bluuebunny/crossref_metadata_2025 huggingface dataset:
Effects of leptin signaling on the development of Alzheimer's Disease pathology
Authors: Platt Thomas L. , Niedowicz Dana M. , Beckett Christina L. , Murphy M. Paul
Published: June 2021

Alzheimer's Disease (AD) is the most common neurodegenerative disease associated with age. Hallmarks of AD pathology are the accumulation of amyloid plaques (containing Aβ) and fibrilization of the microtubule‐associated protein tau into tangles. Type II diabetes mellitus (T2DM) is a metabolic disorder commonly associated with obesity. Many individuals who suffer from T2DM develop resistance to leptin, a hormone released from adipose tissue to regulate food intake and energy metabolism. It has been observed that T2DM confers an increased risk for AD. Therefore, we set out to determine if a loss of leptin signaling affects AD pathogenesis. Previous data indicate that as plasma leptin levels rise, presenilin expression decreases along with total Aβ. To expand on these findings, a diabetic mouse line (db) was crossed with an AD mouse line (APP x PS1) to create a model to observe the effects of leptin resistance on levels of Aβ. In addition, AAV2/1 was used to express the tau mutant P301L in db mice to observe tau phosphorylation. Data were analyzed by western blot, ELISA, RT‐PCR, and IHC to detect pathologic Aβ and tau in the mouse brain. These data suggest that a deficiency in leptin signaling, as with leptin resistance, may promote AD pathogenesis. Future studies will focus on revealing the mechanism by which leptin modulates AD pathology. Supported by the CART Foundation and the Alzheimer's Association (IIRG‐10‐172905).
Effects Of Orange Juice And Vitamin C On The Expression Of Some Selected Genes ( Tnfr, Sod3, P53, Pgr ) In Sprague‐Dawley Rat Brain
Authors: Ebuehi Osaretin Albert , James Ayorinde , Salami Yusuf , Mgbeadichie Imelda
Published: June 2021

The comparative effects of Vitamin C and Nigerian local orange juice on the gene expression levels of Superoxide dismutase (Sod), Tumour necrosis Factor Receptor (Tnfr), p53 protein, and progesterone receptor (Pgr) genes were evaluated in rat neuronal tissues.

5ml/kg bodyweight of extracted orange juice and 20mg/kg bodyweight Vit C were administered orally to Sprague Dawley rats, which were divided into control, Vit. C and orange groups. After the 7th day of administration, total RNA was extracted from the rat brains and normalized RNA was converted to cDNA. PCR Primer pairs were designed using Primer 3 and NCBI primer blast tool. Sybr green Real time PCR relative quantification was done.

Orange juice extract contains 15.84 mg Vit C, 13.67g total sugar, 0.02 mg Sodium, 0.15 mg Iron, 11.48 mg Magnesium, 44.20 mg Calcium, 4.76 mg Potassium, 0.75 mg Zinc, and 22.9 mg Phosphorus. Relative quantification gene expression assays in the rats show a 400 fold change in Tnfr and 38 fold change in p53 gene in the orange administered group in comparison to the Vit C administered and control groups. Vit C caused a 420 fold change in the expression of Pgr gene in comparison with the orange administered group. Data show that orange juice administered at 5ml/kg body weight show higher levels of gene expressions of Tnfr and p53 genes, thereby indicating brain tumour preventing ability of oranges.
Effect Of Very High Fat Diet On Body Weight, Hormonal Levels And Estrous Cycle
Authors: Eldib Ahmed , Eliscar Theressa , Chakraborty Tandra R.
Published: June 2021

Glucose is the main metabolic fuel of the brain. and its availability is directly linked to neuronal activity. In the absence of glucose i.e. hypoglycemia, there is loss of neurons, impaired IQ and can ultimately lead to morbidity. In the present study we have reported the neuroprotection of estrogen on hypoglycemic injury in hypothalamic cell line. There was a marked decrease in the cell count and mitochondrial function when exposed to hypoglycemia which increased in presence of estrogen (42.8 vs 98.5, 24 hours, p<0.0001). The attenuation of the death pathways was only seen within the first 24 hours and gradually decreased with time. DNA microarray studies showed AKT, a promoter of cell survival, was down‐regulation in the absence of glucose and in the presence of estrogen an increase in gene expression. RTPCR showed decrease in AKT expression with a slight increase in presence of estrogen. PRAS 40, proline rich substrate for AKT was measured. Results show low expression of PRAS40 in hypoglycemic conditions in 24 hours and 72 hours with a trend to increase in presence of estrogen. Therefore, it seems that one of the mechanism by which estrogen shows its neuroprotection is through the Akt‐GSK3b pathway activation.
Vitamin C regulates iron uptake from transferrin – a novel role for ascorbate in iron metabolism?
Authors: Richardson Des R , Chikhani Sherin , Richardson Vera , Lane Darius J.R.
Published: June 2021

We previously demonstrated that ascorbate enhances cellular iron (Fe) uptake from 55Fe‐citrate by transmembrane ascorbate cycling. Here we have examined ascorbate's effect on cellular Fe uptake from the major serum Fe transport protein, transferrin (Tf). Incubation of SK‐Mel‐28 cells with ascorbate (50 μM) resulted in a significant 1.5–2.5‐fold increase in 59Fe uptake from 59Fe2‐Tf. Ascorbate oxidase inhibited ascorbate‐stimulated 59Fe uptake from 59Fe‐citrate, but was ineffective against ascorbate‐stimulated 59Fe uptake from 59Fe2‐Tf. The amount of cellular 125I‐Tf uptake, and the rate of 125I‐Tf cycling, was unaffected by ascorbate. Co‐incubation of cells with ascorbate and Fe2‐Tf also enhanced ferritin expression, ferritin 59Fe incorporation. Heterologous expression studies indicated that TfRs 1/2 are necessary for ascorbate's stimulatory effect. Ascorbate‐stimulated, but not basal, 59Fe uptake from 59Fe2‐Tf was inhibited by the membrane‐permeant thiol donor, N‐acetyl‐l‐cysteine and the antioxidant, ebselen. Moreover, ascorbate/Fe2‐Tf markedly decreased intracellular NADPH, but not total NADP levels and increased intracellular reactive oxygen species production. These data suggest that ascorbate may stimulate Fe uptake from Fe2‐Tf through a pro‐oxidant mechanism of action. Ascorbate's contribution to cellular Fe uptake and metabolism may be more complex than initially realized.

