bluuebunny/crossref_metadata_2025 huggingface dataset:
Determining the retinoic acid response element in the liver glucokinase gene promoter using deletion and linker‐scan analysis
Authors: Li Rui , Chen Guoxun
Published: June 2021

Glucokinase is a key enzyme of hepatic glucose utilization when plasma glucose level is high. We have reported that retinoids in a liver derived lipophilic extract synergized with insulin to induce the expression of the glucokinase gene (Gck) in primary rat hepatocytes. Experiments were designed to determine the retinoic acid response element (RARE) in Gck promoter. We have shown that retinoic acid (RA) activated the minimal luciferase reporter construct containing 234‐bp (−237/−3, Gck‐234‐WT‐LUC) of the Gck promoter sequence both in primary rat hepatocytes and INS‐1 insulinoma cells. A series of reporter constructs containing overlapping 10‐ to 12‐bp scramble mutations covering the nucleotide sequence from −237 to −3 were made to narrow down the RARE to a 20‐bp region. Subsequently, a series of 5‐ to 6‐bp linker‐scan scramble mutations covering the entire 20‐bp region were made to narrow down the RARE to a 5‐bp region. RA induced Gck‐234‐WT‐LUC activity by 2.74±0.25‐fold in INS‐1 cells. The scramble mutation covering the sequence −177/−173 destroyed the RA‐induced luciferase activity. Moreover, single nucleotide mutations at −177, −176, and −175 significantly impaired the response of the reporter constructs to RA treatment. We conclude that the RA‐induced Gck transcription is mediated by a transcription activator associated with the RARE sequence covering −177/−175 of the liver Gck promoter.

Grant Funding Source: American Heart Association
Knockout of CYP4f14 causes severe perturbations in vitamin E metabolism and results in tissue accumulation of vitamin E in the mouse
Authors: Bardowell Sabrina Anne , Duan Faping , Swanson Joy E. , Manor Danny , Parker Robert S.
Published: June 2021

Vitamin E is a family of eight naturally‐occurring lipophilic antioxidants, of which one, α‐tocopherol (TOH), selectively accumulates in vertebrate tissues. Cytochrome P450‐4F2 (CYP4F2), an ω‐hydroxylase, is the only enzyme shown to metabolize vitamin E in humans. We identified CYP4f14 as a functional murine ortholog of CYP4F2, via cloning, cell culture expression and activity assays. We then investigated the effect of its deletion on vitamin E metabolism and status in vivo. Cyp4f14‐null mice exhibited substrate‐specific reductions in microsomal vitamin E‐ω‐hydroxylase capacity ranging from 93% (γ‐TOH) to 48% (γ‐tocotrienol). In vivo data obtained from metabolic cage studies showed whole‐body reductions in metabolism of γ‐TOH by 90% and 68% for δ‐ and α‐TOH. While fecal excretion of TOHs increased in null mice, partially compensating for the metabolic deficit, Cyp4f14‐null mice fed a soybean oil diet for 6 weeks hyper‐accumulated γ‐TOH in tissues but were otherwise apparently normal. We conclude that Cyp4f14 is the major, but not the only, vitamin E‐ω‐hydroxylase in mice. Its disruption significantly impairs whole‐body vitamin E metabolism and alters the widely‐conserved phenotype of selective tissue deposition of α‐TOH. This model and its derivatives should prove valuable in determining the in vivo biological potential of specific TOHs and tocotrienols. Funded by NIH DK067494.

Grant Funding Source: NIH DK067494
Maternal and infant predictors of CRP in exclusively breastfed infants born to HIV‐infected Malawian mothers
Authors: Adair Linda S , Thompson A L , Kayira D , Chasela C , Kacheche Z , Kamwendo D , Bentley M , Jamieson D , Allen L H , Shahab-Ferdows S
Published: June 2021

Little is known about inflammation in young infants. We assessed C‐reactive protein (CRP) in exclusively breastfed (EBF) infants (n=537) born to HIV‐infected Malawian women in the Breastfeeding, Antiretrovirals and Nutrition Study (BAN). BAN mothers were randomized to receive a lipid‐based nutrient supplement (LNS), meeting nutritional needs of lactation, or no LNS and to a maternal, infant or no antiretroviral (ARV) regimen, with counseling for EBF to 24 weeks. Maternal and infant CRP levels were measured at 2 (n=366) or 6 (n=170) weeks, and 24 weeks. Infant CRP was elevated early (geometric mean at 2 weeks= 4.4 mg/L) but declined to 1.6 mg/L at 24 weeks, while maternal CRP increased from 0.65 to 1.82 mg/L. In linear regression models, log infant CRP at 6 weeks was significantly positively related to concurrently measured maternal BMI and CRP and inversely related to maternal CD4>500, but unrelated to treatment arm or infant BMI, sex, or infections. At 24 weeks, infant CRP was significantly related to infant infection and higher maternal BMI. Logistic regression comparing low to medium or high inflammation also showed that inflammation in young, EBF infants is driven mainly by maternal factors. By 24 weeks, while maternal factors still predicted infant CRP, infant infections became an important contributor to elevated CRP. Support: CDC U48‐DP000059‐01, Bill & Melinda Gates Foundation OPP53107, CPC R24 HD050924.
Designing in vitro tools to pattern gene expression using inducible gene expression
Authors: McGuigan Alison P , Javaherian sahar
Published: June 2021

