bluuebunny/crossref_metadata_2025 huggingface dataset:
HEPATITIS C VIRUS: Effect Of NS5A On The NS5B in vitro Replication Reaction
Authors: Quezada Elizabeth M , Kane Caroline M
Published: June 2021

The Hepatitis C virus replicates its viral RNA template by a process that involves a protein complex containing the RNA dependent RNA polymerase (RdRp) NS5B, the helicase NS3, the membrane inserted protein NS4, and the phospho‐protein NS5A along with a number of host proteins. In vitro studies of the replication reaction have shown that the rate of nucleotide incorporation of NS5B is slow when compared to other DNA dependent RNA polymerases, suggesting that other factors such as host or non structural proteins, membrane structures, cis‐elements in the template or a combination of these factors are required to achieve higher rates. To understand how NS5A stimulates NS5B, we have developed an in vitro assay to study the effect of NS5A in the replication reaction.

The NS5A recombinant protein was expressed and highly purified from bacterial cells. Using gel filtration, two isoforms of different molecular weight were isolated. The multimeric form stimulates the in vitro NS5B activity in a concentration and in an order of addition dependent manner. The monomeric isomer also will be tested. A pre‐incubation step with NS5B is required for the enhancement effect of NS5A on the replication reaction. Using the most terminal 98 nucleotides of the viral 3’UTR genome, NS5A stimulates the replication reaction three fold. Further studies using templates containing structural elements of the 3’UTR of the positive strand and negative strand are being tested. Stimulation is also seen with a synthetic RNA template. With this latter template, individual steps in replication can be studied; and preliminary data suggests that the activation by NS5A seems to occur at the elongation phase. In addition, the pause profile changes when NS5A is present in the reaction. We will explore in detail these features of the stimulation mechanism as well as analyze the affinity of the template to the complex NS5A‐NS5B.
Studies on components involved in the Degradation of Misfolded Glycoproteins
Authors: Li Guangtao , zhao Gang , zhou Xiaoke , Schindelin hermann , Lennarz William J.
Published: June 2021

In eukaryotic cells, the endoplasmic reticulum associated degradation (ERAD) pathway is essential for degradation of misfolded glycoproteins. Initially, we found that a higher order complex consisting of interactions between the ER bound E3 ligase gp78, mp97, mPNGase and mHR23B. This complex may serve to route misfolded glycoproteins out of the ER where they are degraded by the proteasome. In this complex, mp97 functions as an organizer to interact with E3 ligase mp78 and mPNGase. Recently, we found a novel protein binding motif of mp97 consisting of its last ten amino acid residues, which is both essential and sufficient to mediate mp97's interaction with mPNGase and Ufd3. Phosphorylation of mp97's highly conserved penultimate tyrosine residue, the main phosphorylation site during T‐cell receptor stimulation, completely blocks binding of either PNGase or Ufd3 to mp97. We also found that overexpression of the tyrosine kinase Src significantly increased the phosphorylation level of p97 and caused accumulation of the ERAD substrate TCRα‐GFP. Furthermore, an in vitro phosphorylation assay showed that Src directly phosphorylated p97. All these results indicate that phosphorylation of p97 may modulate its function. Further studies on the role of the phosphorylation of p97 in its function are underway.
Eukaryotic and retroviral aspartic proteases: similarities and differences
Authors: Gustchina Alla
Published: June 2021

The superfamily of aspartic proteases consists of two large families, eukaryotic and retroviral. Eukaryotic enzymes are encoded as single chains with two‐domain structures, whereas retroviral enzymes are active as homodimers. Both families include enzymes of considerable medical relevance, thus a vast number of structures of their inhibitor complexes have been reported. Whereas some of these inhibitors are very specific for particular members of each family, others are pleuripotent. For example, recent findings of cross‐reactivity of potent inhibitors of HIV‐1 PR and plasmepsin emphasized the need to evaluate the inhibitor complexes of other aspartic proteases from both families in an effort to create novel inhibitors. Such a task is hindered by the large differences in size between proteins from two families, and additionally complicated by the deviation in the symmetry of the functional subunits. We succeeded in generating very accurate overall superposition of these enzymes, including the ligands bound in their active sites. That allows us to evaluate an overall fit and the interactions of any inhibitor, which has been developed for one family member (e.g. HIV‐1 PR), in respect to the other family (e.g. renin). We are also able to characterize the structural similarities and differences between the two families with much higher accuracy, addressing their functional and evolutionary significance.
Substrate specificity and conformational preferences of prolyl 4‐hydroxylase
Authors: Gorres Kelly L , Raines Ronald T
Published: June 2021

Prolyl 4‐hydroxylase (P4H) catalyzes the post‐translational hydroxylation of proline residues in procollagen strands. P4H specifically forms 4(R)‐hydroxyproline (Hyp) from proline residues in Pro–Gly sequences of polypeptides. The presence of Hyp is crucial for the folding, secretion, and stability of the collagen triple helix under physiological conditions.

The current study examines the substrate recognition and specificity of P4H. We have synthesized peptide substrate analogs containing 4(R)‐fluoro‐L‐proline (Flp), 4(S)‐fluoro‐L‐proline (flp), and 4‐thio‐L‐proline (Thp). The peptide containing Flp was not found to interact with P4H, whereas the flp‐ and Thp‐containing peptides were determined to be substrates of P4H, forming products containing 4‐keto‐L‐proline and 4(R)‐sulfoxoproline. These results suggest that the cis/trans conformation of the substrate is a factor in recognition by P4H, and provide evidence for a role of conformational changes in collagen biosynthesis.