Support: National Health and Medical Research Council (NHMRC) of Australia and Cancer Institute New South Wales.
Methionine Sulfoxide Reductase A Regulates Cell Growth through the p53‐p21 Pathway
Authors: Kim Hwa-Young , Choi Seung Hee
Published: June 2021

MsrA is an oxidoreductase that catalyzes the stereospecific reduction of methionine‐S‐sulfoxide to methionine. Although MsrA is well‐characterized as an antioxidant and has been implicated in the aging process and cellular senescence, its roles in cell proliferation are poorly understood. Here, we report a critical role of MsrA in normal cell proliferation and describe the regulation mechanism of cell growth by this protein. Down‐regulation of MsrA inhibited cell proliferation, but MsrA overexpression did not promote it. MsrA deficiency led to an increase in p21, a major cyclin‐dependent kinase inhibitor, thereby causing cell cycle arrest at the G2/M stage. While protein levels of p53 were not altered upon MsrA deficiency, its acetylation level was significantly elevated, which subsequently activated p21 transcription. The data suggest that MsrA is a regulator of cell growth that mediates the p53‐p21 pathway.
Effect of Hepatocyte Growth Factor in Alveolar Epithelial Cells
Authors: Mercado Armando Raúl López , Poonyagariyagorn Kristy , Misono Kaori , Dikeman Dustin , Neptune Enid
Published: June 2021

Lung inflammatory diseases, such as chronic obstructive pulmonary disease (COPD), are the third leading cause of death in United States. The disruption of gas exchange observed in COPD is a consequence of alveolar enlargement. Previous results from our laboratory showed that hepatocyte growth factor (HGF) administration in both an elastase‐induced emphysema mouse model and a genetic emphysema model significantly reduced airspace damage. Since hepatocyte growth factor (HGF) is a pleiotrophic cytokine that promotes epithelial proliferation, morphogenesis, migration, angiogenesis and survival, we investigated the cell‐autonomous effects of HGF in alveolar epithelial cell line. MLE‐12 cells were treated with HGF to study downstream mediators, proliferation, protection against apoptosis and scattering. We found that the prosurvival ERK and AKT pathways were activated in the presence of HGF. Using specific inhibitors, we found that basal and HGF‐induced proliferation was mediated through ERK signaling. However, HGF was shown to confer protection against cell death principally by AKT signaling. Finally, we demonstrated that HGF could induce MLE12 cell scattering after 1 hour of treatment. Together, the results obtained in this study showed that MLE12 cells are a good model system to dissect the role of HGF in the critical events of alveolar septation. Supported by grant to E.R.N (RO1‐HL085312).
Functional characterization of two distinct interferon gamma structurally related protein IFNγrel 1 and IFNγrel 2, in ginbuna crucian carp
Authors: Shibasaki Yasuhiro , Yabu Takeshi , Moritomo Tadaaki , Nakanishi Teruyuki
Published: June 2021

Unlike mammals fish have two type II interferons, IFNγ and IFNγ‐related (IFNγrel). Both IFNγ and IFNγrel are structurally similar to human and mouse IFNγ and induce pro‐inflammatory responses. We previously identified two IFNγrel genes, IFNγrel 1 and IFNγrel 2 from ginbuna crucian carp, Carassius auratus langsdorfii. We produced recombinant IFNγrel 1 and IFNγrel 2 proteins using HEK cells and demonstrated that they possess high antiviral activities against CHNV hematopoietic necrosis virus. Both interferons have highly homologous sequences, however, they differ in the C‐terminus region, where IFNγrel has a nuclear localization site (NLS) and IFNγrel 2 does not. Here, we report that the NLS‐like sequence fused to GFP localized in nucleus of the GTS9 gibuna carp fibroblast cell line. Furthermore, we found that IFNγrel 1 translocated from cytosol to nucleus, despite the fact that IFNγrel 1 did not interact with known IFNγ receptors, IFNGR1‐1 and IFNGR1‐2. Pull‐down assay suggested the existence of a novel receptor for IFNγrel 1. These results indicate that IFNγrel 1 and IFNγrel 2 are functional, but appear to signal through different receptor(s) than IFNγ. It is probable that IFNγrel 1 and IFNγrel 2 use different signal transduction pathway(s).
Prediction of The Risk for Hypertension Among Carrier of C825T Genetic Polymorphism of G Protein Beta 3 Gene (GNB3)
Authors: Hemimi Neveen Salah El Din , salam Mona Mohamed Abd El , Ahmad Deena Jalal
Published: June 2021

Background

GNB3 825T is associated with the occurrence of the splice variant Gß3s, which, despite a deletion of 41 amino acids, is functionally active in reconstituted systems. This may cause enhanced vascular reactivity and increased proliferation of smooth muscle cells and cardiac myocytes, ultimately resulting in vascular and myocardial hypertrophy.

Aim

To predict the risk for hypertension in individuals with C825T genetic polymorphism of G protein β3 gene.

Material and Method

The study consisted of 192 normotensive individuals and 120 hypertensive patients. Individuals were genotyped for C825T genetic polymorphism of G protein β3 gene by using polymerase chain reaction‐ restriction fragment length polymorphism. Statistical analysis was done by SPSS program.