Early embryogenesis involves the sequential patterning of gene expression in different regions of the developing embryo, with the ultimate purpose of guiding initially identical cells into different paths of differentiation and thus facilitating body part formation and patterning. Being able to reproduce patterns of gene expression that lead to formation of a specific tissue in vitro would be useful both for engineering artificial tissues, and for developing simplified in vitro models to study and define the rules that guide tissue morphogenesis. Here we report a system to pattern gene expression in epithelial sheets using a drug inducible gene expression system. We created a sheet of epithelial cells transduced with a doxycycline inducible lentivirus encoding GFP. We then delivered patterns of doxycycline to the cell sheet to create controlled patterns of GFP expression. We show that the stability of the gene expression patterns in vitro heavily depends on the relationship between the dynamics of the inducible gene expression system and the dynamics of cellular rearrangements. By modeling the system we can predict the conditions that allow stable gene pattern formation.
Neural crest derivation of the bony skull of the Mexican axolotl
Authors: Piekarski Nadine , Hanken James
Published: June 2021

Contributions of the cranial neural crest to cartilages and bones of the skull have been revealed in great detail in one amniote model using the quail‐chick‐chimeric system. Yet, the extent to which patterns of embryonic derivation are evolutionary conserved or labile remains an intriguing question, given the extensive variability in skull morphology observed among craniates. Amphibians are a non‐amniote tetrapod group with a highly derived skull morphology and ontogeny. Recent technical advances now enable us to derive fate maps at a level of detail previously known only from avian studies. We employ embryonic transplantations, using GFP‐transgenic axolotls as donors, to document the nature and extent of neural crest contribution to the adult osteocranium. Comparison of axolotl with results available from chicken, mouse and Xenopus reveals that different species show mosaics of conserved and variable patterns of embryonic derivation. The recently reported rostral inversion of neural crest‐derived adult cartilages in Xenopus is not a feature common to all amphibians, but instead may be uniquely associated with the extreme posthatching metamorphosis characteristic of most anurans.

Grant Funding Source: MCZ
VEGF immunoexpression in the pituitaries of pregnant women
Authors: Rotondo Angelo , Rotondo Fabio , Scheithauer Bernd W , Syro Luis V , Cusimano Michael , Kovacs Kalman
Published: June 2021

Previous studies showed that in pregnancy the pituitary enlarges and increases in weight due to lactotroph hyperplasia. We demonstrated that in pituitaries of pregnant women, microvessel density is significantly increased compared to those of age– matched non pregnant women. Vascular endothelial growth factor (VEGF) stimulates new vessel formation in several tissues and neoplasms. The aim of the present study was to reveal whether VEGF is involved in the causation of increased vascularity in the pituitaries of pregnant women. We assessed VEGF immunoexpression in 20 autopsy obtained pituitaries of pregnant women and compared the results with 20 controls. Slides were immunostained for VEGF and pituitary hormones using the streptavidin‐biotin‐peroxidase complex method. VEGF immunoexpression was evaluated in 10 random high‐power fields using an image analyzer and the results compared by chi‐square or Fisher exact tests. Immunoexpression of VEGF was not increased in the adenohypophyses of pregnant women compared with the controls indicating that VEGF plays no major role in the causation of increased vascularity. The cause remains to be investigated.
Using the Click‐It assay to identify the proliferative myofibroblast
Authors: Vaughan Melville B. , Odejimi Tobi D. , Thomas Jaime E.
Published: June 2021

Traditionally, diseases of myofibroblasts have been termed proliferative diseases. Our evidence suggests stimulation of the myofibroblast phenotype decreases cell number, either through apoptosis (cell death) or proliferation inhibition. Proliferation has been difficult to study using bromodeoxyuridine (BrdU) since it requires harsh conditions to demonstrate the stain. A commercially available kit (Click‐It) using a modified nucleotide, 5‐Ethynyl‐2′‐deoxyuridine (EdU), has eliminated the need for harsh conditions. We tested the kit's ability to identify proliferative myofibroblasts using cells grown on coverslips or within collagen lattices. Cells were grown on coverslips for 24 to 48 hours, and in lattices for 5 days, to allow the myofibroblast phenotype to develop. Cells were then incubated in the presence of 10mM EdU for up to 4 hours, then fixed. The recommended staining procedure was modified so that the volume could be reduced tenfold. Both coverslips and lattices demonstrated EdU staining and alpha‐smooth muscle actin incorporated into stress fibers. Four cell populations were identified, including the proliferative myofibroblast. This staining technique with modifications will be a useful and inexpensive method for determining how proliferation plays a role during myofibroblast phenotype modulation.
SV40 Virions Encode Transgenerational Epigenetic Information from Cellular Viral Chromatin
Authors: Milavetz Barry I , Kallestad Les , Gefroh Amanda , Adams Nicholas , Wood Emily , Balakrishnan Lata
Published: June 2021

It is becoming increasingly clear that the epigenetic modification of histones plays a significant role in regulating cellular processes. In order to determine whether epigenetics also plays a role in regulating the life cycle of chromatinized DNA viruses, we have analyzed SV40 chromatin for the presence of modified histones. We found that the modified histones present at late times in wild‐type infection were organized into one of five epigenomes, each of which consisted of a specific combination of co‐localized modified histones. Derepression of early transcription with a mutant, cs1085, which does not bind T‐antigen and cannot down‐regulate early transcription resulted in significant reductions in the levels of H3K4me1, H3K4me2 and H3K9me1, while derepression with a mutant, SM, which fails to produce a repressive miRNA resulted in an increase in H3K9 methylation which was maximal for H3K9me2. Interestingly, the epigenetic changes observed in the mutant minichromosomes as well as in other variant infections could also be observed in chromatin isolated from the corresponding virions. These results indicate that viral minichromosomes utilize epigenetic modifications in their regulatory processes like cellular chromatin and that the viral chromatin is capable of transferring information epigenetically from one infection to a subsequent infection through histone modifications present in virions.
p300‐dependent acetylation of Histone H3 at lysine 56 is required for cardioprotection and DNA damage response
Authors: Jain Sumit , Wei JianQin , Bishopric Nanette H.
Published: June 2021

Background

p300 is important for transcription‐dependent DNA damage repair. Acetylation of Histone H3 at Lysine 56 (AcH3K56) by p300 has been suggested to be important for initiation of DNA damage response.

