bluuebunny/crossref_metadata_2025 huggingface dataset:
Lipoprotein cholesterol uptake in bone marrow‐derived murine macrophages
Authors: Meyer Jason , Cai Lei , Ji Ailing , Wang Zhen , van der Westhuyzen Deneys
Published: June 2021

Macrophage foam cell formation is thought to be a key step in early atherogenesis. However, despite considerable research into this process, the cellular mechanisms responsible are still unclear. We and others have observed that macrophage uptake from lipoproteins exhibits several characteristics that suggest that scavenger receptor (SR)‐independent pathways make a significant contribution. Here we use primary bone marrow‐derived macrophages (BMMs) from wild‐type and SR‐deficient mice to study mechanisms of macrophage lipoprotein uptake. We demonstrate that BMMs take up cholesterol from HDL, LDL and oxLDL in a SR‐independent manner and that accumulation of cholesterol ether occurs selectively compared to apolipoprotein. We also show that lipoprotein uptake is neither saturable nor markedly reduced by unlabeled competitor, suggesting that a low affinity uptake mechanism plays a major role. Using radiolabeled and fluorescently‐labeled ligands, we show that the uptake of native and modified LDL are dependent on actin polymerization and that excess unlabeled competitor does not reduce this process, supporting the hypothesis that macropinocytosis is a major mechanism for LDL internalization in macrophages. We conclude that selective uptake and macropinocytosis are active sources of cholesterol in macrophages and that these processes may be important in foam cell formation in vivo, especially in the absence of SR activity. Supported by HL65730 and HL086670 (DvdW), NIH.
Calcium‐induced permeability transition in rat brain mitochondria is promoted by carbenoxolone through targeting connexin43
Authors: Reiser Georg , Azarashvili Tamara , Baburina Yulia , Grachev Dmitry , Krestinina Olga
Published: June 2021

Carbenoxolone (Cbx), a substance from the medicinal licorice is used for anti‐inflammatory treatments. Cbx was already shown to induce opening of the permeability transition pore (PTP) in rat liver mitochondria (RLM). We investigated the mechanism of action of Cbx on Ca2+‐induced PTP opening in rat brain mitochondria (RBM), and in rat heart mitochondria (RHM) and in RLM, in an attempt to identify the molecular target of Cbx in mitochondria. In RBM, RHM and RLM, threshold Ca2+ load induced PTP opening. This was manifested by sudden Ca2+efflux from the mitochondrial matrix, a drop in membrane potential, and swelling of mitochondria. At 1 μM, Cbx enhanced the Ca2+‐induced, Cyclosporine A‐sensitive PTP opening. A well‐known target of Cbx in cells is the connexin family of proteins, which form gap junctions. Moreover, Connexin43 (Cx43) was previously found in RHM and attributed to the protective mechanism induced by ischemic preconditioning. We hypothesized that Cx43 might be a target for Cbx in brain mitochondria. We clearly detected Cx43 by Western blot in RBM and in mitochondria isolated from astrocytes, less in RHM, but not in RLM. Interestingly, the anti‐Cx43 antibody abolished the Cbx‐enhanced PTP opening in RBM, but not in RLM. In RBM, PTP caused serine‐368 phosphorylation of Cx43. These results show that Cbx facilitates Ca2+‐induced PTP opening, addressing as possible molecular target Cx43 in brain mitochondria.
MicroRNA Expression Analysis of Blood Samples from African Green NHP Exposed to Yersinia Pestis
Authors: Jibitu Meskerem , Miller Stacy Ann , Waddy Edward Byron , Muhie Seid , Hammamieh Rasha , Jett Marti
Published: June 2021

Yersinia pestis, the causative agent of plague, is known to develop strategies to overcome the host immune mechanisms and survive in the host. The molecular changes induced by Y. pestis in the host are not well delineated. Here, we examined the early events triggered after the intracellular infection of Y. pestis in NHPs by analyzing the host microRNA profiles using microRNA microarrays. Monkey Blood samples treated with Yersinia pestis and sham controls were collected at room temperature using the QIAGEN™ protocol. RNA samples were isolated and assessed using the Bioanalyzer. SuperArray miRNA PCR Array were used to hybridize the isolated samples. Data were analyzed to identify miRNAs that were differentially expressed in infected NHPs. Some of the identified miRNAs are known to target transcription factors, molecular transport and apoptosis.
Cloning, Expression and Characterization of Novel Aldo‐Keto Reductase 1B (AKR1B) Proteins ‐ Human AKR1B11 and Murine Akr1b16
Authors: Salabei Joshua Kuimeta , Bhatnagar Aruni , Barski Oleg A
Published: June 2021

The Aldo‐Keto reductase (AKR) superfamily comprises enzymes that catalyze redox transformations. A novel gene locus, tcag7.1260 (AKR1B11) has been predicted to cluster with human members of the AKR1B subfamily, AKR1B1 and AKR1B10. AKR1B11 is predicted to have 10 exons and an open reading frame having 91% and 67% identities with AKR1B10 and AKR1B1 respectively. Cross genome comparison of human and murine AKRs reveals the existence of a murine AKR gene, 2310005E10Rik (Akr1b16), having an open reading frame with 82.9% identity with human AKR1B10 protein. We test the hypothesis that AKR1B11 and Akr1b16 are expressed into functional proteins. Using whole human/mouse tissue mRNA, we demonstrated the existence of spliced AKR1B11 and Akr1b16 mRNAs in human brain and testes, and in multiple murine tissues. cDNAs were cloned into pET28a and pIRES‐hrGFP‐1α vectors for bacterial and mammalian expression respectively. Both genes were expressed as 37 kDA proteins. COS 7 cells transfected with pIRES‐hr‐GFP‐1α/AKR1B11 showed a 4.8 fold (with 500μM p‐NB) and 3.3 fold (with 10mM DL‐glyceraldehyde) increase in enzymatic activity. We raised specific antibody against AKR1B11. Immunoprecipitation and Western blot showed the presence of AKR1B11 protein in the ovary, testes and kidney. AKR1B11 protein was also detected in human ovary sections by immunohistochemistry. We conclude that AKR1B11 is present in human tissues. Further confirmation of its activity in these and other tissues is needed. Protein activity measurements with a broad variety of AKR substrates are required for the characterization of AKR1B11 and Akr1b16.

