acids reduces insulin sensitivity. We found that insulin‐stimulated phosphorylation of Akt was reduced by palmitic acid (PA). The same treatment also increased phosphorylation of protein kinase R‐like ER kinase (PERK) and XBP‐1 mRNA splicing, which are both up‐regulated in ER stress. 4‐phenyl butyric acid (4‐PBA) blocked PA‐mediated PERK phosphorylation and reversed the inhibitory effect of PA on insulin‐stimulated Akt phosphorylation. Furthermore, oleic acid (OA) could also recover PA‐reduced Akt phosphorylation and abolish both PA‐mediated PERK phosphorylation and XBP‐1 mRNA splicing. The observation was verified in rat skeletal muscle using venous fatty acid infusion. In addition, PA stimulated AMP‐activated protein kinase (AMPK) phosphorylation, and both compound C and OA could inhibit this phosporylation and recover insulin sensitivity. These data suggest that PA induces insulin resistance via AMPK‐mediated ER stress. Our data also demonstrate that OA and PA competitively regulate insulin sensitivity by controlling ER stress.
Vav proteins are mechanistically involved in CD36‐dependent atherogenic processes
Authors: Rahaman S Ohidar , Febbraio Maria , Zhou Gang , Silverstein Roy L
Published: June 2021

Activation of a CD36‐dependent signaling cascade is necessary for macrophage uptake of oxidatively modified LDL (oxLDL) and foam cell formation, and hence plays an important role in atherogenesis. We now show that CD36 contributes to activation of Vav family guanine nucleotide exchange factors (GEFs) in aortae from hyperlipidemic mice, and that oxLDL induces activation of macrophage Vav in vitro in a CD36 and Src family kinase‐dependent manner. CD36‐dependent uptake of oxLDL in vitro and foam cell formation in vivo was significantly reduced in Vav null macrophages concomitant with impaired calcium signaling and PLC‐gamma1 activation. Chelation of intracellular Ca2+ or inhibition of PLC‐gamma inhibited Vav activation and reduced foam cell formation. Inhibition of dynamin, a Vav interacting protein involved in endocytic vesicle fission, blocked oxLDL uptake and inhibited foam cell formation. Immunofluorescence microscopy studies showed that Vav1 and dynamin‐2 colocalized with internalized oxLDL in vitro and in atherosclerotic lesions in vivo, that mobilization of dynamin‐2 by oxLDL was impaired in Vav null cells, and that disruption of the CD36/Vav pathway caused a defect in maturation of oxLDL‐containing endocytic vesicle. These studies link CD36 and Vavs in a mechanistic signaling pathway required for macrophage foam cell formation and atherogenesis.
The quantitative monitoring PtdIns(4,5)P2 dynamics in living cells using a specific molecular sensor
Authors: Yoon Youngdae , Lee Park Joo , Cho Wonhwa
Published: June 2021

Phosphatidylinositol‐4,5‐bisphosphate (PtdIns(4,5)P2), which is a minor component of the plasma membrane, plays important roles in many cellular processes, including signal transduction, exocytosis, endocytosis, and ion channel activation. A method for quantitatively monitoring the PtdIns(4,5)P2 concentration and dynamics in living cells is lacking at present. We have developed a new method of quantifying the PtdIns(4,5)P2 concentration in living cells using a specific PtdIns(4,5)P2 sensor made of the engineered ENTH domain of epsin1 labeled with an environment‐sensitive fluorophore. The PtdIns(4,5)P2 sensor allows robust quantitative determination of spatiotemporal fluctuation of PtdIns(4,5)P2 in giant unilamellar vesicles and mammalian cells by ratiometric analysis. The results demonstrate the spatially heterogeneous distribution of PtdIns(4,5)P2 in the plasma membrane and provide new insight into the fluctuation and dynamics of cellular PtdIns(4,5)P2 in response to various stimuli. Furthermore, this strategy is generally applicable to in situ quantification of other membrane lipids.