Hypothesis

Acetyltransferase p300 loss is causally related to reduced DNA damage response during aging and genotoxic stress.

Methods

p300 was knocked down in neonatal cardiac myocytes using siRNA and compared to a non‐targeting sequence. Doxorubicin (Dox) was used to model genotoxic stress. Cycloheximide (CY)(5μg/ml) was used to assess p300 stability.

Results

In cardiac myocytes, DOX caused a p300‐HAT activity dependent rapid and sustained stabilization of p300 protein (3.7±1.9 fold vs. control at 8h; p<0.05). AcH3K56 co localized with p300 after DOX treatment. p300 knockdown increased DOX‐induced myocyte death (26.9±13.5%; p<0.05) and caspase3 cleavage, decreased AcH3K56, blunted phosphorylation of H2AX, ATM, and BRCA1, and reduced phosphorylation and acetylation of p53. Converse effects were seen with activation of p300 by HDAC inhibitors TSA and SAHA (HDACi). Further, p300KD completely abolished HDACi mediated cardioprotection and increase in AcH3K56. This could also explain accelerated aging phenotype of mice with heterozygous p300 loss.

Conclusion

Genotoxic stress causes a post‐transcriptional stabilization of p300 that is required for the DNA damage response and cardioprotection.

The work was supported by NIH R01‐ HL7109, the Florida Heart Research Institute and AHA Pre‐doctoral fellowship to Sumit Jain.
Activation of the DNA Damage Response Pathway and the Role of NBS1 in Response to Hedgehog Signaling Inhibition
Authors: AGYEMAN AKWASI , DeVecchio Jennifer , MAZUMDER TAPATI , SHI TING , HOUGHTON JANET
Published: June 2021

Hedgehog (HH) signaling actively regulates key processes such as cell differentiation and cell proliferation during embryonic development. Aberrant HH pathway activation has been identified in several human cancers, however its role in colon carcinoma remains unclear. The GLI proteins downstream of signaling via PTCH and SMO transcriptionally regulate HH target genes. Pharmacologic inhibition of GLI1 and GLI2 by GANT61, or by a C‐terminus deleted GLI3 repressor, GLI3R, induced DNA damage (marked by increased γH2AX, p‐ATM and p‐Chk2) and extensive cell death in human colon carcinoma cells. Exposure to GANT61 for 24 hr followed by washout allowed DNA repair and reversal of cytotoxicity. NBS1 is a key regulator in intra‐S‐phase checkpoint activation, DNA damage signaling, and DNA repair. Following GLI1/GLI2 inhibition, NBS1 nuclear foci colocalized in single cells with MDC1 but not with γH2AX foci, both engaged in recruitment of DNA damage signaling and repair proteins to DNA break sites. During DNA damage, NBS1 demonstrated limited binding to chromatin, while γH2AX and MDC1 binding was induced and sustained. During DNA repair NBS1 and MDC1 were highly chromatin‐bound while γH2AX was released from chromatin. These findings provide new insight into the critical role of GLI1/GLI2 in driving cellular survival in colon cancer and mechanisms that follow dysregulation of GLI1/GLI2 function that determine cell death.
Interplay between signal sequence recognition and N‐terminal protein modification at the ribosome exit site
Authors: Pool Martin , Nyathi Yvonne , Forte Gabriella , Stirling Colin
Published: June 2021

Proteins destined for secretion are targeted to the endoplasmic reticulum by N‐terminal signal sequences. In contrast, the majority of cytosolic proteins are modified at their extreme N‐termini by methionine‐aminopeptidase (MetAP) and/or N‐acetylation. Both of these processes typically occur co‐translationally soon after the nascent chain emerges from the ribosome exit site.

Proteins targeted by the Signal Recognition Particle‐independent targeting pathway have a strong bias against N‐terminal processing and mutations that induce such processing inhibit translocation. Detailed examination of SRP‐dependent substrates reveals that mutations that induce N‐terminal processing have no effect on ER targeting. In the context of signal anchor proteins with a long N‐terminal cytoplasmic region, methionine processing and acetylation can occur, but has no effect on translocation. In contrast, signal anchor sequences with short N‐terminal regions are inefficiently processed at their N‐termini. This is rationalized by the finding that SRP and MetAP compete for a shared binding site on the ribosome. Furthermore, we find that additional ribosome‐associated factors regulate the differential recruitment of these two factors to secretory and cytosolic ribosome nascent chains. Hence a tightly regulated interplay exists to ensure the fidelity of the early events of nascent chain processing.