Research support: COBRE: P20RR024489
Proteomics analysis of the regulatory network involved in long‐distance signaling in potato
Authors: Shah Shweta , Hannapel David J , Rao Aragula G
Published: June 2021

In potato (Solanum tuberosum), BEL5 mRNA is a mobile RNA which is involved in the signaling system that activates tuber formation. Short day (SD) induces tuberization and long day (LD) inhibits the process. The mobility of StBEL5 RNA is induced by a SD photoperiod. It is likely that the movement of StBEL5 is facilitated by the formation of a RNA‐protein complex. To verify this proposed mechanism we have undertaken a proteomic analysis. Proteins from potato petioles (as the transcriptional source of StBEL5 RNA is leaf veins and petioles) grown under LD and SD photoperiod conditions were separated by 2‐dimensional gel electrophoresis (2‐DE). Proteins which were differentially expressed in response to changes in photoperiods were analyzed by Progenesis SameSpots software and identified by MALDI MS/MS. Phosphoproteins (phosphorylation of proteins is central to signal transduction) and RNA binding proteins were also enriched from LD and SD potato petioles, separated on 2‐DE and analyzed by MALDI MS/MS. We have identified many proteins that are differentially expressed in response to LD or SD photoperiods. A comprehensive profile of the phosphoproteomes of LD and SD potato petioles has been established. Significantly, the presence of the RNA‐binding protein, elF‐5A, has been detected in multiple phosphorylation states only under LD conditions. Numerous RNA‐binding proteins have also been isolated and identified.
Characterization of new peptide epitopes derived from human collagen I and II processing by metalloproteases associated with human dendritic cells
Authors: Clement Cristina Corina , Santambrogio Laura
Published: June 2021

Type I and II collagens together with other extracellular matrix molecules are degraded in normal remodeling associated with physiological processes such as morphogenesis, growth, wound healing and physiological bone turnover. In addition, extracellular matrix is degraded during pathological processes, such as arthristis, osteolysis or metastasis. The goal of this research was to characterize HLA‐DR1 and HLA‐DR 4 restricted epitopes derived from the processing of collagen I and II by extracellular metalloproteases (MMPs), which could be different from the epitopes generated by the action of cathepsins, during the endocytic pathway of antigen processing. Complete sequences of peptides proteolyzed from highly purified human collagen‐I and II were determined using both recombinant enzymes as well as purified dendritic cell (DC)‐derived plasma membranes and endosomal fractions coupled with proteomics methods, such as tandem mass spectroscopy (MS/MS). A wide variety of peptides originated from collagen‐I and II were shown to have cleavage sites specific for metalloproteases and typical hydroxylations of proline and lysine, as posttranslational modifications. IC50 and binding affinities of some peptides epitopes derived from collagen‐II were characterized using recombinant HLA‐DR1 and HLA‐DR4 molecules and their immunogenicity was determined using transgenic mice for these proteins.
Application of a high‐resolution mass spectrometry for estimation of proteome dynamics
Authors: Ilchenko Serguei A , Rachdaoui Nadia , Li Ling , Wiilard Belinda , Previs Stephen , Kasumov Takhar
Published: June 2021

We recently developed a method for estimating proteome dynamics in vivo with deuterated water (2H2O) using MALDI‐TOF MS (Rachdaoui N. et al., MCP, 2009, Epub). Although this is a reliable method for precise measurement of protein kinetics, it requires thorough purification of samples and necessitates the determination of intracellular labeling of amino acids in specific tissues. Another approach for quantifying protein kinetics is to measure the 2H‐enrichments of body water and protein‐bound amino acid. We applied nanospray LTQ FTICR MS for the measurement of the isotopic enrichment of proteins and protein‐bound amino acids in a single run. Measurements of the peptides’ isotopic distribution are performed in SIM mode, while the fragment ions are detected in the SRM mode. The MS/MS analyses permit sequence determination, the FT ICR analyses distinguish the isotopic peaks from contaminating species, and the SIM scan allows precise (±0.8%) and accurate (±2%) measurements of isotopic enrichment. A computational method was developed for data processing. The technique was applied to measure the synthetic rate of albumin in mice treated with 2H2O. After identification of the most common peptides, the isotopic distributions of the precursor, fragment ions, and protein‐bound amino acids were measured. Thus, utilization of this technique enables the study of protein dynamics (Supported by NIH Grant 5R21RR025346‐02).
Development of a urine protein preparation method for 2‐D gel analysis and selection of osteoporotic biomarkers
Authors: Lee Jinwoo , Choi Hyunsu , Kim Jong Sang , Choi Je Yong
Published: June 2021