This work is supported by Grant AR44276 (NIH). KLG was supported by the NIH Chemistry–Biology Interface training program.
Mechanistic Analysis of Beta‐Lactam Synthetase and the Influence of Conformational Fluctuations
Authors: Raber Mary L , Townsend Craig A.
Published: June 2021

β‐lactam synthetase (β‐LS) catalyzes formation of the key β‐lactam ring of the potent β‐lactamase inhibitor, clavulanic acid, in an ATP/Mg2+ dependent reaction. In the present study, β‐LS was subjected to pH, pD, temperature, and viscosity variation. The kcat of β‐LS changes very little with varying pH, while its second‐order rate constant can be fitted to three ionizable groups. A normal SIE on kcat of 1.44 ± 0.17 was found, while an inverse solvent isotope effect of 0.80 ± 0.07 was determined for kcat/Km. A relative viscosity versus reciprocal rate plot of kcat gave a slope of around two. This suggests that a conformational change is rate‐limiting, with the small SIE from the increased viscosity of D2O relative to H2O. The kcat Arrhenius plots are linear and the corresponding parameters represent the enthalpically controlled energetics of the conformational change. In contrast, kcat/Km is significantly enhanced with increasing viscosity, and Arrhenius plots of kcat/Km and Km were non‐linear. From the analyses presented, it is proposed that an equilibrium of enzyme conformers exists before the first irreversible step in the β‐LS catalytic mechanism to match one later in the reaction coordinate corresponding to product release. This research was supported by NIH grant RO1 AI014937.
Mitochondrial oxidative stress does not contribute to cardiac hypertrophy in mice with cardiac specific GLUT4 deletion
Authors: Jalili Thunder , Nunthakungwan Orathai , Li Yan , Jin Huifeng , Hu Eric , Wende Adam R , Abel E. Dale
Published: June 2021

Cardiac specific ablation of insulin sensitive glucose transporters (GLUT4) in mice (G4H−/−) results in cardiac hypertrophy via unknown mechanism(s) we hypothesize are related to oxidative stress, as hearts of G4H−/−mice have greater oxidized (GSSG) glutathione and an attenuated ratio of reduced (GSH) glutathione to GSSG vs. controls. To determine the contribution of mitochondrial oxidative stress to cardiac hypertrophy, male and female control (Con) and G4H−/−mice were treated with a mitochondrial targeted superoxide 2 mimetic, MnTBAP [(Mn(III)tetrakis(4‐Benzoic acid) porphyrin Chloride, 10 mg/kg, i.p.] or saline. Male Con and G4H−/−mice had similar levels of mitochondrial GSSG and GSH:GSSG (n=5–6). MnTBAP altered these levels only in Con mice (GSH:GSSG, Con 41±8, G4H−/−30±4, Con+MnTBAP 56±3, G4H−/−+MnTBAP 37±2). Cardiac hypertrophy (n=6–7) was unchanged after MnTBAP (heart:tibia length, Con 7.2±0.2, G4H−/−8.9±0.5, Con+MnTBAP 7.9±0.3, G4H−/−+MnTBAP 8.6±0.5). In female G4H−/−mice even though GSSG (n=5–11) was reduced by MnTBAP (Con 19±2, G4H−/−47±6, Con+MnTBAP 18±2 vs. G4H−/−+MnTBAP 30±4), there was no reduction in cardiac hypertrophy (n=7–10, Con 6.3±0.2, G4H−/−7.9±0.3, Con+MnTBAP 6.2±0.1, G4H−/−+MnTBAP 7.7±0.3). We conclude that mitochondrial oxidative stress does not contribute to cardiac hypertrophy in this model of cardiac specific insulin resistance. Supported by NIH HL085226.
Novel method for fast immunodetection using vacuum filtration
Authors: Gutierrez Sara , Rapiejko Peter , Emerick Marc , Sharma Ajay
Published: June 2021

Western blotting is a well establish technique used for the routine detection of specific proteins from complex mixtures. The immunodetection step of western blotting is a long and tedious process, involving multiple changes of solutions, and is usually extended over several hours to overnight. This paper describes an innovative, vacuum‐based immunodetection approach, which allows the detection of the protein in less than 45 minutes. The system can be used for rapid antibody optimization studies, antibody screening, simultaneous detection of different proteins in multiples blots, or routine western blot analysis. Vacuum filtration enables efficient blocking and washing of the membrane, speeds up the overall process and make the system easy to use. Blocking of the membrane occurs rapidly in the presence of low concentration of non fat dry milk or other common blocking reagents. Since incubation steps are typically performed in ten minutes, users can sequentially process several blots in a day. The system in compatible with PVDF or nitrocellulose membranes and detection can be accomplished by any chemiluminescence or fluorescence detection method. The SNAP id™ system is a novel immunodetection system that uses the basic steps of the standard immunodetection protocol, is easy to use and offers a substantial reduction in processing time.

This research has been totally funded by Millipore Corporation.
Cross‐platform comparisons of algorithms for calculating real‐time PCR amplification efficiencies
Authors: Rulli Samuel J , Arikawa Emi , Yang Jingping
Published: June 2021

The analysis of real‐time PCR data has become the focus of mathematical modeling to increase quantification precision and accuracy. Methods include: (1) the slope‐derived efficiency calculation from the standard curve method using Ct values determined either from (a) the fit‐point method or (b) the second derivative maximum (SDM) of a four parametric logistic model; or (2) the single amplification plot methods to compute efficiencies from individual PCR kinetic curves using comprehensive algorithms such as (a) mid‐point regression (Data Analysis for Real‐time PCR or DART‐PCR), (b) the window‐of‐linearity algorithm (LinReg) or (c) noise‐resistant iterative nonlinear regression (Real‐time PCR Miner). We directly compared these methods on three commonly used real‐time PCR instruments (BioRad iCycler iQ™, Stratagene Mx3005P™ and ABI 7500 Fast PCR System). SYBR‐Green® based RT2 Profiler™PCR Arrays from SuperArray Bioscience were used with serially diluted DNA templates and the amplification efficiency for each PCR assay was computed. This study demonstrates that Real‐time PCR Miner provides the best precision in efficiency estimation independent of the PCR instrument, while the precisions for other methods are platform‐dependent. Accounting for amplification efficiencies in fold‐change calculations may increase the precision of gene expression measurements using real time PCR.
A genome‐wide, promoter tiling design scheme for high quality real‐time PCR assays to measure genomic DNA in chromatin immunoprecipitation (ChIP) samples
Authors: Blanchard Raymond , Gu Xiujing , Burnette Angela , Huang Jiaqiang , Zeng Xiao
Published: June 2021

