Folic acid and pteroylpolyglutamate contents of archaebacteria.

PubMed Central

Worrell, V E; Nagle, D P

1988-01-01

Cell extracts of methanogens and the thermoacidophile Sulfolobus solfataricus contained little or no folic acid (pteroylglutamate) or pteroylpolyglutamate activity (less than 0.1 nmol/g [dry weight]). However, the halophile Halobacterium salinarum contained pteroylmono- or pteroyldiglutamates, and Halobacterium volcanii and Halobacterium halobium contained pteroyltriglutamates at levels equivalent to those in eubacteria (greater than 1 nmol/g [dry weight]). PMID:3137217

Stimulation of ATP synthesis in Halobacterium halobium R1 by light-induced or artifically created proton electrochemical potential gradients across the cell membrane.

PubMed

Danon, A; Caplan, S R

1976-01-15

The relationship between proton movement and phosphorylation in Halo-bacterium halobium R1 has been investigated under anaerobic conditions. The light-induced changes in the bacteriorhodopsin are accompanied by proton movements across the membrane which result in pH changes in the suspending medium. The initial alkaline shift is shown to be closely paralleled by (and hence correlated with) ATP synthesis. Acidification of the medium in the presence of valinomycin, under conditions of low external potassium, brings about ATP synthesis in the dark.

The taxonomic status of "Halobacterium marismortui" from the Dead Sea: a comparison with Halobacterium vallismortis

NASA Technical Reports Server (NTRS)

Oren, A.; Lau, P. P.; Fox, G. E.

1988-01-01

A Halobacterium strain, isolated by Ginzburg et al. from the Dead Sea in the late 1960's, often referred to as "Halobacterium marismortui" or "Halobacterium of the Dead Sea" (deposited in the American Type Culture Collection as ATCC 43049) was compared with Halobacterium (Haloarcula) vallismortis ATCC 29715. The strains appeared to be very closely related, as shown by the near identity of their 5S and 16S ribosomal RNA's, and a large number of other common properties. Distinct differences exist, however, in cell morphology, and in their potency to utilize different sugars and other compounds.

Transient proton inflows during illumination of anaerobic Halobacterium halobium cells

NASA Technical Reports Server (NTRS)

Helgerson, S. L.; Stoeckenius, W.

1985-01-01

The roles of bacteriorhodopsin (bR), halorhodopsin (hR), and the H(+)-ATPase in the proton uptake in intact cells are examined. The Halobacterium halobium strains and solutions utilized in the experiment, and the techniques for measuring extracellular pH changes and intracellular K(+) concentrations are described. It is observed that in Halobacterium halobium strain R1, containing bR and hR, the light-driven proton uptake is divided into three transient inflows superimposed on the larger proton outflow. Under anaerobic conditions early proton uptake consists of an inflow which can be blocked with Dio-9 and a second inflow that can be eliminated by low concentrations (less than 125 nm) of triphenyltin chloride (TPT). The effects of Dio-9 and TPT on the passive proton-hydroxyl permeability of the cell membrane are investigated. A third transient light-driven proton flow observed at later times of illumination is studied. The data reveal that the first proton inflow correlates with proton dependent ATP synthesis; the second inflow is a passive uptake through an unidentified channel in response to electrogenic chloride pumping by bR and/or hR; and the third inflow correlates with the Na(+)/H(+) antiporter function.

Identification of osmoadaptive strategies in the halophile, heterotrophic ciliate Schmidingerothrix salinarum

PubMed Central

Weinisch, Lea; Kühner, Steffen; Roth, Robin; Grimm, Maria; Roth, Tamara; Netz, Daili J. A.; Pierik, Antonio J.

2018-01-01

Hypersaline environments pose major challenges to their microbial residents. Microorganisms have to cope with increased osmotic pressure and low water activity and therefore require specific adaptation mechanisms. Although mechanisms have already been thoroughly investigated in the green alga Dunaliella salina and some halophilic yeasts, strategies for osmoadaptation in other protistan groups (especially heterotrophs) are neither as well known nor as deeply investigated as for their prokaryotic counterpart. This is not only due to the recent awareness of the high protistan diversity and ecological relevance in hypersaline systems, but also due to methodological shortcomings. We provide the first experimental study on haloadaptation in heterotrophic microeukaryotes, using the halophilic ciliate Schmidingerothrix salinarum as a model organism. We established three approaches to investigate fundamental adaptation strategies known from prokaryotes. First, proton nuclear magnetic resonance (1H-NMR) spectroscopy was used for the detection, identification, and quantification of intracellular compatible solutes. Second, ion-imaging with cation-specific fluorescent dyes was employed to analyze changes in the relative ion concentrations in intact cells. Third, the effect of salt concentrations on the catalytic performance of S. salinarum malate dehydrogenase (MDH) and isocitrate dehydrogenase (ICDH) was determined. 1H-NMR spectroscopy identified glycine betaine (GB) and ectoine (Ect) as the main compatible solutes in S. salinarum. Moreover, a significant positive correlation of intracellular GB and Ect concentrations and external salinity was observed. The addition of exogenous GB, Ect, and choline (Ch) stimulated the cell growth notably, indicating that S. salinarum accumulates the solutes from the external medium. Addition of external 13C2-Ch resulted in conversion to 13C2-GB, indicating biosynthesis of GB from Ch. An increase of external salinity up to 21% did not result

Antagonistic interactions and production of halocin antimicrobial peptides among extremely halophilic prokaryotes isolated from the solar saltern of Sfax, Tunisia.