Biomarker development for osteoporosis from human urine was carried out by optimizing urine protein preparation methods and 2‐D gel analysis method through proteomic protein identification tools including mass spectrometry and bioinformatics. Preparation of urine proteins for reproducible high resolution 2‐D gel analysis has been a troublesome topic in urine proteomic analysis. We developed a reproducible pretreatment method for urine protein preparations by ultrafiltration, dialysis, and desalting. This method efficiently removes interfering molecules for IEF analysis and improves resolution and reproducibility in 2‐D gel analysis. Using this improved protein preparation method we could obtain good quality 2‐D gels for the comparison of protein profiles between normal and patient urine samples. Urine samples from human osteoporosis patients and osteoporotic animal model from ovariectomy were used to develop biomarkers for osteoporosis. We could selected several osteoporosis associated biomarkers, among which vitamine D binding protein, vitronectin, acid‐glycoprotein‐alpha were confirmed by Western blot analysis. The results suggest that those biomarker proteins can be utilized for the development of diagnostic kits detecting osteoporosis simply from urine. The study was supported by a grant from Korean Ministry of Health, Welfare and Family Affairs.
Direct procaspase activation as a therapeutic strategy in the treatment of canine lymphoma
Authors: Peterson Quinn P , West Diana C. , Hsu Danny C. , Botham Rachel C. , Fan Timothy M. , Hergenrother Paul J.
Published: June 2021

General cytotoxins that induce cell death in all rapidly dividing cell types have been the backbone of chemotherapy for over 50 years. These drugs are most properly classified as “anti‐proliferatives” rather than “anti‐cancer” as they exhibit little selectivity for cancer cells versus non‐cancerous cells. A burgeoning area of focus in oncology is the identification of compounds that target a defined molecular defect in cancerous cells. This targeted approach would thus induce death specifically in cancer cells while leaving non‐cancer cells unharmed. As one of the hallmarks of cancer is a resistance to apoptosis, a promising approach is the reactivation of the apoptotic program in cancer cells. A compound called procaspase‐activating compound 1 (PAC‐1) was recently reported that activates procaspase‐3 in vitro and induces death in cancer cells in culture and in mouse xenograft models. Experimental evidence indicates that PAC‐1 activates procaspase‐3 in vitro through chelation of inhibitory zinc ions and that PAC‐1 colocalizes with points of caspase‐3 activity in the cell. Importantly, these results suggest a role for endogenous zinc in the regulation of caspase activity in vivo. PAC‐1 is the first small molecule reported to activate a proenzyme protease, and as such PAC‐1 could be a useful anti‐cancer agent and a model for other procaspase‐3 activating compounds. To this end, a recently initiated canine clinical trial investigates the safety, toxicity and efficacy of a derivative of PAC‐1 in pet dogs with lymphoma.
Sumoylation of Sp1 is increased by betulinic acid and led to RNF‐mediated degradation
Authors: Wang Yi‐Ting , Hung Jan‐Jong
Published: June 2021

Our recent findings show that Sp1 can accumulate during tumorigenesis after it undergoes posttranslational modification, namely, sumoylation, and phosphorylation. We found that Sp1 was the substrate of the ubiquitin E3 ligase, RNF4. Mapping of the domain involved in the interaction between Sp1 and RNF4 revealed 2 regions that interacted with each other. One was a SUMO‐modifier in the N‐terminus of Sp1(Lys16) that interacted with the SUMO‐interacting motif (SIM) of RNF4, and the other was the C‐terminus of Sp1 that interacted with RNF4 in a SIM independent manner. We also found that a previous reported anticancer drug‐betulinic acid (BA) increases Sp1 sumoylation, facilitating interaction between Sp1 and RNF4, which leads to Sp1 degradation through the ubiquitin‐dependent proteasome pathway. Here, we elucidate that Sp1 degradation through BA treatment facilitate sumoylation of Sp1 during cancer therapies.
Molecular and functional characterization of a new tumor suppressor protein ZNF509 in the regulation of cell growth
Authors: Jeon Bu‐Nam , Yoon Jae‐Hyeon , Hur Man‐Wook
Published: June 2021

A variety of cellular biological functions like cell proliferation are precisely controlled by a number of protein‐protein interactions among regulatory proteins. In cancer cells, aberrant expression of oncogenes or/and tumor suppressor genes causes dramatic changes in the cellular regulatory program governing cell proliferation. Recently, POK family transcription factors were characterized as critical oncogenes or tumor suppressors that could regulate expression of cell cycle control genes.

We found that a novel POK family transcription factor, ZNF509, is induced by p53 and may be a tumor suppressor. ZNF509 is a potent transcriptional activator of CDKN1A encoding cell cycle arrest gene, p21. EMSA, oligo pull‐down, and ChIP assays showed that ZNF509 directly binds to proximal promoter region of p21 in vitro and in vivo. Also ZNF509 and MIZ‐1 interact with each other and synergistically activate transcription of p21 gene. The molecular interaction recruits more co‐activator p300 to the p21 proximal promoter. The ZNF509‐MIZ‐1‐p300 complex acetylates histone Ac‐H3 and ‐H4 at the p21 proximal promoter. Knockdown of endogenous ZNF509 expression results in decrease in the transcription of p21 gene, which increases cell proliferation and DNA synthesis. Overall, our data suggest that ZNF509 is a potent transcription activator of the cell cycle arrest gene p21 and may be a tumor suppressor gene.
TAF‐dependent basal transcription function of new Cofactors through the Initiator element
Authors: XU MUYU , Pan Songqin , Malik Sohail , Martinez Ernest
Published: June 2021