This work is funded by the Biotechnology and Biological Sciences Research Council.
Different substrate‐dependent transition states in the active site of the ribosome
Authors: Rodnina Marina V. , Kuhlenkötter Stephan , Wohlgemuth Ingo , Wintermeyer Wolfgang
Published: June 2021

The active site of the ribosome, the peptidyl transferase center, catalyzes two reactions, peptide bond formation between peptidyl‐tRNA and aminoacyl‐tRNA as well as the release factor (RF)‐ dependent hydrolysis of peptidyl‐tRNA. We analyzed the pH/rate profiles, thermodynamic parameters, kinetic solvent isotope effects (KSIE) and the proton inventories of the two reactions (Kuhlenkötter et al., Nature 476, 351–354 (2011)). The proton inventory for peptide bond formation indicates the rate‐limiting formation of three hydrogen bonds with about equal contributions, consistent with a concerted eight‐membered proton shuttle in the transition state. The catalysis is due to a favorable change in the activation entropy of the reaction and does not involve ionizing groups of the ribosome. The transition state of the RF2‐dependent hydrolysis reaction is characterized by the rate‐limiting formation of a single strong hydrogen bond. The reaction is driven by a favorable change in the activation enthalpy and involves an ionizing water molecule at the active site. Thus, the ribosome supports different rate‐limiting transition states for the two reactions that take place in the peptidyl transferase center.
Coordinated movement of eukaryotic translation initiation factors 1, 1A, and 5 within the 43S ribosomal preinitiation complex (PIC) mediates the response to start codon recognition.
Authors: Nanda Jagpreet Singh , Munoz Antonio , Saini Adesh K , Hinnebusch Alan G
Published: June 2021

Eukaryotic translation initiation factor (eIF) 5 acts as a GTPase activating protein for eIF2 that carries the first tRNA onto the ribosome. Recent data have shown that eIF5 antagonizes eIF1, enhancing its release from the PIC in response to start codon recognition. eIF1 release promotes the conversion of the PIC from open conformation that scan the mRNA to closed one arrested on the start codon. Recent data indicate that an interaction occurs between eIF1A and eIF5 upon start codon recognition by PIC. We observed fluorescence energy transfer (FRET) between fluorescently labeled derivatives of eIF1A and eIF5 within the PIC that is strongly enhanced upon start codon recognition. Our data indicate that the C‐terminal tail (CTT) of eIF1A moves closer to the N‐terminal region of eIF5 upon start codon recognition. This conformational change is involved in stabilizing the closed state of PIC. The kinetics of this FRET change mirror the kinetics of eIF1 release from the PIC on start codon recognition, suggesting two events are coupled. Mutations in eIF1A CTT that decrease the fidelity of start codon recognition in vivo uncouple eIF1 release from eIF1A CTT movement towards eIF5 NTD as well as from release of inorganic phosphate from eIF2. Our results suggest a model in which eIF1 release from PIC promotes movement of the eIF1A CTT towards eIF5 NTD that in turn promotes Pi release from eIF2, converting it to its inactive, GDP‐bound state.
Allosteric regulation of protein kinase PKCζ by the N‐terminal C1 domain and small compounds to the PIF‐pocket
Authors: Biondi Ricardo M. , Schulze Jörg O. , Fröhner Wolfgang , Zhang Hua , Weber Nadja , Navratil Jeanette , Amon Sabine , Hindie Valerie , Zeuzem Stefan , Jørgensen Thomas J.D. , Alzari Pedro M. , Neimanis Sonja , Engel Matthias , Lopez-Garcia Laura A.
Published: June 2021

Protein kinases are key mediators of cellular signaling and de‐regulation of protein kinases is the cause of diseases such as cancer and diabetes. AGC kinases are regulated by phosphorylation and by N‐ and C‐terminal regions.

Objective

Understand the molecular mechanism of regulation and develop novel drugs. The PIF‐pocket, present in AGC kinases, plays a central role in the mechanism of activation by phosphorylation and small compounds binding to the PIF‐pocket can activate the protein kinase PDK1 in vitro. Here, we studied the mechanism of inhibition of atypical PKCζ using molecular biology, biochemistry, synthetic chemistry of small compounds and crystallographic methods. We found that the inhibition by the N‐terminal region cannot be explained by a simple pseudosubstrate inhibitory mechanism, that the C1 domain allosterically inhibits PKCζ activity and identified an allosteric communication between the binding site of the C1 domain and the PIF‐pocket of atypical PKCs. Finally, we developed low‐molecular‐weight compounds that bind to the PIF‐pocket and allosterically inhibit PKCζ activity.

Conclusion

the work establishes a central role for the PIF‐pocket on the regulation of PKCζ and allows us to envisage development of drugs targeting the PIF‐pocket that can either activate or inhibit AGC kinases. Supported by BMBF Go‐Bio, DFG (BI 1044/4‐1 and BI 1044/2‐1) and Europrofession Foundation (Germany).
Annexin A1 derived peptide Ac2‐26 promotes epithelial wound healing through p120‐Vav2‐Rac1 signaling and ROS generation
Authors: Leoni Giovanna , Mohammad Alam , Perretti Mauro , Parkos Charles , Neish Andrew , Nusrat Asma
Published: June 2021

Barrier properties of the intestinal epithelium play a pivotal role in mucosal permeability and thus regulate exposure of the mucosal immune system to commensal, pathogenic microbes and endogenous anti‐inflammatory proteins like Annexin A1. We have recently reported that the N‐formyl peptide receptor‐1 (FPR‐1) is expressed in the intestinal epithelium and mediates G‐protein coupled signaling to regulate intestinal epithelial cell motility and wound closure. However, the mechanism by which FPR‐1 transduces signals to control epithelial functions is not understood. In the current study, we observed that exposure of epithelial cells to FPR‐1 ligand, Ac2–26, the N terminal cleavage product of endogenous Annexin A1, induces rapid generation of reactive oxygen species (ROS). Analysis of the signaling pathways identified a role of Src, p120, Vav2 and Rac1 GTPase in mediating NOX1 (NADPH oxidase 1) dependent ROS generation, inactivation of protein tyrosine phosphatase (PTP)‐PEST and phosphorylation/activation of focal adhesion kinase (FAK). Taken together, these results support an important role for endogenous anti‐inflammatory ligands of FPR‐1 to stimulate signaling pathways in promoting critical homeostatic properties of intestinal mucosa.
Peroxisome proliferator coactivator 1a (PGC‐1a) deficiency accelerates endothelial dysfunction during chronic angiotensin II treatment by increasing mitochondrial oxidative stress and vascular aging
Authors: Jansen Thomas , Kroeller-Schoen Swenja , Daiber Andreas , Muenzel Thomas , Schulz Eberhard
Published: June 2021