Results

Frequency of C and T alleles were 47.5% and 52.5% in hypertensive group compared to 62% and 38% in control group. The carriers of T allele exhibited a significant greater risk for hypertension compared to carriers of C allele (OR= 1.8, 95%CI= 1.3–2.6).

Conclusion

T allele is a risk factor for hypertension.

Source of Fund: DMCG
Biochemical requirements for suppressing a constitutively active allele of Gs alpha
Authors: Tobar-Rubin Raquel , Sultan Dahlia , Janevska Daniela , Rylaarsdam Robin Pals
Published: June 2021

Inappropriate activation of the Gs alpha subunit by mutation of modification of Arg201 is associated with the human diseases of McCune‐Albright Syndrome and cholera. Previous work in our laboratory identified Asp223 as a site where substitution to valine could reverse the constitutive activity of an Arg201His mutation. In this study, Asp223 was substituted with a variety of other amino acids, including glutamic acid, asparagine, glutamine, alanine, leucine, and glycine. HEK cells transfected with plasmids carrying the different Gs alpha subunit alleles were used to measure basal levels of cAMP. The Arg201His allele significantly elevates basal cAMP. Substitution of Asp223 with nonpolar residues suppressed the elevation in basal cAMP, while substitution with acidic or polar residues had no effect on the constitutive activity of the Arg201His mutation. This characterization of which amino acids at residue 223 can suppress the Arg201His mutation may provide a foundation for identifying small molecules that can be used as effective therapies against McCune‐Albirght Syndrome.

Supported by NIH grant1R15DE02109‐01
Define a novel role for Giα proteins in differentially regulating RTK‐mediated signaling in response to growth factors
Authors: Chu Wen-Ming , Wang Zhanwei , Cruz Rica Dela , Chu Catherine H , Jiang Meisheng
Published: June 2021

The activation of the ERK and the PI3K/Akt/mTORC1 pathways by growth factors is mediated by the receptor tyrosine kinases (RTKs), and plays a crucial role in physiological and pathological processes. However, the molecular mechanism of this activation is still unclear. Herein we demonstrated that Giα proteins differentially regulate RTKs‐mediated activation of the ERK and the Akt/mTORC1 pathways.

Loss of Giα proteins impaired activation of ERK by EGF, IGFI and Insulin, but not bFGF. Intriguingly, while Akt and mTORC1 activation by EGF and bFGF was severely defective in Giα‐deficient cells, this activation by insulin and IGFI was only slightly reduced. Further, Giα proteins altered Akt, mTORC1 and ERK activation by serum and PDGF. Also, growth factors induced the association of Giα with RTKs and their downstream adaptors. Loss of Giα proteins impaired RTKs’ association with their adaptors indicating that Giα proteins act downstream of RTKs but upstream of the adaptors.

Finally, we found that knockdown of Giα proteins in human cancer cell lines diminished Akt, mTORC1 and ERK activation by growth factors leading to a defect in cancer cell growth and migration. Given the fact that growth factors and their receptor pathways play pivotal roles in tumorigenesis and metastasis of cancer, our findings will promote further understanding of RTK signaling regulation in cancer and may have implications for cancer therapy.
Regulator of G Protein Signaling 6 (RGS6) ensures coordination of motor movement by modulating GABAB Receptor (GABABR) signaling
Authors: Stewart Adele , Maity Biswanath , Yang Jianqi , Loo Lipin , Shepherd Andrew J , Mohapatra Durga P , Fisher Rory A
Published: June 2021

Inhibitory interneurons located within the cerebellar cortex release GABA, which limits neuronal excitation in part through activation of metabatropic GABABRs. Here, we identify RGS6, a member of the R7 subfamily of RGS proteins, as a key modulator of GABABR signaling in cerebellum. RGS6−/− mice exhibit abnormal gait and ataxia characterized by impaired rotarod performance that can be partially rescued by administration of a GABABR antagonist. In addition, RGS6−/− mice treated with baclofen, a GABABR agonist, show exaggerated motor coordination deficits on the rotarod compared to their wild type counterparts. Stimulation of GABABRs triggers a number of signaling events downstream of G protein activation including Gβγ subunit activation of G protein‐coupled inwardly rectifying potassium (GIRK) channels. Indeed, RGS6 is enriched in the granule cell layer of the cerebellum along with neuronal GIRK channel subunits 1 and 2 where RGS6 forms a complex with known binding partners Gβ5 and R7‐binding protein (R7BP), which stabilize and promote membrane association of R7 family members, respectively. Cerebellar granule neurons isolated from RGS6−/− mice showed a significant delay in the deactivation kinetics of baclofen‐induced GIRK currents. These results establish RGS6 as a key modulator of GABABR signaling, providing the first demonstration of an essential role for RGS proteins in cerebellar function.
Regulation of estrogen receptors and CREB‐binding protein (CBP) following traumatic brain injury
Authors: Zee Isabelle , Duncan Kelli A.
Published: June 2021