Specific transcription initiation by eukaryotic RNA polymerase II is controlled by core promoter DNA elements that recruit general/basal transcription factors (GTFs) and by distal promoter and enhancer sequences that bind regulatory factors. The TATA box (at −30) and the Initiator (INR) at the transcription start site (+1) are core promoter elements that are variably present in eukaryotic promoters, and are bound, respectively, by the TBP and TAF components of the TFIID complex. TATA and INR elements synergize to enhance TFIID binding and basal transcription and their presence or absence influences the activation of core promoters by specific regulators and enhancers. While the INR is recognized by the TAF1 and TAF2 subunits of TFIID, INR‐dependent transcription and TATAINR synergy are not observed in purified transcription systems reconstituted with TFIID, the other GTFs (TFIIA, TFIIB, TFIIE, TFIIF, TFIIH) and RNA polymerase II, but requires additional TAFs and Initiator‐dependent cofactors (TICs) that have remained elusive. Here, we report the identification of several of these new Cofactors that potentiate the basal transcription function of TFIID/TAFs through the INR element and allow TATA‐INR synergy.
mRNAs with a complex: The long‐term consequences of a nuclear upbringing
Authors: Moore Melissa J. , Singh Guramrit , Kucukural Alper , Weng Zhipeng
Published: June 2021

As pre‐mRNAs mature into mRNAs in the nucleus, they are decorated with proteins that impact their subsequent fate in the cytoplasm. Pre‐mRNA splicing stably deposits a set of proteins (the exon junction complex, EJC) 20‐24 nucleotides upstream of exon junctions. EJCs thus assembled preserve the splicing history of an mRNA and also serve as binding platforms for other proteins mediating downstream events such as mRNA surveillance, translation and mRNA localization. Work in recent years has elucidated the protein composition of the EJC and the molecular interactions required for EJC assembly as well as its molecular architecture at the atomic level. Nonetheless, many fundamental questions regarding the EJC remain, such as: Are EJCs deposited after splicing on all exon‐exon junctions of an mRNA? Are EJCs always deposited at the same position in relation to every exon‐exon junction? Are EJCs exclusive to exon‐exon junctions, or do they bind to other sites on mRNAs or unspliced RNAs? To address these questions, we purified EJCs from cell lines stably expressing FLAG‐tagged EJC proteins at near endogenous levels. EJCs thus purified exhibit a defined RNA footprint that was used to generate short cDNA libraries for Deep Sequencing. These genome‐wide deposition profiles will enhance and broaden our understanding of the EJC's contribution to post‐transcriptional gene regulation.
Novel extracellular‐signal regulated kinase (ERK) substrate inhibitors mediate apoptotic signaling
Authors: Boston Sarice R , MacKerell Alexander D , Shapiro Paul
Published: June 2021

ERK proteins promote cancer cell proliferation and survival through substrate phosphorylation events. Thus, selective inhibition of substrates involved in these mechanisms provides promising targets for anti‐cancer therapies. Small molecules designed to interfere with ERK docking domains involved in substrate interactions were identified using computer‐aided drug design (CADD). A total of 184 compounds were tested in HeLa cells. Upon treatment, 35 compounds caused the induction of apoptotic signaling as measured by poly (ADP‐ribose) polymerase (PARP) cleavage. Importantly, these effects were not observed in non‐transformed cells. To evaluate the mechanism of apoptosis induction, ERK‐mediated Caspase‐9 phosphorylation, which prevents activation of the intrinsic apoptotic pathway, was inhibited by several of the active compounds. However, other active compounds inhibited Bim phosphorylation, a pro‐apoptotic protein whose ERK‐mediated phosphorylation results in proteosomal degradation. Moreover, induction of apoptotic signaling by the active compounds did not appear to be due to off target effects on Akt kinase, which also mediates pro‐survival mechanisms. Taken together, selective inhibition of ERK substrates promote apoptosis in HeLa cells, suggesting a novel approach in the development of new anti‐cancer therapeutics. (Supported by NIH Grant # CA120215‐01 to PS & CA120215‐02S to SB)
Characterization and Inhibition of a Novel Diterpene Virulence Factor from Mycobacterium tuberculosis
Authors: Mann Francis Michelle
Published: June 2021

Mycobacterium tuberculosis (Mtb) remains a widespread and devastating human pathogen whose ability to infiltrate the human immune system is an active area of investigation. We have recently reported the discovery of a novel diterpene from Mtb, edaxadiene, which has the ability to arrest phagosome maturation at an early stage, presumably contributing to the early survival of Mtb in the immune system. Importantly, Mtb knockout in the committed step of edaxadiene synthesis are impaired in their ability to survive in the phagosome. In addition, the closely related but less infectious Mycobacterium bovis has a frameshift mutation in this enzyme, resulting in an inactive enzyme in vitro, further indicating involvement of edaxadiene in early infection. Together, these results indicate that this enzyme is a suitable target for drug design. In an attempt to evaluate this enzyme as a drug target, we have kinetically characterized the committed enzyme in edaxadiene synthesis and evaluated it with two sub‐nM inhibitors, which are not suitable for clinical applications, but offer a platform for further pharmacological design. Further, our kinetic characterization has indicated a mechanism for tight regulation of this pathway via divalent cation starvation during phagosomal engulfment, allowing timely and potent regulation of edaxadiene synthesis during infection.
Influence of Hydroxylation and Copper Binding on Poly (L‐Proline) Type II Helix Formation in the N‐terminal Domain of the Prion Protein
Authors: Dow Katelyn Marie , Eberhardt Eric S
Published: June 2021