PCR is the standard for measuring DNA from ChIP because of its ability to simultaneously amplify and detect from limited samples. Compared to end‐point PCR, real‐time PCR (qPCR) provides faster data acquisition, greater dynamic range and increased sensitivity. However, the traditional “targeted” assay design scheme (primers flanking a binding site) hinders the design of amplicons meeting the performance criteria of specificity and amplification efficiency. To avoid this issue, we have applied an alternative “tiling” assay design scheme that allows greater flexibility in positioning the primers away from poor PCR sequences. Using this approach on a genome‐wide scale, 30 kb regions of promoters have been successfully “tiled” at a 1 kb interval with assays that display consistent, high‐quality qPCR performance. When used to measure p53 binding interactions in the CDKN1A promoter of HCT116 cells after treatment with 5‐FU, the ChIP‐qPCR assays showed that p53 binding increased from a few fold to 50 fold above background. Tiling assays corresponding to p53 binding sites in other genes show similar enrichments. The functional consequences of the p53 binding was also confirmed by RT‐qPCR measurement of mRNA. The genome‐wide availability of promoter tiling ChIP‐qPCR assays allows an increased number of binding sites to be surveyed simultaneously and facilitates ChIP‐chip microarray validation.
Room temperature storage and stabilization of complex biological samples
Authors: Muller‐Cohn Judy , de Rozieres Sohela , Ohgi Sharron , Faix Peggy Ho , Muller Rolf
Published: June 2021

We have developed a novel stabilization technology designed to protect biological samples such as DNA, RNA, proteins, cells and blood from degradation during storage at room temperature (RT). The storage medium is based on the natural principles of anhydrobiosis (“life without water”), a mechanism employed by some organisms that enables their survival while dry for >100 years, and synthetic chemistry. The objective of this study is to evaluate and develop an alternative handling, storage and shipping strategy to facilitate biological research; the development of such technologies would provide a useful alternative to current cold storage procedures. Results demonstrate stabilization of DNA for >2 yrs at RT and for an equivalent of up to 14 years under accelerated aging conditions. Storage of bacterial cultures in the medium allows for rapid recovery of bacterial genomic and plasmid DNA that can be used directly in transformations, PCR, electrophoresis and restriction analysis. Data for the inhibition of degradation even at elevated temperatures will be presented, including stability of RNA, blood and other protein‐based samples. Results indicate that recovered samples can be used directly in downstream assays without interference or inhibition, including cloning, sequencing, PCR, restriction analysis, hybridization, electrophoresis, microarray analysis, genotyping and transcription. Data will also be presented demonstrating improved PCR amplification with inclusion of a polymerase enhancer. Results indicate that technology advances in sample preservation can prevent the degradation process of biological samples used in life science research.
Cerium Oxide Nanoparticles Reduce Free Radical‐Mediated Toxicity in Drosophila melanogaster
Authors: Cohen Courtney Alicia , Karfakis Julie A , Kurnick Matthew D , Rzigalinski Beverly
Published: June 2021

According to The Free Radical Theory of Aging, cellular damage induced by oxidative stress is associated with multiple neurodegenerative disorders. Prior studies portray cerium oxide nanoparticles (CeONP) as potent free radical scavengers and neuroprotective agents. These studies were designed to evaluate an effective dosing regimen for CeONP in achieving lifespan extension and motor function preservation in Drosophila. We also determined the efficacy of CeONP when challenged with the toxic redox cycling agent paraquat (PQ), often used in Parkinson's disease modeling. Cohorts of 100 flies were fed with varying concentrations of CeONP (0–100μM) throughout adulthood or the larval stage. For PQ studies, flies were starved for 3 hours, and exposed to PQ for 24 hours. Motor function was evaluated by measuring activity in a Trikinetics monitoring system at various lifespan or post‐PQ intervals. In females, treatment with CeONP increased median lifespan by 18 days, or 32% (p<0.001), maximum lifespan by 19 days, or 25.3% (p <0.001), and overall activity in aged females increased by 150%. When exposed to PQ, average survival was prolonged 8 days, or 31% (p = 0.002), and females were 40 times more active than control flies. Male lifespan responded poorly to neuroprotective effects, and yet motor function was increased up to 900%! Our results suggest that nanoparticles may provide effective future treatment for free radical‐mediated disorders, opening a new realm of nanopharmaceutical development.
Macromolecular‐crowding induced protein stabilization at the residue level
Authors: Barnes Christopher O’Neil , Charlton Lisa M. , Pielak Gary J.
Published: June 2021

Macromolecular crowding has significant thermodynamic and kinetic consequences for biological macromolecules. Experimental evidence has shown that crowding enhances protein association, increases the rate of folding and refolding, and affects diffusion in the cytoplasm. Statistical thermodynamic models predict that macromolecular crowding increases protein stability, yet quantitative, residue‐level experimental evidence supporting these predictions is largely absent. Here we report the first residue‐specific information about the effects of macromolecular crowding on protein stability. We used NMR‐detected amide proton exchange to quantify the effects of 300 g/L polyvinyl pyrrolidone (PVP, 40 kDa) on the stability of the globular protein chymotrypsin inhibitor 2. Crowding increases the equilibrium constant for folding by up to 100‐fold compared to dilute solution and the model of the PVP monomer. We also show that, consistent with the local unfolding model, the magnitude of the increase depends on the location of the residue in the structure. Our results demonstrate that the increase in stability upon addition of PVP is the result of macromolecular crowding.
Creating a Chick Embryo Model of Spinal Muscular Atrophy
Authors: Murphy Kelley J. , Li Ling , Swoboda Kathryn J. , Schmid Aloisia
Published: June 2021