Following brain injury, estradiol is neuroprotective and promotes neuronal survival and cell proliferation. Transcription of estrogen‐mediated targets occurs through the dimerization of the estrogen receptor alpha and beta (ERα, ERβ), to DNA following recruitment of coregulators and heat shock proteins. Coregulators (coactivators and corepressors) remodel chromatin via histone‐DNA contact stabilization and allow for the binding of additional transcription factors and RNA polymerase II recruitment. The role of coregulators following traumatic brain injury (TBI), though, is unknown. In this study, we investigated the interaction between ERα, ERβ, and CREB‐binding protein (CBP) following neurotrauma. CBP is a nuclear coactivator that interacts with both the amino‐terminal domain (AF‐1) and carboxy‐terminal ligand‐binding domain (AF‐2) of ER. Adult male and female zebra finches (Taeniopygia guttata) were given a single penetrating injury and collected at 1 and 7 days post injury. Transcriptional activity and expression of ERα, ERβ, and CBP were examined using quantitative polymerase chain reaction and immunohistochemistry. Preliminary data suggest that CBP is upregulated following injury and play a role in estrogen receptor mediated neuroprotection following injury, and suggest a novel role for estrogen receptor coactivators.
Zoanthus sociatus extract blocks the nifedipine sensitive‐Ca+2 influx in beta cells and impairs glucose stimulated insulin secretion
Authors: Diaz-Garcia Carlos Manlio , Sanchez-Soto Carmen , Fuentes-Silva Deyanira , Leon-Pinzon Carolina , Dominguez-Perez Dany , Varela Carlos , Rodriguez-Romero Adela , Castañeda Olga , Hiriart Marcia
Published: June 2021

Crude extracts from coelenterates have been a source of neurotoxins that still possesses a great potential in the discovery of novel drugs. Studies about the effect of cnidarians venoms upon insulin release are not common, despite the coincidences in ionic mechanisms of glucose stimulated insulin secretion and neurotransmitter release. In the present work we tested the effect of a crude extract of Zoanthus sociatus on insulin secretion in isolated rat β‐cells and its toxic effects in vivo.

We observed that 50 ug/ml of the extract inhibited insulin release in a 40 % when incubated at 15.6 mM glucose and that a low molecular weight fraction was responsible of this effect. Moreover, the active fraction impaired intracellular Ca+2 rises in response to high glucose and high K+‐induced depolarization. The results suggest the presence of voltage‐gated Ca+2 channels inhibitors in the active fraction, which cause glucose intolerance when rats are intraperitoneally inoculated with the extract.

This work was supported by Gobierno del Distrito Federal PICDS08‐72, CONACYT CB2009‐131647, and DGAPA‐PAPIIT IN215611, Universidad Nacional Autónoma de México. Carlos Manlio Díaz‐García was supported by a doctoral fellowship from Consejo Nacional de Ciencia y Tecnología (CONACyT).
Gene identification in Pseudomonas aeruginosa: from bioinformatics to experimental analysis
Authors: Oden Steve , Zhang Ying , Jin Shouguang , Tornaletti Silvia , Brocchieri Luciano
Published: June 2021

Computational prediction of short genes and experimental identification of small proteins is problematic due to the intrinsic weakness of the coding signal in short sequences and to the difficulties in detecting small proteins often expressed at low levels and in specific conditions. Nevertheless, prokaryote genome annotations include a large fraction of “hypothetical” short gene predictions that are often not conserved and are functionally uncharacterized. We developed algorithms to detect in DNA or RNA sequences localized compositional contrasts signaling presence of coding sequences and to associate statistical significance to each prediction. Our procedures identify previously undetected potential coding regions and provide statistical support to previously annotated hypothetical genes. To validate the results of computational analyses in Pseudomonas aeruginosa we developed a protocol for the application of the technique of ribosomal profiling to this prokaryotic organism, combining polysome profiling and high throughput RNA sequencing technologies for sequencing and mapping actively translated mRNA regions.
Improving a software system for protein active site determination
Authors: Dodge Greg James , Corwin Cyprian , Bernstein Herbert J. , Craig Paul A.
Published: June 2021

ProMol is a plugin for the PyMOL molecular graphics system that is designed to locate the active site on a query protein. ProMol does this by comparing the three dimensional distances of all of the atoms in a protein to the three dimensional positions of a library of known active site residues. This is a computationally intensive process, and it would take months to run the entire PDB (Protein Data Bank) through the program. Recently however, advances in GPGPU (General Purpose Graphics Processing Unit) computing and GPU design have allowed simple computations, such as those used by ProMol, to be performed rapidly across hundreds of cores. Utilizing GPGPU computing, the time needed to run proteins through our program could be drastically reduced. Due to the rapid turnover of authors on the project however, ProMolˈs codebase was quite fragmented. Over the course of summer 2011, our group worked to streamline ProMolˈs source in preparation for implementing GPGPU computing. This work consisted of bug hunting, re‐implementing the PDB loading functionality, and cleaning up the user interface, among other things. Ideally, with the changes in place, we will be able to adapt PyMOL and ProMolˈs selection algebra to run in parallel across many GPU cores.
Protein Structure Initiative:Biology‐Materials Repository: Developing a Public Resource for Structural Biology Plasmids
Authors: Cormier Catherine , Park Jin , Fiacco Michael , Steel Jason , Kramer Jason , LaBaer Joshua
Published: June 2021

The Protein Structure Initiative: Biology‐Materials Repository (MR; http://psimr.asu.edu) is a centralized repository that sequence‐verifies, store and distributes plasmids created as part of the high throughput structural genomics efforts of the PSI. Each plasmid is fully sequence‐verified and stored, along with plasmid annotation such as the full length sequence, vector information, and associated publications, in a freely available, searchable database called DNASU (http://dnasu.asu.edu). Each plasmid links to external resources, including the PSI Structural Biology Knowledgebase (http://sbkb.org) facilitating cross‐referencing of plasmids to additional protein annotations and experimental data. Recent developments to DNASU include automation of our Expedited Material Transfer Agreement (MTA) process, which eliminates delays caused by MTA processing. As of November 2011, over 50,000 protein expression plasmids and 85 empty vectors from the PSI are available upon request from DNASU. By providing expression‐ready plasmids to the research community, we are facilitating the study of the biological function of proteins whose structures have been solved by the PSI.