Objective

PGC‐1a is an important mediator of mitochondrial biogenesis and function. We have previously shown that the AMP‐activated protein kinase (AMPK) protects endothelial cells against oxidative stress by preservation of mitochondrial function in a PGC‐1a dependent manner. Since dysfunctional mitochondria might be involved in the pathogenesis of vascular disease, we investigated the effects of in vivo PGC‐1a deficiency during chronic angiotensin II (ATII) treatment.

Methods and Results

Deletion of PGC‐1a had no effect on endothelial function or mitochondrial mass in aortic tissue under basal conditions. However, chronic ATII infusion resulted in mild endothelial dysfunction, which was significantly impaired in PGC‐ 1a knockout mice. In parallel, oxidative stress was increased in aortic rings from ATII‐treated PGC‐1a knockout mice while serum antioxidative capacity was decreased. By using the mitochondrial specific superoxide dye mitoSOX and complex I inhibitor rotenone, we identified the mitochondrial respiratory chain as the major PGC‐1a dependent ROS source in vivo. In accordance with the prominent role of mitochondrial ROS for the signalling events leading to vascular aging and apoptosis, we found increased expression of p16INK4 and Chk‐2 as markers of cell senescence as well as increased TUNEL‐staining accompanied by activation of the JNK pathway in aortas from ATII treated PGC‐1a knockout mice.

Conclusions

PGC‐1a deficiency impairs endothelial function during chronic ATII infusion by increasing mitochondrial ROS production, resulting in premature vascular aging and apoptosis.
The effect of in vivo PBDE treatment on hepatic phosphoenolpyruvate carboxykinase (PEPCK) enzyme kinetics in male wistar rats
Authors: Nash Jessica T , Szabo David T , Carey Gale B
Published: June 2021

Xenobiotics such as phenobarbital, TCDD and PCB significantly suppress on the activity of a key gluconeogenic enzyme, phosphoenolpyruvate carboxykinase (PEPCK), suggesting that xenobiotics could disrupt glucose metabolism. The effect of polybrominated diphenyl ethers (PBDEs), a family of synthetic flame‐retardant chemicals, on PEPCK is unknown. This study investigated the effect of PBDE treatment on PEPCK. Forty‐eight, one month‐old, male Wistar rats were gavaged daily with corn oil or corn oil containing 14 mg/kg DE‐71 for 3, 14 or 28 days (N = 8 per group). At each time point, fasting plasma glucose, insulin and C‐peptide were measured and hepatic PEPCK and CYP enzyme activities were assayed. PBDE treatment significantly decreased PEPCK Vmax (μmol/min/g liver weight) by 22% (3 days) to 44% (28 days). CYP1A, −2B and −3A Vmax were increased by 6‐, 6‐ and 30‐fold, respectively, in treated rats compared to control. There was a significant inverse and temporal correlation (r = −0.74) between CYP3A and PEPCK Vmax for the treatment group. Fasting plasma glucose, insulin and C‐pep levels were unaffected by treatment, but the glucose:insulin ratio tended (P=0.07) to be higher in treated compared to control rats. These data suggest that 3 days of in vivo PBDE treatment is sufficient to compromise liver glucose metabolism and 28 days of treatment may influence insulin sensitivity. Supported by NH Agr. Exp. Stn. grant #NH00541.
Ambient ultrafine particles (UFP) inhibit eNOS activity via S‐glutathionylation
Authors: Hill James , Shen Melody , Li Rongsong , Ning Zhi , Sioutas Constantinos , Hsiai Tzung
Published: June 2021

Atherosclerosis is a systemic disease, and its development is dependent upon a complex interplay among numerous genetic, epigenetic, and environmental risk factors. It is increasingly recognized that ambient UFP exposure promotes vascular oxidative stress and inflammatory responses. In this study, we tested the hypothesis that UFP attenuates eNOS activity via S‐glutathionylaiton. Treatment of human aortic endothelial cells (HAEC) with ambient UFP (Dp<100nm) significantly inhibited nitric oxide (NO) production as measured by Griess assay (Control: 3.645, UFP: 1.662, P<.01, n=4). To assess whether oxidative stress induced eNOS uncoupling via S‐glutathionylation, we demonstrated that UFP treatment increased eNOS S‐glutathionylation in HAEC by a modified ELISA procedure. To inhibit S‐glutahionylation, we over‐expressed glutaredoxin‐1 (Grx‐1), and showed a significant attenuation of UFP‐mediated decrease in NO production (LacZ+UFP: .338, Grx1+UFP: .630, P<.01, n=6). Thus, our findings suggest that UFP inhibited eNOS activity via an increase in S‐ glutathionylation.
To Gibberellins and Beyond! Insights into the Evolution of Diterpenoid Metabolism
Authors: Peters Reuben J.
Published: June 2021