A family of neurodegenerative disorders including scrapie, chronic wasting disease, bovine spongiform encephalopathy and Creutzfeldt‐Jakob disease are thought to be associated with the conversion of normal cellular prion protein (PrPc) to a protease resistant prion isoform, PrPSc (prion protein scrapie). The N‐terminal region of PrP is highly flexible, containing poly (L‐proline) type II (PPII) helical content. The five octarepeat sequences in the N‐terminal region of the prion protein (amino acids 53–90) are also involved in metal ion binding. While the biological role of the N‐terminal region in the conversion of PrPc to PrPSc remains unclear, a recent report indicates that Pro 44 is post‐translationally modified to a 4‐hydroxyproline residue (4‐Hyp) and that the PPII helix content is increased in N‐terminal domain peptides that are hydroxylated. Circular dichroism spectroscopy has been used to examine the effect of hydroxylation at Pro 44 on PPII helix formation in unmodified and modified (4‐Hyp) model peptides of the N‐terminal domain containing zero, one, or two octarepeat sequences (amino acids 37–53, 37–61, 37–69). In all cases, hydroxylation of Pro 44 increases PPII helical content. The addition of copper to these peptides also alters PPII helical content, with different effects observed for each pair of peptides.
Biological activity of a stabilizing peptide fusion protein of leptin and ATS‐leptin in vivo and in vitro
Authors: Park Hyunju , Lee Saet‐byul , Kim Jongsun
Published: June 2021

Protein aggregation is a major stability problem of therapeutic proteins, particularly in the biomedical and biopharmaceutical. ATS‐leptin of the fusing the C‐terminal acidic tail of α‐synuclein (119?40 residues of α‐synuclein, ATS hereafter) appeared to be very resistant to aggregation induced by agitation, freeze/thaw, and heat stresses. In this study, we investigated the biological activity of ATS‐Leptin fusion protein in vivo and vitro. Biological activities of Leptin and ATS‐Leptin proteins were determined by MCF7 cell proliferation assay in vitro. Mice (female ICR) were injected i.p. 1μg/g twice daily of Leptin and ATS‐Leptin and saline for 14 d. Human Leptin and ATS‐Leptin stimulated the proliferation and the survival of breast cancer cell. Mouse Leptin and ATS‐mouse Leptin significantly decrease body weight, food intake in female ICR mice. Serum GOT, GTP levels of mLeptin and ATS‐mLeptin treated mice were higher compared with saline mice. The protein of mLeptin or ATS‐mLeptin reduced serum TG levels. The groups also induced colon shortening. The decreased abdominal fat was observed compared with the control group. These results demonstrate that ATS‐Leptin fusion protein also have Biological activity in vivo and vitro. The ATS‐Leptin will be used the therapeutic protein to treat a variety of diseases.
Protein Targeting to the Albino3 Insertase
Authors: Rogers Jennifer D , Marty Naomi , Lewis Nathaniel , Kight Alicia , Kumar TKS , Goforth Robyn , Henry Ralph
Published: June 2021

The chloroplast thylakoid membrane is the site of photosynthetic light capture and oxygenic electron transport. Nuclear encoded light harvesting chlorophyll‐binding proteins (LHCPs), which bind chlorophyll a/b, provide much of the light absorbing capacity in plants. Following their import into the chloroplast, LHCPs are routed to the thylakoid where they are inserted into the lipid bilayer by the Albino3 (Alb3) insertase. Intra‐chloroplast targeting of LHCPs to Alb3 is accomplished by a chloroplast signal recognition particle (cpSRP), which binds LHCPs in the soluble stroma and directs them to the membrane where they are thought to be passed to Alb3 for post‐translational insertion. CpSRP is composed of a conserved 54 kD subunit (cpSRP54) and a chloroplast‐specific 43 kD subunit (cpSRP43). Data from protein localization and protein interaction assays shows that cpSRP43 binding to Alb3 is critical for LHCP localization. Alb3 is also thought to function in the co‐translational insertion of chloroplast synthesized proteins by a co‐translational targeting/insertion mechanism. This presents the possibility that the post‐ and co‐translational targeting pathways converge at Alb3 for insertion into the membrane. Results of studies presented here may provide an ability to understand how the relative level of post‐ and co‐translational insertion activity by Alb3 is regulated during thylakoid biogenesis.
Recent Advances in Metabolomics in Providing Mechanistic Insight and Discovering Biochemical Markers
Authors: Evans Anne
Published: June 2021

Metabolomics is defined as “the non‐biased quantification and identification of all metabolites present in a biological system” but in practice the term metabolomics is used in a rather broad sense and covers many different analytical methodologies. To address the challenges associated with metabolomics, an integrated analytical and data handling platform was developed that provides a chemo‐centric global metabolomics analyses of biological systems. The analytical platform incorporates two separate UHPLC/MS/MS2 methods and a GC/MS method which increases the overall coverage of small molecules in a biological sample. The resulting MS and/or MS2 data are searched against an in‐house generated authentic standard library which includes retention time, molecular mass to charge (m/z), preferred adducts and in‐source fragment information as well as the associated MS fragmentation spectra for all biochemicals in the library. The library enables the rapid and accurate identification of the experimentally detected small molecules based on a multi‐parameter match without need for time consuming further analysis. This integrated platform allows the robust, high‐throughput collection and analysis of analytical data and accurately identifies a large number and broad spectrum of biochemicals thereby facilitating biochemical interpretation of metabolomic experiments.
Small molecule inhibitors of fatty acid uptake in 3T3‐L1, primary human adipocytes, INS‐1E and C2C12 cell lines
Authors: Jesch Elliot Denver , Wang Zhigang , Black Paul N , DiRusso Concetta C
Published: June 2021

Previous work in this lab identified 5 chemically distinct small molecules that inhibit the uptake of fatty acid (C1‐BODIPY‐C12) through a series of screenings in yeast expressinghomo sapiens fatty acid transport protein (FATP) 2 and Caco2 cells. Subsequently, we looked to examine the effectiveness and toxicity of each compound in a variety of cells that accumulate lipid. We examined two adipocyte cell lines (mouse 3T3‐L1 and primary human), one pancreatic beta cell line (INS‐1E) and one skeletal muscle cell line (mouse C2C12). Cells were seeded into 96 well (black wall/optically clear bottom) plates and differentiated or allowed to incubate until firmly attached. Cells were subsequently starved for one hour in serum free MEM, one hour incubation with a dilution series of each compound (1nM‐1mM, range dependent on individual cell line), a 5–15 minute incubation with the fluorescent fatty acid C1‐BODIPY‐C12 and finally measured fluorescence intensity using 485/20 nm excitation and 530/20 nm emission filters. Sigmoid dose‐response curves for C1‐BODIPY‐C12uptake were generated in each cell line and EC50 values were from the low nM to high μM range, depending on the particular compound and cell line. The compounds were determined to benon‐toxic to each cell line by utilizing the MTT cytotoxicity assay. Specificity to fatty acid uptake was confirmed by measuring acyl‐CoA synthestase activity and glucose uptake.