This work was supported by the National Institute of General Medical Sciences.
Identifying targets and characterizing the role of aPKCs in murine embryonic stem cells
Authors: Duarte Mariana Lemos , Zeri Ana Carolina , Krieger Jose Eduardo , Schechtman Deborah
Published: June 2021

The protein kinase C (PKC) family of isoenzymes plays a pivotal role in maintaining undifferentiated embryonic stem cells (ESC) and in differentiation to specific cell types. We saw that aPKCs are highly expressed in undifferentiated ESC, to elucidate their role in these cells we aimed at identifying direct and indirect aPKC substrates. Using 2‐D phosphoproteomics and a ζ/λ pseudo‐substrate inhibitor peptide we were able to identify 68 proteins whose phosphorylation decreased in the presence of the inhibitor peptide. 34% of the identified proteins were proteins involved in protein folding, stability and synthesis, 27% in metabolism and 13% cytoskeletal elements and associated proteins involved in cell/cell adhesion processes, and cell polarization such as RhoGDI. Most of the identified proteins involved in metabolism were members of the glycolytic pathway. In addition, glucose consumption was reduced in cells treated with the pseudo‐substrate peptide. Metabolomic analysis combined with our proteomics results will help elucidate metabolic processes in which aPKC is involved in undifferentiated ESCs. Together our study indicates that aPKC is involved in cell/cell adhesion and glucose metabolism in undifferentiated ESCs, processes which are known to contribute to ESC self‐renewal.
Role of cystein residues in the normal operation and assembly of K+‐Cl− cotransporter isoform 2 (KCC2)
Authors: Carpentier Gabriel , Caron Luc , Jacob-Wagner Mariève , Isenring Paul
Published: June 2021

K+‐Cl− cotransporters (KCCs) regulate intracellular K+ and Cl− concentration as well as cell volume. Their transport activity is sensitive to N‐ethylmaleimide (NEM), a thiol reagent that alkylates available sulfhydryl groups, and their C‐terminus can interact with other CCCs to form oligomers. These features suggest that a number of cystein residues within the KCCs play an important role in carrier activity and/or assembly. To determine whether this is the case, we have exploited a mutagenic approach using rat KCC2 as a model. Substitutions (C8G, C742G, C854G and C982G) were generated by site‐directed mutagenesis and expressed in X. laevis oocytes. Substitution C854G increased while C982G decreased Rb+ transport without altering cell surface protein expression. They responded as did wild type (wt) carrier to the inhibitory effect of 250 μM furosemide but were more sensitive to the inhibitory effect of 1 mM NEM. Coimmunoprecipitation studies showed that the quantity of carriers precipitated with wt KCC2 was lower for C854G than for wt KCC2 or C982G. Lastly, FRAP studies in KCC2‐transfected HEK‐293 cells showed that NEM increased post‐bleach fluorescence recovery time in a concentration‐dependent manner. Collectively, the data suggest that C854 and C982 play an important role in KCC2 function, perhaps by supporting homo‐ and/or heterooligomeric structure formation. This work was funded by CIHR and CFI.
Isolation and mass spectrometric analysis of native protein complexes in rat liver mitochondrial contact sites
Authors: Lee Kwangwon , Kerner Janos , Hoppel Charles
Published: June 2021

Contact sites (CS) are structures where the mitochondrial inner and outer membrane are fused and can be isolated by discontinuous sucrose gradient centrifugation of crude outer membrane derived from swollen/shrunk rat liver mitochondria. We used a combination of Blue‐Native PAGE (BNE) and LC‐MS/MS to isolate and characterize the protein complexes present in CS.

CS proteins were extracted with Triton X‐100 and separated into eleven different size bands by BNE. All eight bands, analyzed by LC‐MS/MS, contained voltage‐dependent anion channel (three isoforms), long‐chain acyl‐CoA synthetase 1, phosphate carrier. Four bands between 600 kDa and 960 kDa contained carnitine palmitoyltransferase 1a, very long‐chain acyl‐CoA dehydrogenase, and the trifunctional protein (both Î± and Î2). In the same four bands, adenine nucleotide translocase (two isoforms) and apoptosis‐inducing factor 1 were identified. Of interest, carnitine‐acylcarnitine translocase was found only in the bands between 140 kDa and 400 kDa. Based on the proteins identified, liver mitochondrial CS from fed rats contain protein complexes of various high molecular weight.

We propose these protein complexes of various sizes represent carnitine transport system as well as very long chain fatty acid oxidation enzymes, nucleotide transport system and apoptosis system.

Supported by the NIH grant DK‐066107
Microtubule stability, Golgi organization, and transport flux require dystonin‐a2/MAP1B interaction
Authors: Ryan Scott D , Bhanot Kunal , De Repentigny Yves , Chu Alphonse , Blais Alexandre , Kothary Rashmi
Published: June 2021

Loss of function of dystonin cytoskeletal linker proteins causes neurodegeneration in dystonia musculorum (dt) mutant mice. While much investigation has focused on understanding dt pathology, divergent functions of dystonin isoforms remain unclear. Here, we highlight novel functions of the dystonin‐a2 isoform in mediating microtubule (MT) stability, Golgi organization, and flux through the secretory pathway. Using dystonin mutant mice combined with isoform‐specific loss of function analysis, we find dystonin‐a2 is bound to MT associated protein 1B (MAP1B) in the centrosomal region, where it maintains MT acetylation. In dt neurons, absence of the MAP1B/dystonin‐a2 interaction results in altered MAP1B perikaryal localization, leading to MT deacetylation and instability. Deacetylated MTs result in Golgi fragmentation and prevent anterograde trafficking via motor proteins. Maintenance of MT acetylation through trichostatin A (TSA) administration or MAP1B overexpression in vitro, mitigates the observed defect. These aberrations are apparent in pre‐phenotype dorsal root ganglia (DRG) and primary sensory neurons, suggesting they are causal in the dt disorder.
Differential sensitivity of melanoma cell lines with differing B‐Raf mutational status to the new oncogenic B‐Raf kinase inhibitor UAI‐201
Authors: Lee Michael , Ahn Jun-Ho , Ahn Soon Kil
Published: June 2021