The requisite production of gibberellins as hormones necessary for higher plant growth and development has provided a genetic reservoir of biosynthetic genes whose duplication and divergence is thought to underlie the widespread production of more specialized diterpenoids. These natural products occur throughout the plant kingdom, although examples of such metabolism are found more broadly in fungi and bacteria as well, with a total of almost 7,000 such natural products already known. Here I present insights into the evolution of such diterpenoid natural products biosynthesis that arise from our systematic approach to biochemical investigations of such metabolism. In particular, using rice as a model system, we have significantly increased understanding of the underlying biosynthetic processes. This includes identification of all the responsible diterpene synthases and number of the cytochrome P450 mono‐oxygenases. Our investigations have further led to evidence for plasticity in these enzymes and, hence, the corresponding metabolic pathways in not only rice, but the related cereal crop plant wheat, providing insights into the facility with which more specialized metabolism can be derived from gibberellin biosynthesis.
Structural and Functional Evolution of Isopropylmalate Dehydrogenases in the Leucine and Glucosinolate Pathways
Authors: Balogun Sherifat F. , Galant Ashley , Jez Joseph M.
Published: June 2021

Glucosinolates are sulfur‐containing molecules produced as either defense compounds or chemical attractants in the Brassicales order of plants, which includes Arabidopsis thaliana. In humans, some of these molecules function as cancer‐prevention agents because of their ability to promote apoptosis and induce detoxification enzymes. Although the oxidative decarboxylation catalyzed by isopropylmalate dehydrogenase (IPMDH) is needed for the synthesis of leucine and glucosinolates, little is known about this family of enzymes in plants. To provide structural insight on the differential function of IPMDH, three Arabidopsis IPMDH isoforms were expressed and purified for protein crystallization. To date, crystals of AtIPMDH2 and AtIPMDH3 have been obtained. Importantly, the 2.25‐Å resolution structure of AtIPMDH2 was solved by molecular replacement and is the first detailed molecular architecture of a plant IPMDH. Through substrate modeling and site‐directed mutagenesis of AtIPMDH2, the molecular basis for the glucosinolate chain‐elongation activity of AtIPMDH1 is derived from the variation of a single amino acid in the active site. Future work will include crystallization of AtIPMDH2 with its substrates and further mutagenesis of the key residues in the active site to better understand how this enzyme catalyzes the decarboxylative formation of glucosinolates and leucine.
Assay of Vitamin C in Red Blood Cells
Authors: Li Hongyan , Tu Hongbin , Wang Yaohui , Levine Mark
Published: June 2021

Although vitamin C (ascorbate) is present in whole blood, accurate measurements in red blood cells (RBCs) are problematic because of assay interferences, limited sensitivity, and sample volume requirements. We describe a new technique for ascorbate measurement in RBCs of humans, wild type mice, and mice unable to synthesize ascorbate. Exogenously added ascorbate was fully recovered even when endogenous RBC ascorbate was below detection threshold (25 nM). Twenty Âμl of whole blood or 10 Âμl of packed RBCs were sufficient for assay. RBC ascorbate was stable for 24 hours from whole blood samples at 4°C. Processed, stored samples were stable for > one month at −80°C. Unlike other tissues, ascorbate concentrations in human and mouse RBCs were linear in relation to plasma concentrations (R = 0.8, 0.9 respectively). In healthy humans, RBC ascorbate concentrations were 9–57 ÂμM, corresponding to ascorbate plasma concentrations of 15–90 ÂμM. Mouse data were similar. In human blood stored as if for transfusion, initial RBC ascorbate concentrations varied approximately 6 fold, and decreased 50% after 6 weeks of storage under clinical conditions. With this assay, it becomes possible for the first time to characterize ascorbate function in relation to endogenous concentrations in normal RBCs and those stored for transfusion.
Purification of MBP‐ and Strep‐tag II‐tagged proteins
Authors: Carlsson Marianne , Heijbel Anna , Karlsson Anneli , Lilja Lydia
Published: June 2021

Recombinant tagged proteins can be purified to high purity in one step using affinity chromatography. Two chromatography media are presented, StrepTactin™ Sepharose™ High Performance (HP) which has high affinity for the small 8 amino acid peptide, Strep‐tag II, and Dextrin Sepharose HP, that binds to Maltose Binding Protein‐tag (MBP). The specificity of the two media towards their respective tag gives high purity of the target proteins. In this study, simple purifications using MBPTrap™ HP (prepacked with Dextrin Sepharose HP and StrepTrap™ HP, (prepacked with StrepTactin Sepharose HP) are presented. Reproducibility during repeated usage of StrepTrap HP was tested by running 6 repeated purifications of a Strep‐tag II‐tagged protein expressed in E. coli on the same StrepTrap HP column including regeneration with 0.5 M NaOH between each run. No tendencies in reduction of final purity and yield of the target protein were observed. Equivalent results are obtained when scaling up showing the simplicity to purify larger quantities of pure target protein.
Preparation and characterization of polysaccharides microparticles loaded with cisplatin
Authors: Ribeiro Camyla Mara S , Gomes Anderson J
Published: June 2021

The nanotechnology applied into systems for controlled release of drugs in cancer treatment intended assiste drug release, in vivo, to achieve clinical benefit. Among the polymers used in the preparation and characterization of microparticles, the highlights are the polysaccharides, including sodium alginate and guar‐gum. This study produced and characterized the polysaccharide microparticles to encapsulate anticancer drugs, such as cisplatin, in order to reduce adverse effects and enhance bioavailability with sustained delivery. The microparticles were prepared using the method of emulsification/ionic crosslinking. The microparticles of alginate obtained around 70% efficiency of encapsulation, the mean particles size was 4,2 ± 0,4 μm, and zeta potencial of −2.19 mV. The microparticles of guar‐gum obtained 70% efficiency of encapsulation, the mean particles size was 2,9 ± 0,3 μm, and zeta potencial of 5,16 mV. Consequently, microspheres have a good physico‐chemical stability, low aggregation and high efficiency of encapsulation. Therefore, polysaccharides microspheres appear, technologically, a promising anticancer drugs delivery system. Supported by FAPDF, CAPES, CNPq, FINATEC and DPP‐UnB.
Temporal hierarchy of membrane restructuring during cold acclimation in fish
Authors: Ward Kyle , Williams E Eugene
Published: June 2021