[Supported by NIH DK071076]
Mechanism of Sterol Transport by Cyclodextrin
Authors: Xu Zhi , McCauliff Leslie , Storch Judith
Published: June 2021

We examined the regulation and mechanism of sterol transport by cyclodextrin(CD) using in vitro model systems and fluorescence spectroscopy, and NPC2‐deficient fibroblasts. We demonstrate that cholesterol transport from the lysosomal cholesterol binding protein‐NPC2, to three different species of CD occurs via aqueous diffusional transfer, which is very slow; the rate limiting step appears to be dissociation of cholesterol from NPC2, suggesting that specific interactions between NPC2 and CD do not occur. In contrast the transfer rate of the fluorescent cholesterol analogue dehydroergosterol(DHE) from CD to phospholipid membranes is very rapid, and is directly proportional to the acceptor membrane concentration; this suggests that sterol transfer from CD to membranes occurs by a collisional transfer mechanism, involving direct interaction of CD with membranes. Marked enhancement of sterol transport by CD was also found in cells, where addition of CD rapidly rescued the cholesterol accumulation phenotype of NPC2‐deficient fibroblasts, as quantified by filipin staining. Thus, the recent observations of CD efficacy in mouse models of NPC disease are likely the result of CD enhancement of cholesterol transport between membranes, with rapid sterol transfer occurring during CD‐membrane interactions. Supported by the Ara Parseghian Medical Research Foundation and the American Heart Association.
Hen egg yolk LDL as novel substrate for cholesterol oxidation assays
Authors: Palmere Shaina Lynn , Patel Purvi , Chen Feng , Spiegelberg Bryan D.
Published: June 2021

When low density lipoprotein (LDL) is oxidized it is taken up by macrophages in an unregulated manner. The resulting foam cell formation increases risk for atherosclerosis and cardiovascular disease. The goal of this project is to test the hypothesis that oxidation of LDL can be inhibited by antioxidants, including epigallocatechin‐3‐gallate (EGCG) from green tea. We present data concerning the HPLC separation of biologically‐active compounds from green tea extracts. In addition, LDL from hen egg yolk is being developed as a readily accessible and consistent substrate for Cu2+‐mediated oxidation assays. A spectroscopic assay measuring the conjugated diene lipid oxidation product, λmax = 234, has been designed as a potentially high‐throughput technique. We expect that the antioxidant properties of EGCG will considerably delay formation of the conjugated diene. In order to test the specificity of the simpler spectroscopic assay, a binding assay using a fragment of Aspergillus fumigatus hemolysin is also in development. This peptide is known to bind specifically to a byproduct of LDL oxidation, lysophosphatidlycholine, in a concentration‐dependent manner. The use of hen egg yolk LDL and a verified spectroscopic assay to quantify antioxidant activity of green tea extracts and other nutritional compounds represents an important advance to the study of cholesterol oxidation.
Plant nuclear lipid signaling
Authors: Dieck Catherine B. , Im Yang Ju , Boss Wendy F.
Published: June 2021

Our goal is to characterize the nuclear PI pathway in plants and understand the role of phosphatidylinositol 4,5 bisphosphate (PtdIns(4,5)P2) in regulating nuclear signaling. Because PtdIns(4,5)P2 is so low in plants, our approach was to generate a plant cell line with increased nuclear PtdInsP kinase activity. We expressed the human phosphatidylinositol 4‐phosphate 5 kinase 1α (HsPIP5K1) in Nicotiana tabacum cells, isolated nuclei from the transformed and non‐transformed cells and compared the effect of expressing HsPIP5K1 on nuclear lipids. Lipid kinase activity was assayed. The PIPK specific activity was 20‐fold higher in HsPIP5K1–expressing cell nuclei. Transformed nuclei contained an average of 32.4 pmoles of PtdInsP(4,5)P2 per mg protein while the PtdInsP(4,5)P2 in WT nuclei was not detectable. Nuclear lipids in HsPIP5K1‐expressing cells showed a decrease in total LysoPC and LysoPE; however, the percentage of unsaturated fatty acids increased in several phospholipids e.g. 18:1 in LysoPC and LysoPE. In spite of the increase in nuclear PtdInsP(4,5)P2, the HsPIP5K1 cells continue to divide and grow although the rate of DNA synthesis is less. We are currently assessing the status of the posttranslational modifications of histones in anticipation that these will be affected by the high levels of PtdInsP(4,5)P2. Funded in part by grant from NSF (WFB) and NIH training grant (CBD).
Effects of Cnidium oficinale Makino Extract on Adipogenic Mode of Action in 3T3‐L1 Preadipocytes
Authors: Abanto Oliver Diaz , Yang Seung Hak , Hwang Seong‐Gu
Published: June 2021