The antiproliferative effect of the new oncogenic B‐Raf targeting drug UAI‐201 on 2 types of melanoma cell lines with differing B‐Raf mutational status was examined, and the underlying mechanisms were investigated. In cellular assays, UAI‐201 displayed high selectivity for tumor cells bearing B‐Raf(V600E), showing more than 1,000‐fold higher inhibition of their proliferation than wild‐type B‐Raf‐bearing cells. As expected, UAI‐201 completely abolished MEK‐ERK phosphorylation in A375P cells harboring B‐Raf(V600E). In SK‐MEL‐2 cells expressing B‐Raf(WT), UAI‐201 caused the paradoxical activation of the MAPK pathway but to a much lesser extent than that observed of other oncogenic B‐Raf inhibitors. These data suggest that UAI‐201 may be a more ideal B‐Raf inhibitor capable of preserving potency against oncogenic B‐Raf while minimizing the paradoxical activation of MAPK signaling. In addition, we showed that UAI‐201 treatment of A375P cells simultaneously induced cellular autophagy and apoptosis, as determined by GFP‐LC3 and caspase‐3 assays, respectively. Moreover, our results suggested that apoptosis and autophagy may cooperate in the induction of cell death in UAI‐201‐treated cells, which is dependent on Raf‐1 signaling. Collectively, our data suggest that UAI‐201 may be an effective B‐Raf inhibitor and a potential therapeutic strategy for B‐Raf( WT) and Ras mutant melanoma.
A novel RNA detection technology for gene expression analysis in living cells
Authors: Weldon Don S , Ko Alex , Johnston Grace , Hsu Matthew
Published: June 2021

Detecting gene expression has traditionally been limited to technologies that examine expression in lysed or fixed cell populations. The ability to detect gene expression in live cells would allow for more physiologically relevant information based on a cell's response to given stimuli. Determining which genes were up or down regulated in those cells provides insight into complex gene regulatory networks and cell function. Here we present a novel RNA expression detection technology capable of detecting specific mRNA and miRNA in live, intact cells. This technology allows for carrier‐free cellular uptake of the reagent, followed by detection of target RNA, with the ability to perform downstream analysis in the same sample. This reagent has no toxic effects on cells, and we have demonstrated the ability to isolate and further propagate live cell populations based on gene expression level. This would allow researchers to detect RNA and subsequently protein levels within the same cell.

Since current methods to interrogate the cellular RNA examine non‐native, amplified RNA targets, the relevance of such data often does not correlate to in vivo results. Furthermore, these methods do not provide RNA information from individual cells, and cells probed by these methods cannot be further studied following detection. Our technology presented here leaves the cells unchanged following detection, and therefore this same cell population can be used for downstream experiments. This research was funded by EMD Millipore.
In vitro budding assay to study folding and export of CFTR
Authors: Gupta Vijay , Balch William E.
Published: June 2021

Cystic fibrosis (CF) is one of the most common Caucasian autosomal recessive misfolding diseases leading to loss of lung and organ function. CF is caused by mutations in the cystic fibrosis transmembrane conductance regulator (CFTR) gene encoding the CFTR protein, a chloride (Cl−) channel. The most common mutation, a missing phenylalanine at position 508 (ΔF508) leads to misfolding and retention of the mutant protein in the endoplasmic reticulum (ER) where it is efficiently degraded, thereby reducing normal channel‐activity resulting in the disease phenotype. Protein trafficking out of ER is mediated by COPII vesicles and regulated by many factors including kinases and GTPases. We have developed an assay that reconstitutes COPII vesicle formation from the ER in vitro. ER export of ΔF508 CFTR occurs in a temperature, energy and time dependent manner that requires cytosolic factors. Our results provide a biochemical foundation for determining the critical factors that are responsible for failure and restoration of mutant ΔF508 CFTR trafficking to the cell surface in CF. This study is supported by Cystic Fibrosis Foundation.
Exploiting Adaptive Laboratory Evolution of Streptomyces clavuligerus for Antibiotic Discovery and Overproduction
Authors: Charusanti Pep , Fong Nicole Liu , Nagarajan Harish , Pereira Alban R , Li Howard J , Abate Elisa A , Su Yongxuan , Gerwick William H , Palsson Bernhard O
Published: June 2021

Adaptation is normally viewed as the enemy of the antibiotic discovery and development process because adaptation among pathogens to antibiotic exposure leads to resistance. We present a method here that, in contrast, exploits the power of adaptation among antibiotic producers to accelerate the discovery of antibiotics. A competition‐based adaptive laboratory evolution scheme is presented whereby an antibiotic‐producing microorganism is competed against a target pathogen and serially passed over time until the producer evolves the ability to synthesize a chemical entity that inhibits growth of the pathogen. When multiple Streptomyces clavuligerus replicates were adaptively evolved against methicillin‐resistant Staphylococcus aureus N315 in this manner, a strain emerged that acquired the ability to constitutively produce holomycin. In contrast, no holomycin could be detected from the wild‐type strain. Moreover, genome re‐sequencing revealed that the evolved strain had lost pSCL4, a large 1.8 Mbp plasmid. This and other strains also acquired numerous single nucleotide polymorphisms, some of which are located within genes that have been shown to affect secondary metabolite biosynthesis. These results demonstrate that competition‐based adaptive laboratory evolution can constitute a platform to create mutants that overproduce known antibiotics and possibly to discover new compounds as well.
Chiral Hydrolysis of G‐type Nerve Agents by Human Prolidase
Authors: Kajih Takwen , Cerasoli Douglas , Harrison Joshua , Norris Joseph , di Targiani Robert
Published: June 2021