Supported by NIH GM 68849 and GM76581
Phosphorylation of yeast phosphatidate phosphatase by protein kinase C
Authors: Su Wen‐Min , Han Gil‐Soo , Carman George M
Published: June 2021

The yeast PAH1‐encoded phosphatidate (PA) phosphatase, which catalyzes the Mg2+‐dependent dephosphorylation of PA to yield diacylglycerol, plays a major role in the regulation of lipid metabolism. In fact, mutants defective in the enzyme show defects in phospholipid synthesis regulation, an abnormal expansion of the nuclear/ER membrane, and a 90 % reduction in triacylglycerol. PA phosphatase has been identified as a phosphoprotein in vivo. Studies with recombinant enzyme isolated from Escherichia coli have shown that PA phosphatase is a substrate for protein kinase C. Truncation analysis of PA phosphatase followed by site‐directed mutagenesis experiments revealed that Ser677, Ser769, and Ser788 are the major sites for protein kinase C phosphorylation in vitro. Yeast cells that carry a S677A S769A S788A triple mutation in PA phosphatase exhibit a 40 % reduction in enzyme phosphorylation. These results indicated that protein kinase C phosphorylates PA phosphatase in vivo. Supported by NIH grant GM 50679.
Decreased apolipoprotein E and VLDL receptors cause lipidemia in nephrotic syndrome
Authors: Wang Limin , Walzem Rosemary L , Kaysen George
Published: June 2021

Hyperlipidemia in nephrotic syndrome (NS) is due to decreased clearance of triglyceride rich lipoproteins (TGLs). Lipoprotein lipase (LPL) hydrolyzes plasma TG, promotes the binding and catabolism of TGLs. Apolipoprotein E (apo E) facilitates binding by TGLs both to their receptor and LPL. A reduced plasma LpL level and reduced VLDL apo E were found in nephrotic rats. It is unknown whether low levels of LPL and/or VLDL apoE may contribute to the defective clearance. Lipoprotein particle size measured by Microtrac, showed no significant difference in nephrotic VLDL compared with control one. This finding indicated that the increased lipids in NS are due to increased particles accumulation in plasma instead of defective TGLs lipolysis via LpL. To study whether this accumulation is from decreased clearance and/or uptake by cells and organs, heart perfusion study demonstrated a significant decrease of VLDL uptake in NS heart. Endothelial cell VLDL uptake using fluorescent labeling technique showed a significant decreased cell uptake VLDL in NS compared with control. However, two groups had a similar increase when adding exogenous LpL. ApoE significantly increased VLDL cell uptake in NS compared with control at low concentration, but not at high concentration. Further, Western showed a significant decreased VLDL receptor in NS heart. These findings indicate that decreased LpL in nephrotic is not the major cause of defective VLDL clearance, decreased apoE and VLDL receptor contribute to the defect. The study was supported by the Department of Veteran's Affairs.
GDP‐mannose transporter oligomer formation in the filamentous fungus Aspergillus nidulans
Authors: Johnson Laura Raquel , Jackson‐Hayes Loretta , Hill Terry W. , Loprete Darlene M.
Published: June 2021

GDP‐mannose transporters (Gmt) carry nucleotide sugars from the cytosol across the Golgi apparatus membrane in various eukaryotic organisms. Some fungal species like Saccharomyces cerevisiae express a single gmt, while others including A. nidulans express two (gmtA and gmtB). GmtA and GmtB display a punctate pattern of distribution indicative of localization within the Golgi apparatus. Gmt in S. cerevisiae localizes to the Golgi as well, and proper localization is dependent on oligomerization. Here we report progress on determining if GmtA and GmtB form oligomers and the preferred combinations of GmtA and GmtB for Golgi localization. In addition, C‐ and N‐ terminal truncated versions of GmtA and GmtB were created in order to designate regions responsible for Golgi localization and possible oligomer formation.
Ca ‐ signaling activation by complex nucleotide substance (PriMed) in mouse myoblast cells c2c12
Authors: Orlova Elena V. , Orlova Valentina S. , Baeva Julia I. , Schemyatovsky Alexandr N.
Published: June 2021

In present paper PriMed was investigated as a potential therapeutic agent in stem cells therapy and particular metabolic intervention.