Climate change may have dramatic affects on the diversity and abundance of fish. It has been shown fish can restructure membrane phospholipids to aid in their survival in response to a change in water temperature. Biochemical evidence suggests these changes do not occur at the same time or rate. Some reactions occur rapidly, whereas others are delayed. We are currently trying to elucidate this hierarchy for mRNA expression for reactions involved in membrane restructuring. We have developed primers for many of the enzymes involved in these restructuring pathways including de novo phospholipid synthesis, in situ phospholipid modifications and phospholipid head group alterations. Our goal is to develop additional real time probes based on PCR products obtained from primers designed for enzymes in these restructuring pathways. To date, five such probes have been developed and preliminary data show that changes in mRNA expression are occurring at different rates. These data will help us further understand temperature acclimation of fish at the molecular level. This work is supported by the Department of Biological Sciences and the Henson School of Science and Technology at Salisbury University.
Characterization of rOrf8/EscI of the enteropathogenic Escherichia coli as an inner rod protein
Authors: Sal-Man Neta , Deng Wanyin , Finlay B. Brett
Published: June 2021

The type III secretion system (T3SS) is a multi‐protein complex that plays a central role in the virulence of many Gram‐negative bacterial pathogens. This apparatus spans both bacterial membranes and transports virulence factors from the bacterial cytoplasm into eukaryotic host cells. While the inner rod and the needle structures are being assembled, translocators and effectors cannot be secreted. In this study, we characterized a small protein, designated rOrf8, that functions as the inner rod protein of enteropathogenic E. coli (EPEC). We demonstrated that rOrf8, is essential for type III secretion and is secreted by the T3SS, as previously reported for inner rod proteins of other T3SS. Furthermore, we showed that EscI self‐associates and interacts with the outer membrane secretin, EscC, further supporting its function as an inner rod protein. Overall, our data suggest that EscI is the YscI/PrgJ/MxiI homolog in the T3SS of attaching and effacing pathogens.
Thrombospondin‐1 signaling via CD47 regulates T lymphocyte glycosaminoglycan biosynthesis
Authors: Kaur Sukhbir , Kuznetsova Svetlana A , Pendrak Michael L , Sipes John M , Roberts David D
Published: June 2021

T cells express two cell surface proteoglycans that bind thrombospondin‐1: CD47 and APLP2. CD47 mediates inhibition of T cell signaling by thrombospondin‐1, which requires glycosaminoglycan modification of CD47 at Ser64. This prompted us to examine how T cell activation modulates CD47 glycosylation. T cell activation increased both heparan and chondroitin sulfate biosynthesis by globally inducing mRNA levels of the respective glycosyltransferases and sulfotransferases. Activation resulted in larger glycosaminoglycan chains with a decreased density of sulfation, a net shift towards increased chondroitin sulfate, and increased release of proteoglycan into the medium. These changes resulted in altered modification of CD47. T cell activation in the presence of thrombospondin‐1 inhibited these changes, but not in cells lacking CD47. Remarkably, CD47 null T cells exhibited delayed induction of mRNAs for glycosaminoglycan biosynthetic enzymes upon anti‐CD3 stimulation. Re‐expression of CD47 in CD47 mutant cells restored activation‐dependent mRNA induction and sensitivity to thrombospondin‐1. These data reveal a negative feedback loop by which thrombospondin‐1 controls the level of an essential post‐translational modification of its own signaling receptor in T cells.
The role of N‐linked glycosylation during Drosophila embryonic development
Authors: McCague Allison , Selva Erica M.
Published: June 2021

N‐linked glycosylation is a key post‐translational modification for many secretory proteins. It has roles in cell recognition and adhesion, protein folding, and cellular signaling. Endoplasmic reticulum glycosyltranferases construct a 14‐sugar precursor on a dolichol carrier, which is then transferred en mass to proteins on consensus sites. The function of N‐glycosylation during Drosophila embryogenesis was investigated in alg9 and alg10 germ line clone mutant embryos. alg10 encodes the enzyme catalyzing terminal glucose addition to the sugar‐substrate prior to protein transfer, while the alg9 encoded enzyme acts five steps earlier in sugar synthesis. alg9 and alg10 displayed severe and pleotrophic defects in cuticle secretion. Expression of Wingless (Wg), a model secreted glycoprotein of the Wnt pathway, was examined to identify earlier effects of these mutations. Normal Wg secretion was disrupted in alg9 and alg10 glc embryos. However, defects in secretion did not block downstream Wg signaling, as Engrailed (En) was expressed fairly normally in both backgrounds. The central nervous system (CNS) develops later and is highly dependent on glycosylation, alg10 loss caused severe defects in CNS development, but alg9 glc embryos lack CNS. Hence, alg10 may cause global underglycosylation or is only needed for some glycoproteins, while alg9 may be required for N‐glycosylation of most or all protein substrates.
rLosac Induces Pro‐survival Mechanisms In Dorsal Root Ganglia Neurons
Authors: Alvarez-Flores Miryam Paola , Remuzgo Cesar Manuel , Cury Yara , Chudzinski-Tavassi Ana Marisa
Published: June 2021