Human prolidase is a bioscavenger of organophosphorus nerve agents that catalyzes the hydrolysis of GA, GB, GD, and GF. Chiral gas chromatography/mass spectrometry was used to measure the rate of hydrolysis of racemic nerve agent, yielding a relative measure of the stereopreference of prolidase for the enantiomers of GA, GB, GD, and GF. Each of the nerve agents analyzed consists of P(+) and P(−) isomers, of which the P(−) isomer is more inhibitory of AChE in vitro and more toxic in vivo. Both skin and kidney isoforms of prolidase were expressed as recombinant proteins in insect larvae and purified (>95% by Chesapeake‐PERL); no functional differences between the wild‐type isoforms were detected. Catalytic efficiencies for the different G agents varied between 104 to 106 M−1min−1, and preference for the P(+) isomer was detected for each agent. Based on these results, wild‐type prolidase is not predicted to be capable of affording protection in vivo against G agents. We also attempted to enhance the OP hydrolyzing activity of prolidase by producing three variants: R395S, R398S and T299Y. None of the variants improved the catalytic efficiencies sufficiently to be predicted to afford protection. Future efforts to improve the hydrolytic activity of prolidase toward OP nerve agents will utilize the knowledge obtained here in the generation of a directed evolution library.
Structural and functional roles of potassium ions on aldehyde dehydrogenases
Authors: MUÑOZ-CLARES ROSARIO ADELAIDA , MÚJICA-JIMÉNEZ CARLOS , DÍAZ-SÁNCHEZ ÁNGEL GABRIEL
Published: June 2021

The homotetrameric betaine aldehyde dehydrogenase from Pseudomonas aeruginosa (PaBADH) dissociates into inactive monomers at low ionic strength in the absence of K+ ions while the association state of the homodimeric enzyme from spinach is not affected, in spite of both enzymes having the same intrasubunit and intersubunit K+‐binding sites. The prevention of PaBADH dissociation by K+ ions is due to non‐specific effects of ionic strength, since the enzyme did not dissociate in the presence of tetraethylammonium (TEA) chloride. Cation‐binding sites unique to PaBADH, located in the negatively charged central cavity of this enzyme, are responsible for its dissociation in the absence of cations. In addition to their non‐specific, ionic‐strength effects, K+ ions have specific effects on enzyme structure, which result in increased affinity for NAD(P)+ as indicated by equilibrium binding studies. The latter effects probably arise from binding of the cation to the intrasubunit K+‐binding site, which is a common structural feature of aldehyde dehydrogenases. The result of pre‐steady state kinetic studies, carried out at very high protein concentration, to prevent enzyme dissociation, and at high NAD(P)+ concentration, to ensure saturation by the nucleotide, showed that the enzyme activity in non‐salt or TEACl buffers is similar to that in KCl buffer. Therefore, K+ ions are not required for the catalytic steps. Supported by CONACYT 16712 grant to RAMC.
Protection against hepatic steatosis and systemic insulin resistance by liver‐specific depletion of p70 S6 kinase
Authors: Bae Eunju , Xu Jianfeng , Olefsky Jerrold
Published: June 2021

Obesity‐associated hepatic steatosis is a manifestation of selective insulin resistance whereby lipogenesis remains sensitive to insulin but the ability of insulin to suppress glucose production is impaired. We created a mouse model of liver‐specific knockdown of p70 S6 kinase (S6K) (L‐S6K‐KD) by systemic delivery of an adeno‐associated virus carrying a shRNA for S6K and examined the effects on steatosis and insulin resistance. High fat diet (HFD) fed L‐S6K‐KD mice showed improved glucose tolerance and systemic insulin sensitivity compared to controls, with no changes in food intake or body weight. The induction of lipogenic gene expression was attenuated in the L‐S6K‐KD mice with decreased sterol regulatory element‐binding protein (SREBP)‐1c expression and mature SREBP‐1c protein, as well as decreased steatosis in HFD. Our results demonstrate the importance of S6K: 1. as a modulator of the hepatic response to fasting/refeeding, 2. in the development of steatosis, and 3. as a key node in selective hepatic insulin resistance in obese mice
EAR: OBSERVATION OF AN ENLARGED, DISMANTABLE ANATOMICAL MODEL OF THE MIDDLE OF THE 20TH CENTURY
Authors: Le Floch-Prigent Patrice Pierre , BARBET PATRICK J.
Published: June 2021

The aim of the study was to appreciate the anatomical veracity of a teaching model of ear. The model measured 47cm in length (encumbrancy), 35cm of total length and 50cm high. It was made in plaster and brightly colored. It presented a dismantable external ear and a dismantable upper pat of the petrous pyramid. It could be separated in two parts in the frontal plane. The model was studied on the morphological point of view and photographed in its whole and for each of its five components after their separation. The sample laid on a squared, wooden basis (48cm). A label was sticken “Sciences et Pédagogie, 35 rue des petits champs, Paris 1er”. The anatomy of each component was well respected especially the one of the tympanic membrane and of the ear's ossicles. The cochleo‐vestibular apparatus was dismountable and the upper part of the cochlea also. The representation of the nerves was good but the vascular representations were limited to the internal carotid artery and to the intra‐cranial, middle meningeal artery.
A novel type of dietary and metabolic glutamate transporter (dmGLUT) plays a crucial role in megamitochondrial formation in Drosophila
Authors: Shim Myoung Sup , Kim Jin Young , Lee Kwang Hee , Jung Hee Kyoung , Carlson Bradley A. , Xu Xue-Ming , Hatfield Dolph L. , Lee Byeong Jae
Published: June 2021