Methods

c2c12 was cultured by typical experimental protocol. NO‐synthase in c2c12 was determined by EPR‐spectroscopy. For the fluorescence in c2c12 0.005% Pluronic was used.

Results

It was shown that:

In c2c12 PriMed (0.005μg/μl) activated Ca2+‐dependent NOS in 47‐fold vs base level and in 4‐fold vs dinitrosyl iron complexes (DNIC) (1.8μM) and in more than 6‐fold vs ionomycin (20μM).

PriMed (0.005μg/μl) gave 1.5‐fold Ca2+ increase in cytoplasm vs DNIC(1.8μM) and 3‐fold vs base level in c2c12.

Thus, the below experiments suggest that there is a direct influence of PriMed on biochemical and metabolic pathways of myoblast cells.
Identification of regulatory binding sites on the Glut1 glucose transporter
Authors: Reyes Alejandro , Ojeda Paola , Pérez Alejandra , Valenzuela Ximena , Vera Juan C
Published: June 2021

Facilitative glucose transport through plasma membrane is facilitated by GLUT1, transporter protein expressed in most cells. To identify regions on the protein involved on sensitivity towards inhibitors of transport, we identify three short sequences that show homology with typical ATP‐binding sites, as putative targets for inhibitor interaction. We probe the functional role of these regions by site directed mutagenesis and expression on Xenopus oocytes. For each mutant we analyze their kinetic properties for transport and their abilities to be affected by cytochalasin B, quercetin and tyrphostin A47. We identify mutants of exofacial orientation that suffers complete loss of sensitivity towards quercetin. Besides, we detected two groups of mutants of endofacial orientation that exhibit a decreased sensitivity to tyrphostin A47 or cytochalasin B. These data suggests that there are at least two regulatory regions, one accessible from the external side and another located on the internal surface of the protein and identify amino acid residues implied on the interaction with these ligands (Financed by FONDECYT 1060198 and FONDEF D07I1117).
Instrumentation Grant Savvy – What's the Best Fit for Your Institution?
Authors: Svoboda Kathy K H
Published: June 2021

Large instruments (expensive) are necessary for many types of investigations. Most institutions use private, foundation or federal funds to purchase these instruments. In this presentation, the federal sources for instrumentation will be discussed and the specific requirements for each grant will be outlined. National Science Foundation, National Institutes of Health Shared Instrument and High End Instrument programs will be featured. The grant structure for the different grants varies, but has common elements that reviewers seek in evaluating the grant applications. Tips for finding the best funding mechanism and writing the best grant possible will be covered.
Team Building: Growing Your Research Career through Institutional Awards
Authors: Dechow Paul C.
Published: June 2021

When looking for that first faculty job, it is important to consider the quality of the prospective institution, department, and research group. High quality institutions will have funded institutional awards that may help you jump start your career. Examples of such grants are U24, U54, and various P and R grants among others. Examples and tips for how young faculty can make use of some of these mechanisms to grow their careers will be described.
Structural and Functional Remodeling of the Cardiac Muscle in the Hypertensive Profilin1 Transgenic Mouse Model
Authors: Hassona Mohamed Darwish Hessein , Abouelnaga Zeinb Ahmed , Abdulrahman Basant , Elnakish Mohammed , Hassanain Hamdy
Published: June 2021