Spinal Muscular Atrophy (SMA) is the most common neurodegenerative disease in live births, and is caused by the homozygous deletion of the SMN1 gene and modulated by the amount of protein product of the SMN2 gene. Higher primates are the only animals to carry both the SMN1 and SMN2 genes; all other organisms have the single copy of SMN1 and its deletion causes an embryonic lethal condition. Chick embryos are ideally situated for (1) detection of distinct stages of development, (2) manipulation via electroporation of an interfering RNA (RNAi) construct into the one copy of SMN in the developing chick, and (3) analysis of the resulting deficiency within a short period of time (48 hours) after RNAi introduction. We show that electroporation alone or electroporation of the GFP reporter construct do not cause phenotypic abnormalities which are present with the electroporation of the SMN/GFP RNAi, and that the presence of the SMN/GFP RNAi reduces the amount of SMN protein in the chick as demonstrated by Western Blot and immunohistochemistry. Creating this new model of a common neurodegenerative disease allows us to modulate the severity, and thus more closely mimic the clinical presentation of the disease, by targeting the microinjection/electroporation to earlier or later stages of development; test drug and other interventions; and determine some of the earliest embryonic manifestations of SMA. 1RO1HD054599‐01 to KJS
Centromere assembly requires selective recognition of centromeric chromatin by centromere protein N
Authors: Straight Aaron , Carroll Christopher
Published: June 2021

The kinetochore is a unique chromosomal locus that binds to the mitotic spindle so that chromosomes can segregate in mitosis. Kinetochore assembly requires the specialized chromatin of the centromere that is distinguished by the replacement of histone H3 with the histone H3‐variant centromere protein A (CENP‐A). CENP‐A is thought to epigenetically mark the centromere and is required for kinetochore assembly but it is not known how this epigenetic mark is recognized to initiate kinetochore assembly. We have explored the molecular basis for centromere recognition by developing a simple and rapid in vitro assay designed to identify human kinetochore components that directly and specifically interact with reconstituted CENP‐A nucleosomes. This approach led to discovery that centromere protein N (CENP‐N) is a DNA‐sequence independent CENP‐A‐nucleosome binding protein. Nucleosomes reconstituted with a chimeric histone H3 that contains only the centromere‐targeting domain of CENP‐A were sufficient for CENP‐N binding. Point mutations that disrupted binding of CENP‐N to CENP‐A nucleosomes in vitro blocked localization of CENP‐N to the centromere in vivo, indicating that nucleosome binding is required for the centromere‐specific localization of CENP‐N. Thus CENP‐N recognition of centromeric chromatin is an essential step in the initiation of centromere and kinetochore assembly.
The pK's of Cardiolipins Are Altered by the Number and Position of their Specific Fatty Acid Esters
Authors: Haines Thomas H. , Epand Richard , Epand Raquel
Published: June 2021

We’ve confirmed that the 2 phosphate groups of tetrastearoyl‐cardiolipin (CL) have distinctly different pK values, one above neutrality. Although CL's chemical structure, suggests that it has 2 identical negative charge at neutral pH, our data shows it does not. Previously (Kates et al. (1993) Lipids, 28, 877–882), showed the pK2 was >8.0 with the tetrastearoyl CL, we have examined the dependence of the titration of cardiolipin on the number and nature of the acyl chains. The high pK2 varies, with the chainlength and desaturation of the chains on the lipid. As the chain structure is varied, the pK2 ranges from 3–8. The latter dominates with C18 chains. Short chains yield lower pK2's e.g., E. coli CL. Typical mitochondrial CL's have 4 linoleoyl chains. The high pK2 is explained by a bicyclic, H‐bonded conformation of CL's headgroup. Evidence for this has been provided by showing that deoxycardiolipin, lacking the central OH of the connecting glycerol, has a pK2 below 4.0. This data supports the proposal that CL has a role in ATP synthesis by providing a pool of protons on both sides of the membrane for proton pumping (Haines & Dencher (2003) FEBS Lett.528, 35–39). Our results show the high pK2, present in tetramyristoyl cardiolipin is missing in myristoyl lysocardiolipin. A chemical test for the often fatal Barth's Syndrome is lysocardiolipin. Our results may be relevant to this genetic disease which may have defective ATP synthesis.
Influence of Complex Nucleotide Substance from baking yeasts on selective cells cytotoxicity.
Authors: Orlova Elena V. , Orlova Valentina
Published: June 2021

It was shown that Complex Nucleotide Substance (CNS) from Saccharomyces cerevisiae been added in final concentration 0.2 ‐1.0 mg/ml to HeLa, U937, MCF7 and L929 cells culture gave cytotoxic effect in all these cell types but not on human leucocytes.

CNS also activated caspase‐3 in dose‐dependent manner in all the above cancer cell lines.

Under cell‐cytometry investigations CNS increased apoptotic cells quantity and decreased the number of G2/M of all cancer cells lines.