The present study was conducted to determine the effects of Cnidium oficinale Makino (CO) extract on adipocyte differentiation, and to explore its mode of action in adipocyte differentiation. Post‐confluent mouse 3T3‐L1 cells were treated with differentiation medium containing 0, 50, 100, 200, and 400 ppm of CO methanol extract. The extent of adipocyte differentiation was evaluated by Oil Red O staining and measurement of glycerol‐3‐phosphate dehydrogenase (GPDH) activity. The IRS‐1, GLUT4, PPARγ and C/EBPα mRNA expressions were measured using RT‐PCR and protein expression by Western Blot. The results of Oil Red O staining and GPDH activity analyses showed that CO extract stimulated 3T3‐L1 adipocyte differentiation dose‐dependently up to 200 ppm, but reversed the effect at 400ppm. It has been shown that the CO extract may induce adipocyte differentiation through stimulation of PPARγ and C/EBPα expression. The CO extract also exhibited insulin‐like activity by inducing IRS‐1 and GLUT4 expressions. The results of the present study indicated bi‐potential effects of CO on adipogenesis, a plausible candidate for regulating obesity. The CO extract also showed some potential for regulating blood glucose level.
Glucosylceramide synthase is essential in MDCK cell differentiation
Authors: Sterin‐Speziale Norma Beatriz , Pescio Lucila Gisele
Published: June 2021

We previously observed that the synthesis of glycosphingolipids (GSLs) increased under hypertonic conditions. The increase was PDMP sensitive but cycloserine (CS) and fumonisin B1 (FB1) resistant. Now we demonstrate that GCS is up‐regulated by hypertonicity at the transcriptional level. The analysis of differentiation markers showed that MDCK cells developed a hypertonicity‐ induced differentiated phenotype even in the presence of CS or FB1. Since FB1 can inhibit both the de novo and salvage pathways, we used other strategy to determine the pathway involved in the hypertonicity‐ induced glucosylceramide (GC) increase. MDCK cells were labeled with [14C] palmitic acid for 1 or 24h. [14C] GC was significantly increased under hypertonic conditions in long‐term labeling but not in pulse conditions. All together, these results indicate that hypertonicity induces an increase in the synthesis of GSLs by the regulation of GCS expression and the activation of the salvage pathway of GC formation, and that such mechanism is specific and essential to MDCK cell differentiation. This work was supported by the University of Buenos Aires (UBACyT B076), CONICET (PIP 5557) and ANPCyT (PICT 33154) grants.
Role of the transcription factor Opi1p and phosphatidic acid in the formation of lipid droplets in Saccharomyces cerevisiae
Authors: Chang Yu‐Fang , Henry Susan A
Published: June 2021

Phosphatidic acid (PA) is a signaling molecule that plays an important role in mediating yeast phospholipid biosynthesis. In cells grown in the absence of inositol, the transcriptional repressor Opi1p is localized to the endoplasmic reticulum (ER) through interactions with Scs2p via its FFAT motif (two phenylalanines in an acidic tract) and the binding with PA. Under these conditions, the UASINO‐containing phospholipid biosynthetic genes are expressed at the maximum level through activation via the Ino2p‐Ino4p activation complex. When inositol is added to the medium, the increased synthesis of phosphatidylinositol causes a rapid depletion of PA in the ER. Opi1p then translocates to the nucleus and represses the transcription of target genes (Loewen et al. 2004. Science 304, 1644–1647). Here we present studies utilizing Opi1p reporter constructs to monitor the change in PA metabolism. After the cells grew under inositol starvation for 2 hrs, novel punctate structures were recognized by Opi1p‐GFP in the FFAT and scs2 null mutants. Interestingly, these structures are highly associated with ER. Furthermore, by using Tgl4p‐RFP as a lipid droplet marker to identify these structures, we observed colocalization of Tgl4p‐RFP with the punctate structures. These results suggest a role for both Opi1p and PA in the formation of lipid droplets.

Supported by NIH grant GM019629.
Regulation of Hepatic Lipid Metabolism by a Natural Health Product
Authors: Enns Jennifer Emily , Wu Nan , Siow Yaw L. , Karmin O
Published: June 2021

Hypercholesterolemia is a major risk factor for cardiovascular disease. Current treatment for lipid lowering is provided primarily by the statin drugs, which block HMG‐CoA reductase, the rate‐limiting enzyme of cholesterol synthesis, and improve clearance of plasma LDL‐cholesterol. However, many patients cannot tolerate the statin dosages recommended to reach their target lipid levels, due to side effects such as muscle pain and decline in liver function. Berberine, an herbal product used in traditional Chinese medicine, has great potential as a new regulator of lipid metabolism. The objective of this study was to determine whether berberine had an effect on HMG‐CoA reductase and to elucidate the underlying mechanism by which it might act. HMG‐CoA reductase activity was decreased in HepG2 cells after treatment with berberine. The inhibitory effect of berberine on HMG‐CoA reductase was due to post‐translational modification of the enzyme. Western immunoblotting analysis revealed that berberine treatment resulted in a significant increase in the phosphorylation of HMG‐CoA reductase, leading to inactivation of the enzyme. Cells that were treated with berberine exhibited a decrease in cholesterol storage as a result of reduced HMG‐CoA reductase activity. The molecular mechanism by which berberine regulated posttranslational modification of HMG‐CoA reductase was also investigated. Funding: NSERC, CIHR, HSF and MHRC.
Yeast diacylglycerol kinase is essential for the resumption of growth from stationary phase
Authors: Fakas Stylianos , Carman George
Published: June 2021

The Saccharomyces cerevisiae DGK1 gene encodes a novel diacylglycerol (DAG) kinase that catalyzes the formation of phosphatidate (PA) from DAG. This enzyme is unique in that it uses CTP, instead of ATP, as the phosphate donor. DGK1 is a major regulator of PA levels in yeast and its overexpression causes PA overproduction and a nuclear membrane expansion by inducing the formation of PA‐enriched membranes around the nucleus. In this work, we discovered that DGK1 is essential for the resumption of growth from stationary phase when de novo fatty acid synthesis is blocked with cerulenin. Lipid analysis showed that under these conditions wild type cells mobilized triacylglycerol (TAG) reserves to release fatty acids needed for phospholipid synthesis and growth, but dgk1Δ cells were unable to mobilize TAG. Supplementation with choline or ethanolamine resulted in the synthesis of phospholipids, the hydrolysis of TAG, and suppression of the growth inhibition phenotype of dgk1Δ cells.