Pressure overload to the heart, via hypertension, results in cardiac hypertrophy which induces heart failure. We have developed a novel transgenic mouse model by over‐expressing the cDNA of human profilin 1 in the blood vessels of transgenic mice to study cardiovascular dysfunction. Our previous results showed significant increased medial thickness of the microvessels of profilin1 and elevated blood pressure in profilin1 mice starting at age 6 months. Our current study investigates the effects of hypertension on the cardiac muscle structure and function of older profilin 1 mice. Western blotting showed no expression of profilin1 in young profilin 1 mice, but significant activation of profilin1 in older profilin1 mice. The hypertrophic signaling cascade was activated in the cardiac muscle of old profilin 1 mice as demonstrated by increased, ERK1/2, JNK and ROCK II kinase (312.3%, 271.4% and 350% respectively, P<0.05). Our Echocardiography demonstrated decreased LV ejection fraction and fractional shortening (35% and 25%, respectively. P<0.05) and increased LV mass in old profilin 1 mice. In conclusion, the cardiac hypertrophy in profilin 1 transgenic mice is due to activation of profilin 1 in cardiac muscle as a secondary effect of pressure overload. This study will enable us to understand the role of profilin‐associated signaling mechanisms in triggering cardiac hypertrophy in hypertensive humans.
Association of Angiotensin Converting Enzyme Insertion/Deletion and Angiotensinogen T235 Polymorphisms with Risk of Essential Hypertension
Authors: Hemimi Neveen Salah , Oda Naglaa Raafat , Hemedi Samira Ali
Published: June 2021

Background

polymorphism of renin angiotensin system has been proved to be implicated in the pathogenesis of several multigenetic diseases including hypertension.

Aim of the work

to investigate the linkage of I/D polymorphism of angiotensin converting enzyme (ACE) and Met to Thr variant at position 235 of angiotensinogen (AGT) to the incidence of essential hypertension.

Method

Samples of blood were collected from 203 subjects with matched age and sex (110 hypertensive and 93 normal controls). The polymorphism of ACE and AGT were investigated by polymerase chain reaction techniques.

Results

AGT polymorphism showed significantly increased incidence of hypertension in TT genotype as compared to (MT& MM) genotypes (OR= 2.2, 95% CI= 1.0–5.1, P<0.05). Similarly the study of ACE polymorphism declared a minor risk for hypertension in DD compared to (DI& II) genotypes (OR= 1.2, 95% CI= 0.7–2.2). No significant association was found between DD genotype and other risk factors of hypertension, while there was association of TT genotype and increased serum creatinine and fasting blood sugar.

Conclusion

TT genotype of angiotensinogen polymorphism is a possible risk factors for hypertension.

Source of Fund: Dubai Medical College
Effect of Hypotonic Osmotic Stress on Apoptosis in Cultured Human Skin Keratinocytes
Authors: Nalbandian Ari Dikran , McCauley Michael , Brum Gabriella , Fredette Rebekah , Lu Shan , Wan Yinsheng
Published: June 2021

While it is known that a cell in a hypotonic environment swells from an influx of water due to an osmotic imbalance, the pathways activated inside the cell are not as clearly observable or thoroughly investigated. To determine whether apoptotic and cell survival signaling pathways are activated, cultured human skin keratinocytes (HaCaT cell line) were exposed to increasing levels of hypotonicity from 0% dilution up to 50%. Western blotting analysis was used after 30 minutes of incubation. The results show that AMPK is activated when the dilution is increased, and the activity is peaked at 40% dilution and then dropped slightly at 50% dilution. Similarly, AKT is activated and the activity is peaked at 40% and 50% dilutions, suggesting that at this level of hypotonicity these pathways are activated for cell survival. The results also demonstrate that p38 and JNK are activated as the dilution is increased, and the activity is peaked at 40% dilution. Interestingly, the data from cell migration assay show that cell migration is increased gradually as dilution is increased, but dropped significantly at 50% dilution. Collectively, our data suggest that hypotonic stress affect both apoptotic and survival pathways and to certain degree enhance cell migration.