Thus, we can conclude that CNS is a selective apoptotic agent and it could give good results in cancer therapy.
EVI5 is a cytokinesis‐associated protein with dynamic subcellular localization
Authors: Faitar Silviu Leonard , Dabbeekeh Jeremy TS , Cowell John K
Published: June 2021

The EVI5 gene was originally identified in a screen for integration sites of ectopic viruses that gave rise to tumors in mice. The product of the gene is an 810 amino acids long ubiquitously expressed protein. It carries two functional domains, a Tre2/Bub2/Cdc16 (TBC) consensus sequence in the N‐terminal region, which is known to have a GTPase activating function, and an extensive coiled coil in the C‐terminal region. The goal of the present study was to elucidate the cellular localization and function of the EVI5 protein. A highly specific antibody against the C‐terminal section of the protein was used in an immunocytochemical analysis, and demonstrated that EVI5 is a nuclear protein, that is also present in the pericentriolar region of interphase cells. EVI5 is localized to the mitotic spindle during chromosome migration, and is also found in the midzone and midbody in late mitosis and during cytokinesis. Knockdown of EVI5 using siRNA results in a high incidence of binucleate cells with evidence for incomplete septation. The role of EVI5 in cytokinesis is further supported through the demonstration of its interaction with members of the chromosome passenger complex (INCENP, aurora B kinase and survivin). Taken together, these results suggest that EVI5 is involved in the completion of cytokinesis.

This work was supported by NINDS grant NS35791 from the National Institutes of Health.
Influence of vitamin D on androgen receptor‐mediated signaling in LNCaP prostate cancer cells
Authors: Rybarczyk Megan Michelle , Russell Nicholas , Mordan Lawrence , Welsh JoEllen , Tenniswood Martin
Published: June 2021

The efficacy of the anti‐androgen Casodex relies on the interaction with the androgen receptor (AR). This study is designed to elucidate the interactions between AR signaling and the vitamin D receptor (VDR) signaling pathways. Using the LNCaP prostate cancer cell line as a model of early stage prostate cancer, we have shown that Casodex induces apoptosis in a time dependent manner which is co‐incident with the loss of the AR from the nucleus, in agreement with previous studies. While 1,25(OH)2D3 induces apoptosis in this cell line in the absence of exogenous testosterone, in the presence of testosterone, treatment with 1,25(OH)2D3 induces cell cycle arrest, but even at doses as high as 100 nM there is no evidence of apoptosis. Furthermore, in the presence of testosterone, 1,25(OH)2D3 blocks the Casodex‐mediated degradation of the AR and abrogates the induction of apoptosis by Casodex. These data suggest that there is significant cross talk between the AR‐mediated signaling and VDR‐mediated signaling that influences the susceptibility of the cells to anti‐androgen induced cell death. Changes in the expression of several p73 isoforms, monitored by Western blotting, correlate with the sensitivity of the cells to Casodex, suggesting that the responsiveness of tumors to anti‐androgens may be inversely related to vitamin D status.

(Supported by USPHS RO1 CA101114‐04 and the Coleman Foundation)
Structural and functional analysis of cell cycle control genes in Cryptococcus neoformans
Authors: Kawamoto Susumu , Virtudazo Eric , Ohkusu Misako , Sonoda Tomoko , Miyagi Yohei , Okuda Kenji , Takeo Kanji
Published: June 2021

Our group has reported the unique cell cycle pattern of the pathogenic yeast Cryptococcus neoformans, different from that of the model budding yeast Saccharomyces cerevisiae. To clarify the cell cycle mechanisms, homologues of cell cycle control genes in C. neoformans were cloned. We have reported the cloning of the CDC28/Cdc2 homologue, CnCdk1 and three Cdk1 cyclin homologues: two B‐type G2/M cyclins and a single G1 cyclin, CnCln1. Further search of the C. neoformans genome database however did not yield additional ORFs with G1 cyclin similarities. Sequence analysis and comparison with other cyclin sequences showed that CnCln1 possesses the typical amino acid residues conserved among G1 cyclins. Analysis of putative amino acid sequences of the C. neoformans CDC28/Cdc2, CnCdk1, revealed that it possesses the conserved motifs found in CDC28/Cdc2 homologues and is highly similar to the fission yeast Schizosaccharomyces pombe Cdc2 and S. cerevisiae CDC28. Notable however, is the difference in an amino acid residue in the well conserved Cdk PSTAIRE motif known to be involved in cyclin binding; in CnCdk1 alanine is changed to serine to become PSTSIRE. Further studies are currently being done to confirm their role and function in C. neoformans cell cycle. In order to analyze at the protein level, we have also been performing the proteomic analysis of the yeast cells.
The Importance of Being CoA: Generation of a Pank1 Knockout Mouse with Reduced CoA Levels
Authors: Leonardi Roberta , Jackson Pamela , Wang Jina , Charles O. Rock , Jackowski Suzanne
Published: June 2021

Coenzyme A (CoA) is an essential cofactor involved in energy metabolism. CoA is assembled in five steps from pantothenate, and pantothenate kinase (PanK) catalyzes the first and most regulated step. Mammals possess four PanK isoforms, PanK1α, 1β, 2 and 3, encoded by three separate genes. The murine PanK1α and 1 arise from alternate transcriptional start sites of the Pank1 gene, and their transcript levels are particularly abundant in liver, kidney and heart. We derived a Pank1 knockout mouse to investigate the effect on CoA and the physiological consequences. The Pank1 knockout mice were viable and fertile, but their livers contained 40% less CoA, and the hearts of knockout males, but not females, showed a 60% decrease in the cofactor level. CoA is required for gluconeogenesis and ketone body production during fasting. The steady state glucose and ketone body levels after a 48 h fast were comparable to wild type, but knockout mice showed a blunted response to a pyruvate challenge. Serum triglycerides were higher, and Pank1 knockout mice were 10–15% larger than wild type controls. These results indicate that a 40–60% reduction in CoA is not lethal but impairs the ability of the liver to quickly produce glucose from pyruvate. The higher serum triglycerides and weight of the knockout animals suggest that the reduction in CoA decreases the efficiency of fatty acid β‐oxidation. Supported by NIHGM062896 and ALSAC.
Concerted Regulation of the Corepressor SMRT by Cdk2 and Pin1
Authors: Stanya Kristopher J. , Liu Yu , Means Anthony R. , Kao Hung‐Ying
Published: June 2021