Supported by NIH grant GM‐28140.
A novel microdissection model for probing cellular interactions. II. LCA and PSA binding sites identified
Authors: Allen Jenieke , Dreyfuss Justin , Coyle‐Thompson Catherine , Oppenheimer Steven B.
Published: June 2021

We use two lectins Lens culinaris agglutinin (LCA) and Pisum sativum agglutinin (PSA), both with alpha D‐mannose and alpha D‐glucose preferential binding specificities, to probe the binding of archenterons and blastocoel roofs microdissected out of 52–54 hr Lytechinus pictus sea urchin embryos, a NIH designated model system. The 24 hour embryos were incubated in each of the lectins (0 (control) – 1 microgram/ml artificial sea water, ASW) for 24–30 hrs in pH 8.0 ASW at 15 degrees C, fixed in 3.7% formaldehyde, dissected and the roofs of the blastocoel and the tips of the archenterons were placed together to determine if the components would bind to each other. Results were either 100% binding or no binding in 15–57 trials for each experiment. LCA treated archenterons bound to untreated roofs, while LCA treated roofs did not bind to untreated archenterons. PSA treated archenterons did not bind to untreated roofs, while PS treated roofs did not bind to untreated archenterons. Experiments were also performed on roofs and archenterons that were both treated with the same or different lectins. In 204 total trials it became clear that lectins with the same general sugar specificities produced sometimes the same and sometimes different results. Although factors such as steric hindrance might account for some effects, the results suggest that alpha D‐mannose and/or alpha D‐glucose may be involved in this cellular interaction and that the saccharide structures in vivo may be complex, binding to one of the lectins and not the other (supported by NIH NIGMS SCORE S0648680, MARC, RISE, the Joseph Drown Foundation, the Sidney Stern Memorial Trust, and CSU Northridge Biology Full Immersion Research Experience (FIRE) course funding; JA and JD equal contributions).
A novel microdissection model for probing cellular interactions. III. mapping with Con A and mannan beads
Authors: Coyle‐Thompson Catherine , Badali Oliver , Askari Tina , Mkrtchian Mary , Portillo Georgina , Manukyan Mariam , Frederick Robert , So Sokuntheavy , Oppenheimer Steven B.
Published: June 2021

Adhesion between the archenteron tip and blastocoel roof in the NIH designated sea urchin embryo model is a cellular interaction that has interested investigators for over a century, but its molecular basis is not understood. Here we microdissect the two components of this cellular interaction and use concanavalin A (Con A) and mannan derivatized agarose beads to map molecular groups that may be involved in mediating the cellular interaction. In over 50 separate trials, the results were either 100% binding or no binding, and indicated that Con A derivatized and mannan derivatized agarose beads bound to formaldehyde fixed dissected archenterons and roofs in both sterile distilled water (ddH2O) and pH 8.0 artificial sea water (ASW), 15°C, while 0.2 M alpha methyl mannose blocked the binding. Previous work indicated that fixed tissue displayed the same cell surface properties as live tissue. The results suggest that both Con A binding ligands and mannan binding partners are present on the two components of the cellular interaction. These studies offer a novel approach to map glycans and glycan binding partners that may be functionally important. By microdissecting the components of cellular interactions out of the embryo proper, these components can be probed in pristine media away from factors in intact embryos that could confuse results (Supported by NIH NIGMS SCORE S0648680, MARC, RISE, the Joseph Drown Foundation, the Sidney Stern Memorial Trust, and CSU Northridge Biology Full Immersion Research Experience (FIRE) course funding).
A novel microdissection model for probing cellular interactions. IV. Con A bead binding parameters
Authors: So Sokuntheavy , Budiyono Herry , Allen Jenieke , Askari Tina , Coyle‐Thompson Catherine , Oppenheimer Steven B.
Published: June 2021

We developed a novel method to probe mechanisms of cellular interactions by microdissecting the archenteron and blastocoel roof out of 48–54 hr Lytechinus pictus sea urchin ermbryos. These are the two components of a classic cellular interaction in the NIH designated model sea urchin embryo. Here we test the ability of isolated fixed archenterons to bind to agarose beads derivatized with concanavalin A (Con A) in artificial sea water (ASW) at pH 4,6,8 (control), 9 or 10 or in the presence of 4M NaCl or 2M or 4M alpha methyl glucose. The binding of a total of 204 isolated archenterons was only inhibited in 4M alpha methyl glucose (p less than 0.05 compared with controls). All other conditions gave p values of greater than 0.05. As Con A beads bound to the isolated archenterons and alpha methyl glucose inhibited the interaction, the results suggest that Con A binding ligands are present on the isolated archenterons. While 4M sugar is high, the archenterons were fixed, reducing osmolarity concerns. High monosaccharide concentrations are often required to inhibit lectin‐involved binding interactions. Part I of this series provides evidence for glycan involvement in the actual cellular interaction. This microdissection method allows probing of isolated components of cellular interactions outside of the embryo proper, to avoid complex problems that may occur in whole embryos (supported by NIH NIGMS SCORE S0648680, MARC, RISE, the Joseph Drown Foundation, the Sidney Stern Memorial Trust, and CSU Northridge Biology Full Immersion Research Experience (FIRE) course funding).