Previously, we found that intracellular glutamine accumulation leads to the formation of megamitochondria (MMT) in a Drosophila cell and that two proteins, glutamine synthestase 1 and l(2)01810, play a critical role in the process. However, the function of l(2)01810 has not been elucidated. In this study, we studied the function of l(2)01810 during MMT formation. The overexpression of l(2)01810 and the inhibition of glutamine synthesis showed that l(2)01810 is involved in the accumulation of glutamate. l(2)01810 was predicted to contain transmembrane domains and was found to be localized to the plasma membrane. By using 14C‐labeled glutamate, l(2)01810 was found to uptake glutamate into Drosophila cells in a Na+‐independent manner with high affinity (Km = 69.4 μM). l(2)01810 has a conserved functional domain found usually in vesicular type glutamate transporters and Arg146 in the domain was found to play a key role in glutamate transport and MMT formation. In addition, the analysis of expression patterns of l(2)01810 in adult organs reveals that digestive system, such as the gut, Malpighian tubule and salivary gland, is primarily site where it uptakes glutamate, unlike any other known glutamate transporters which is mainly expressed in the central nervous system (CNS). These results indicate that l(2)01810 is a novel type of glutamate transporter and that glutamate uptake is a rate limiting step for MMT formation.
Inhibitory Effects of Green Pepper Water Extract on Lipoprotein Lipase activity in 3T3‐L1 Cells
Authors: Kim Kyoungkon , Lee Jaesung , Heo Jeongwon , Baek Jongmi , Kim Taehyuk , Kim Daejung , Kim Taewoo , Kim Cheonan , Ochir Sarangowa , Kanazawa Tsutomu , Choe Myeon
Published: June 2021

To investigate inhibitory effects of green pepper water extract on lipoprotein lipase activity, the extract were supplemented to 3T3‐ L1 cells and changes of lipoprotein lipase(LPL) activities were measured by reverse transcription polymerase chain reaction (RT‐PCR). We also subfractionated water extract of green pepper by using columns packed with sephadex LH20 and MCI CHP‐20. Subfractions were checked by UPLC and LPL activity was also investigated.
Effects of Capsicum L. var. annuum and Capsicum annuum L. Species Water Extracts on Lipid Metabolism‐Related Enzymes Activities in 3T3‐L1 Cell
Authors: Kim Kyoungkon , Lee Jaesung , Heo Jeongwon , Baek Jongmi , Kim Taehyuk , Kim Daejung , Kim Taewoo , Kim Cheonan , Choe Myeon
Published: June 2021

Obesity was long been known as a negative factor for improving average life span. Therefore, a therapeutic approach using natural compounds has been proposed as a novel strategy for preventing metabolic disorders. In the present study, we investigated the anti‐obesity effect of Capsicum L. var. annuum and Capsicum annuum L. species water extract supplemented to 3T3‐L1 cells. The gene expression of lipid metabolism‐related enzymes, such as lipoprotein lipase (LPL), hormone sensitive lipase (HSL), acyl CoA synthetase(ACS) and carnitine palmitoyltransferase‐1(CPT‐1), were measured by reverse transcription polymerase chain reaction (RT‐PCR).
Rhodopsin Interacts With Retinal Guanylate Cyclase; Involvement In Phototransduction
Authors: Bondarenko Vladimir
Published: June 2021

We expressed bovine Rho and intradiscal domain of retGC in HEK293 and used membrane and soluble fractions pull down assay to demonstrate interaction between these proteins. We also co‐expressed fluorescently‐tagged proteins in cultured cells to analyze their co‐localization in cell.

It has been previously shown that retGC activity in light‐exposed rod outer segment membranes was much higher than activity of the same membranes isolated under dark conditions suggesting that communication of retGC with activated rhodopsin is required for its full activation by GCAP. To study interaction between Rho and retGC we used pull down assay and colocalization experiments. Fluorescent protein‐tagged bovine Rho and intradiscal domain of retGC were expressed in HEK293 cells and membrane fraction containing Rho and soluble fraction containing intracellular domain of retGC were prepared. Membrane and soluble fractions were mixed together and upon incubation, membranes were separated from soluble fraction by centrifugation and the presence of both recombinant proteins in washed membranes has been analyzed by western blot. Intradiscal domain of retGC was present only in cell membranes containing Rho suggesting direct interaction between these proteins, which was also supported by colocalization experiments.
LGI3 increases human keratinocyte migration
Authors: Kim Dong-Seok , Jeong Yun-Mi , Kim Myo-Kyoung , Baek Kwang Jin , Kwon Nyoun Soo , Yun Hye-Young
Published: June 2021

We recently found that that leucine‐rich glioma inactivated 3 (LGI3) is expressed in human skin. However, the physiological roles of LGI3 in human keratinocytes remain unknown. In the present study, it was demonstrated that LGI3 acts as a stimulator for human HaCaT keratinocyte migration without affecting cell viability and proliferation. Western blot analysis showed that LGI3 induced FAK activation, Akt phosphorylation, and GSK‐3β phosphorylation in HaCaT cells. Further study demonstrated that LY294002, a selective PI3K inhibitor and LiCl, a selective GSK‐ 3β inhibitor abrogated the increased cell migration induced by LGI3. Next, we examined β‐catenin levels after LGI3 treatment, because the Akt‐GSK‐3β pathway is known to regulate accumulation of β‐catenin, which is an important mediator of cell migration. LGI3 treatment increased β‐catenin protein level and nuclear localization, whereas LY294002 prevented LGI3‐induced FAK and Akt activation as well as β‐catenin accumulation. Taken together, it is suggested that LGI3 stimulates HaCaT cell migration via the accumulation of β‐catenin through the Akt pathway. This work was supported by the National Research Foundation of Korea (NRF) grant funded by the Korea government (MEST) (No. 2011‐0026587).