Transcriptional repression is required for normal mammalian development. SMRT (silencing mediator of retinoid and thyroid receptors) is a corepressor that mediates active repression by nuclear receptors and various transcription factors and thus may act as a signaling integrator. SMRT is thought to achieve this repressive effect through recruitment of histone deacetylases (HDACs). Using a yeast two‐hybrid screen we found that SMRT interacts with the peptidyl‐prolyl isomerase Pin1, which recognizes phosphorylated serine/threonine‐proline motifs and isomerizes proline residues. We have demonstrated that SMRT and Pin1 associate in vitro and in vivo. We have also shown that the SMRT‐Pin1 interaction depends on phosphorylation of SMRT and have identified several in vivo phosphorylation sites within SMRT. We have shown that several of these residues are required for Pin1 interaction. Pin1 also has a negative effect on the half‐life of SMRT; concomitantly, SMRT mutants deficient in Pin1 binding have longer half‐lives. These residues are putative cyclin‐dependent kinase (Cdk) phosphorylation sites as identified by sequence analysis. Following this, we further show that Cdk2 phosphorylates SMRT and coimmunoprecipitates SMRT in vivo. In vitro phosphorylation of SMRT by Cdk2 facilitates a direct interaction with Pin1. ErbB2, an upstream activator of both Pin1 and Cdk2 can modulate SMRT half‐life in a Pin1 and Cdk2‐dependent manner. We hypothesize that Pin1 and Cdk2 are critical regulators of SMRT‐dependent signaling and may promote degradation of SMRT in a cell cycle and phosphorylation‐dependent manner. NIH (T32GM08056), ACS (RSG‐04‐052‐01‐GMC) and OCRA (5302)
Kinetics of EGF receptor dimer formation
Authors: Yang Katherine S , Pike Linda J
Published: June 2021

The EGF receptor is thought to exist in the membrane as a monomer that dimerizes in response to ligand binding. Studies on the relationship between ligand binding and dimer formation have been hampered by the lack of an assay that can follow receptor dimerization in living cells in real time. Here we report the use of a split‐luciferase enzyme complementation assay to study the kinetics of EGF receptor dimerization in intact cells. For the assay, the EGF receptor was C‐terminally fused to the N‐terminal or C‐terminal half of firefly luciferase and both constructs transfected into CHO cells. In the absence of ligand, significant basal light production was observed. Addition of EGF led to a rapid decrease in light production during the first two minutes, followed by a rebound in light production over the ensuing 3 to 5 min. The data suggest that under resting conditions, the C‐terminal tails of the EGF receptor are in proximity to each other, possibly in pre‐formed dimers, and that ligand binding induces a conformational change that disrupts these interactions but ultimately allows the formation of an alternative oligomeric state of the receptor.

This work is supported by NIH grant GM64491to LJP.
Parathyroid Hormone (PTH) Regulation of Non‐Canonical Wnt‐4: A Stimulator of Differentiation in Osteoblasts and Bone Marrow Stromal Stem Cells
Authors: Bergenstock Marika Katherine , Partridge Nicola C
Published: June 2021

Parathyroid hormone (PTH) is the only clinically available anabolic agent for treating osteoporosis. PTH increases bone formation by directing changes in osteoblast function. We identified PTH‐induced gene expression profile changes using DNA microarray analysis in osteoblastic cells and in osteoblast‐rich regions of bone. This uncovered a novel, PTH‐regulated gene, Wnt‐4. Wnt proteins are essential in development and are necessary for the acquisition of high bone mass. Wnts are divided into Wnt‐1 and Wnt‐5a signaling classes, which activate canonical (Wnt/β‐catenin) and non‐canonical intracellular pathways (Wnt/Ca2+ and Wnt/PCP), respectively. Wnt‐4 is a non‐canonical protein. Real‐time RT‐PCR analysis shows significant PTH stimulation of Wnt‐4 both in vitro and in vivo. The stimulation is a primary response to PTH and PKA pathway dependent. Primary osteoblastic cells treated with WNT‐4 show greater alkaline phosphatase activity and real‐time RT‐PCR shows early stimulation of osteoblast marker genes and genes of the Wnt pathway. WNT‐4 treatment stimulates the non‐canonical signaling pathways, and only minimally activates the canonical pathway. Most significantly, real‐time RT‐PCR analysis of WNT‐4 treated stromal stem cells shows induction of osteoblastogenesis‐markers and inhibition of adipogenesis‐markers, as well as increased colony forming units. We hypothesize that PTH acts on mature bone‐lining osteoblasts to stimulate their expression and production of Wnt‐4, which then acts on immature stromal stem cells in the bone marrow to stimulate their differentiation into the osteogenic lineage and thereby preventing differentiation into adipocytes.
Protein kinase C ζ‐dependent LKB1 phosphorylation at serine 428 induces LKB1 nuclear export and apoptosis in endothelial cells
Authors: Song Ping , Xie Zhonglin , Wu Yong , Xu Jian , Dong Yunzhou , Zou Ming‐Hui
Published: June 2021

LKB1 is a serine‐threonine protein kinase that, when inhibited, may result in unregulated cell growth and tumor formation. However, the mechanisms underlying regulation of LKB1 are unknown. The aim of the present study was to define the upstream signaling molecules responsible for peroxynitrite (ONOO‐)‐induced LKB1 phosphorylation. Exposure of cultured human umbilical vein endothelial cells (HUVECs) to pathological concentrations of ONOO‐ significantly increased the phosphorylation of LKB1 at Ser428 and protein kinase C [zeta] (PKC[zeta]) at Thr410. These effects were accompanied by, increased activity of the lipid phosphatase PTEN, decreased activity and phosphorylation (Ser473) of Akt, and induction of apoptosis. In LKB1‐deficient HeLa S3 cells or in HeLa S3 cells transfected with kinase‐dead or inactive LKB1, ONOO‐ enhanced Akt‐Ser473 phosphorylation. Overexpression of wild‐type LKB1 in these cells mimicked the effects of ONOO‐ in HUVECs. Further analysis revealed that PKC[zeta] directly phosphorylated LKB1 at Ser428 in vitro and in HUVECs, resulting in increased PTEN phosphorylation at Ser380/Thr382/383. ONOO‐ also enhanced PKC[zeta] nuclear import and LKB1 nuclear export. We conclude that PKC[zeta] mediates LKB1‐dependent Akt inhibition in response to ONOO‐, resulting in endothelial apoptosis.