Losac (a protein from Lonomia obliqua caterpillar) shows high similarity with the four first Ig‐like domains of NCAMs from human which are crucial for embryonic development of neural system. Previous studies known that Losac is capable to inhibit endothelial cell death. Using a model of serum deprivation, we investigated the molecular mechanism by which recombinant Losac (produced in bacteria), exerts its protective effect in dorsal root ganglia (DRG) neurons. Cell survival (MTT assay) was better observed after 48h of treatment. Immunoblot analysis showed an increase in phospho‐p44/42 MAPKs after 6h of treatment with Losac. It was observed a reduction of apoptotic DNA and the increasing of the number of cells in the S phase (48 and 72h). In addition, it was observed an increase of the mitochondrial membrane potential (rhodamine‐123) and KI‐67 proliferation marker. All these results indicate that rLosac trigger cell viability through the activation of survival pathways in the early hours of cell contact with Losac and subsequent DNA synthesis and proliferation. It will be further investigated the potential application of Losac on neurodegenerative diseases. Support: FAPESP, CAT‐CEPID.
Physiologic Cardiac Hypertrophy and Cardiac Dilation: A Comparative Study Using ZmRacD Transgenic Mouse Model
Authors: Elnakish Mohammad T. , Hassona Mohamed D. H. , Alhaj Mazin A. , Moldovan Leni , Khan Mahmood , Hassanain Hamdy H.
Published: June 2021

The pathways inducing the critical transition from compensated hypertrophy to cardiac dilation and failure remain poorly understood. The goal of our study is to examine the role of Rac‐induced signaling in this transition process. Previously, we have shown that Thyroxin (T4) treatment resulted in increased myocardial Rac expression in wild‐type mice and a higher level of expression in ZmRacD mice. Currently, we showed that T4‐treated wild‐type mice exhibited physiologic cardiac hypertrophy, as indicated by echocardiography and histopathology analyses. This was coupled with significant increases in myocardial O2.−, ERK1/2, and JNK activities. However, there was significant decrease in myocardial connexin‐43 expression. Conversely, T4‐ treated ZmRacD mice exhibited cardiac dilation. This was linked with further increases in myocardial O2.− and ERK1/2 activity. Also, there were significant increases in caspase‐8 expression and caspase‐3 activity. However, there were significant decreases in p38‐MAPK activity and myocardial connexin‐43 expression. In conclusion, T4‐induced ZmRacD represents a novel mouse model for cardiac dilation that is similar to that of the human disease. Moreover, Rac, ERK and p38‐MAPK seem to play a central role in the transition from cardiac hypertrophy to cardiac dilation and failure and they could represent valuable therapeutic targets for heart failure in the future.
CARDIOTONIC STERIODS INDUCE STRESS SIGNALING IN PREECLAMSIA: A TRANSLATIONAL APPROACH WITH IN VIVO, IN VITRO, AND PATIENT STUDIES
Authors: Uddin Mohammad Nasir , Horvat Darijana , Allen Steven R , Jones Richard O , Zawieja David C , Kuehl Thomas J
Published: June 2021

Objective

Cardiotonic steroids (CS): marinobufagenin (MBG), ouabain (OUB) and cinobufatalin (CINO) are endogenous inhibitors of Na+/K+ ATPase. MBG is associated with preeclampsia (preE). We evaluated the effect of CS on stress signaling in pregnant rats, cytotrophoblast (CTB), and preE patients.

Methods

(i) Five groups of rats were studied: nonpregnant (C, n= 10); normal pregnant (NP, n = 10); pregnant rats given saline to drink and desoxycorticosterone acetate injection (PDS; preE rat model, n = 10); NP injected with MBG (NPM, n = 10); and NP injected with CINO (NPC, n = 10). We analyzed the stress signaling in all groups. (ii) Effects of CS on stress signaling in CTB were assessed. (iii) MBG and stress proteins were assayed in urine samples from 17 PreE and 23 NP patients.

Results

(i) Placental expression of Gadd45a, caspase 3 & 8, sFlt‐1, sFlt‐1R, & p38 were significantly higher in PDS, NPM, and NPC rats compared to NP. (ii) Expression of Gadd45a, sFlt‐1, IL‐6, 8‐IP & p38 were significantly upregulated in ≥1nM CS‐treated cells compared to vehicle‐ and 0.1 nM‐ treated cells. RNAi‐mediated inhibition of Gadd45a attenuated CS‐induced CTB stress signaling. (iii) Urinary MBG levels were significantly higher in preE patients compared to NP. Angiogenic imbalance was observed in preE patients compared to NP.

Conclusions

We demonstrated that CS induce the stress signaling in (i) NP rats; (ii) CTB cells; and (iii) preE patients.
Mathematica's computable document format (CDF) player: a versatile tool for displaying interactive graphs and 3‐D structures
Authors: Sears Duane William , Clayton Nicholas K
Published: June 2021

Considering that biochemical research often aims to establish structure/function correlations, biochemistry students need exposure to the methodology biochemists use for analyzing biomolecular structures and their functions. The free Jmol web browser plug‐in is particularly effective as a molecular visualization tool for introducing students to interactive structure exploration but the options for engaging students in functional analysis are limited. For example, interactive data plots promote student understanding of biochemical reactions but existing computer applications tend to be technically cumbersome and/or expensive. A notable exception is the computable document format (CDF) player, a web browser plugin designed to “play” content or “notebooks” created with Wolfram's Mathematica 8 program. Although Mathematica requires certain programming sophistication, notebooks, once created, can be readily disseminated online with the free CDF player. As described with this poster, we have created a series of interactive CDF plots that aim to facilitate student understanding and exploration of various types of reactions typically encountered in a biochemistry course. In addition, we evaluate the new 3‐D molecular rendering capability of Mathematica, which provides an interesting alternative for projecting interactive PDB‐based structures with a web browser.