This work was supported by NIH grants (HL079584, HL07439, and HL080499).
GMF overexpression causes apoptosis in neuroblastoma cells
Authors: Zaheer Asgar , Knight Scott , Zaheer Ashna , Ahrens Marcus , Sahu Shailendra K. , Yang Baoli
Published: June 2021

In the present study we report that a replication‐defective adenovirus construct of GMF cDNA (GMF‐V) induced overexpression of GMF protein in neuroblastoma (N18) cells. Overexpression of GMF in N18 cells is correlated with the increase in toxicity and loss of cell viability. A significant increase in GSK‐3beta activity occurred after infection with GMF‐V compared to the no infection (mock) and lacZ controls. Overexpression of GMF also increased caspase‐3 activity, an early marker of apoptosis. Depletion of GMF mRNA by introducing GMF‐specific siRNA completely blocked GSK‐3beta and caspase‐3 activation whereas control scrambled siRNA had no effect. A cell‐permeable specific inhibitor of GSK‐3beta and lithium completely prevented GMF‐dependent activation of caspase‐3. Our results demonstrate that GMF is involved in the signaling leading to the activation of GSK‐3beta, caspase‐3 and inactivation of Akt in neuroblastoma cells and strongly suggest the involvement of GMF in neuro degeneration. This is the first study to demonstrate that GMF overexpression causes apoptosis through activation of the GSK‐caspase‐mediated signaling mechanism.
Mechanism of action of iejimalide, a novel marine macrolide, in prostate and breast cancer cells
Authors: McHenry Peter Ryan , Kluesner Nicholas , Schweitzer Dirk , Helquist Paul , Tenniswood Martin
Published: June 2021

Iejimalides are macrocyclic lactones (macrolides) composed of a 24‐member ring with two methoxy groups and an N‐formyl‐L‐serine side chain. We have tested iejimalide for biological activity in the NCI panel of 60 tumor cell lines and have established that the compound is active at low nanomolar concentrations. Using p53‐wild type or ‐null prostate tumor cell lines we have shown that iejimalide B induces apoptosis in LNCaP cells, which express wild type p53, and S‐phase arrest but not apoptosis in p53‐null PC‐3 cells. In p53‐wild type MCF‐7 breast cancer cells iejimalide B induces nuclear accumulation of p53, p21WAF1/CIP1, and AIF (apoptosis inducing factor). The signaling pathway responsible for the increase in nuclear p53 levels has not been fully elucidated; however, we have shown that iejimalide blocks the acidification of lysosomes, probably through the inhibition of the vacuolar H+‐ATPase (V‐ATPase) as early as 3 h after treatment. This precedes the depolarization of the mitochondrial membrane, cytochrome c release and DNA fragmentation, which occur after 24 h. Whether this delay is due to slow signaling from lysosomes to the nucleus and mitochondria or due to a second site of action of iejimalide, possibly through the inhibition of the mitochondrial F‐ATPase, remains to be determined. Nevertheless these data suggest that the macrolactone ring may be a useful pharmacophore for the development of new chemotherapeutic drugs for the treatment of some hormone dependent cancers. (Supported by the Walther Cancer Institute and the Coleman Foundation)
Tissue plasminogen activator blocks ISOFLURANE‐mediated neuronal apoptosis in developing neurons
Authors: Head Brian Patrick , Chin‐Lee Blake , Patel Hemal H. , Drummond John C. , Roth David M. , Patel Piyush M.
Published: June 2021

An important contributor to synaptogenesis during neuronal development is the neurotrophin BDNF (brain derived neurotrophic factor). BDNF is secreted from synaptic vesicles as a precursor or proneurotrophin (proBDNF) and is proteolytically cleaved to become a mature BDNF (mBDNF) by the protease plasmin in the synaptic cleft. Plasminogen, the precursor to plasmin, is proteolytically cleaved by tissue plasminogen activator (tPA), a protease released from pre‐synaptic vesicles. mBDNF binds to TrkB receptors to promote neuronal survival and synaptogenesis, in contrast to the uncleaved proBDNF which binds with high affinity to p75NTR resulting in reduced dendritic spines and enhanced neuronal apoptosis. Isoflurane (ISO‐1.4%; 4 hr) significantly increased cleaved caspase‐3 (Cl‐Csp3) in postnatal day 5 (PND5) neurons but not in PND14 or PND21, and Cl‐Csp3 was elevated in the hippocampus in vivo in PND5 animals exposed to ISO. ISO exposure significantly reduced the amount to tPA in the neuronal media compared to control media. Application of tPA (0.03 to 3 μg/ml) attenuated ISO‐mediated Cl‐Csp‐3 in primary neurons as assessed by immunoblot and immunofluorescence microscopy. In addition, tPA application led to increased levels of phosphorylated Akt, a downstream enzyme of TrkB signaling. Pep5, a TAT‐conjugated p75NTR inhibitor, significantly attenuated ISO‐mediated Cl‐Csp3. Application of either plasmin or tPA prior to ISO‐exposure increased dendritic spine numbers and complexity as assessed by the dendritic spine marker, drebrin. These findings show that tPA attenuates ISO‐mediated reduction in dendritic spines and enhanced apoptosis in primary neurons. (Supported by NIH)
Gonadotropin‐Regulated Testicular Helicase (GRTH/Ddx25): A Negative Regulator of Apoptosis in Male Germ Cells
Authors: Gutti Ravi Kumar , Tsai‐Morris Chon‐Hwa , Dufau Maria L.
Published: June 2021

GRTH/DDX25 is an essential post‐transcriptional regulator of spermatogenesis. In GRTH null mice severe apoptosis was observed in spermatocytes entering the metaphase of meiosis. Pro‐ and anti‐apoptotic factors were found to be under GRTH regulation in comparative studies of spermatocytes from wild type and GRTH −/− mice (KO). KO displayed decreased levels of Bcl‐2 and Bcl‐xL (anti‐apoptotic factors) an increase in Bid Bak and Bad (pro‐apoptotic), reduced phospho‐Bad, and release of Cytochrome C. Also, an increase on Smac, a competitor of inhibitor apoptotic proteins that release Caspases, was observed. These changes caused an increase in cleavage of Caspases 9 and 3, activation of Caspase 3 and increases in cleavage products of PARP. The half‐life of Caspase 3 transcripts was markedly increased in KO, indicating GRTH negative role on its mRNA stability. IκBα, which sequesters NFκB from its transcriptional activation of pro‐apoptotic genes was highly elevated in KO, and its phospho‐form, which promotes its dissociation, was reduced. Increase of HDAC1 and abolition of p300 expression in KO indicated a nuclear action of GRTH on the NFκB‐mediated transcription of anti‐apoptotic genes. It also regulates the associated death domain pathway and Caspase 8 mediated events. GRTH‐mediated apoptotic regulation was further indicated by its selective binding to pro‐ and antiapoptotic mRNAs. These studies have demonstrated that GRTH, as a component of mRNP particles acts as a negative regulator of the TNFR1 and Caspase pathways and promotes NFκB function to control apoptosis in spermatocytes of adult mice.
Identification of a New Target in the Interactions between Staphylococcal Enterotoxin B with the Renal Proximal Tubule Epithelial Cells
Authors: Hammamieh Rasha , Sumaida Dena , Miller Stacy , Lin Yixin , Morris Sam , Jett Marti
Published: June 2021

Staphylococcal eneterotoxin B (SEB) a monovalent T cell mitogen and inducer of T suppressor cells, is known to form complexes with the major histocompatibility complex class II and T‐cell receptor in a manner that is quite different from the way the natural ligands bind to the same receptors and direct normal cellular responses it bypasses the normal route of antigen processing by binding as an intact protein to the complex formed by the MHC class II receptor on the antigen‐presenting cell and the T cell receptor.

It is estimated that within 90 min. post‐exposure to SEB, >75% of the toxin is cleared to the renal tubule epithelial cells. We hypothesize that SEB binds to these cells using a mechanism that does not include MHCII.

Binding assays carried out in vitro in proximal tubule epithelial cells suggests that glycosphingolipids and lipid rafts play a major role in SEB interactions with these cells.

CD1d is an MHC class I homologous protein that is present in the plasma membrane lipid rafts and is proposed to be involved in presenting the antigen to the NKT cells.

We have studied SEB interactions with the proximal tubule epithelial cells using imaging and direct protein‐protein interaction studies and showed that SEB can interact with CD1d on the proximal tubule epithelial cells.
Sensors of mitosis
Authors: Morris May Catherine , Scheper Johanna , Pellerano Morgan
Published: June 2021

Detection of cell cycle regulators whose aberrant expression or activation influences cell cycle progression and cell proliferation is critical for the development of efficient diagnostic and therapeutic strategies. In particular Cdk1‐cyclin B1 and Cdc25 phosphatases are central players and partners that coordinate entry into mitosis and whose function is critical for cell proliferation. We have developed non‐genetically encoded fluorescent sensors which specifically recognize Cyclin B1 and Cdc25 phosphatases through molecular interfaces distinct from ATP‐binding pockets or active sites. In vitro characterization of these sensors reveals significant changes in their fluorescence upon specific detection of their target. The combination of these sensors with non‐covalent peptide‐based delivery systems enables efficient delivery into cultured mammalian cells. Characterization of the specificity and efficiency of these biosensors in a cellular environment provides promising perspectives for in vivo applications. Source of Research Support: CNRS & ANR Grant to MCM
JL13, a clozapine‐like potential antipsychotic agent, reduces sympathetic cutaneous vasomotor alerting responses (SCVARs) in rats, but the drug is less potent than clozapine
Authors: Blessing William W. , Capuano Ben , Crosby Ian T. , Taylor David A.
Published: June 2021

Clozapine is an atypical antipsychotic drug used to treat schizophrenia. However clozapine has serious side effects and there is a search for new antipyschotics. One such agent is JL13 [5‐(4‐methylpiperazin‐1‐yl)‐8‐chloro‐pyrido[2,3‐b][1,5] benzoxazepine fumarate], structurally related to clozapine but postulated to have less haematological and cardiological side effects because of reduced sensitivity to oxidation. Clozapine reduces SCVARs in rats. We have evaluated JL13 in this model. Sprague Dawley rats (250–450 g) were instrumented (Fluothane anesthesia) with Doppler blood flow probe at the base of the tail artery and the effect of JL13 (0.0625–5 mg/kg s.c.) on SCVARs (1) was evaluated in the conscious freely moving animal. JL13 significantly reduced SCVARs, (linear regression between log dose and SCVAR index P<0.001, R2=0.60, n=6 rats at each dose). Prior blockade of dopamine D2 receptors with spiperone (25 μg/kg) did not reduce the effect of JL13. Comparison with previous results (1) indicates that the SCVAR‐reducing action of JL13 is only approximately one‐fifth as potent as clozapine, and dopamine D2 stimulation does not contribute to the action of JL13. Supported by NHMRC.