PubMed

Ghanmi, Fadoua; Carré-Mlouka, Alyssa; Vandervennet, Manon; Boujelben, Ines; Frikha, Doniez; Ayadi, Habib; Peduzzi, Jean; Rebuffat, Sylvie; Maalej, Sami

2016-05-01

Thirty-five extremely halophilic microbial strains isolated from crystallizer (TS18) and non-crystallizer (M1) ponds in the Sfax solar saltern in Tunisia were examined for their ability to exert antimicrobial activity. Antagonistic assays resulted in the selection of eleven strains that displayed such antimicrobial activity and they were further characterized. Three cases of cross-domain inhibition (archaea/bacteria or bacteria/archaea) were observed. Four archaeal strains exerted antimicrobial activity against several other strains. Three strains, for which several lines of evidence suggested the antimicrobial activity was, at least in part, due to peptide/protein agents (Halobacterium salinarum ETD5, Hbt. salinarum ETD8, and Haloterrigena thermotolerans SS1R12), were studied further. Optimal culture conditions for growth and antimicrobial production were determined. Using DNA amplification with specific primers, sequencing and RT-PCR analysis, Hbt. salinarum ETD5 and Hbt. salinarum ETD8 were shown to encode and express halocin S8, a hydrophobic antimicrobial peptide targeting halophilic archaea. Although the gene encoding halocin H4 was amplified from the genome of Htg. thermotolerans SS1R12, no transcript could be detected and the antimicrobial activity was most likely due to multiple antimicrobial compounds. This is also the first report that points to four different strains isolated from different geographical locations with the capacity to produce identical halocin S8 proteins.

Transcription start site associated RNAs (TSSaRNAs) are ubiquitous in all domains of life.

PubMed

Zaramela, Livia S; Vêncio, Ricardo Z N; ten-Caten, Felipe; Baliga, Nitin S; Koide, Tie

2014-01-01

A plethora of non-coding RNAs has been discovered using high-resolution transcriptomics tools, indicating that transcriptional and post-transcriptional regulation is much more complex than previously appreciated. Small RNAs associated with transcription start sites of annotated coding regions (TSSaRNAs) are pervasive in both eukaryotes and bacteria. Here, we provide evidence for existence of TSSaRNAs in several archaeal transcriptomes including: Halobacterium salinarum, Pyrococcus furiosus, Methanococcus maripaludis, and Sulfolobus solfataricus. We validated TSSaRNAs from the model archaeon Halobacterium salinarum NRC-1 by deep sequencing two independent small-RNA enriched (RNA-seq) and a primary-transcript enriched (dRNA-seq) strand-specific libraries. We identified 652 transcripts, of which 179 were shown to be primary transcripts (∼7% of the annotated genome). Distinct growth-associated expression patterns between TSSaRNAs and their cognate genes were observed, indicating a possible role in environmental responses that may result from RNA polymerase with varying pausing rhythms. This work shows that TSSaRNAs are ubiquitous across all domains of life.

Resistance of the Extreme Halophile Halobacterium sp. NRC-1 to Multiple Stresses

DOE Office of Scientific and Technical Information (OSTI.GOV)

Gygli, Patrick E.; Prajapati, Surendra; DeVeaux, Linda C.

2009-03-10

The model Archaeon Halobacterium sp. NRC-1 is an extreme halophile known for its resistance to multiple stressors, including electron-beam and ultraviolet radiation. It is a well-developed system with a completely sequenced genome and extensive post-genomic tools for the study of a variety of biological processes. To further understand the mechanisms of Halobacterium's, radiation resistance, we previously reported the selection for multiple independent highly resistant mutants using repeated exposure to high doses of 18-20 MeV electrons using a medical S-band Linac. Molecular analysis of the transcriptional profile of several of these mutants revealed a single common change: upregulation of the rfa3more » operon. These genes encode proteins homologous to the subunits of eukaryotic Replication Protein A (RPA), a DNA binding protein with major roles in DNA replication, recombination, and repair. This operon has also been implicated in a somewhat lesser role in resistance of wild type Halobacterium to ultraviolet radiation, suggesting common mechanisms for resistance. To further understand the mechanism of radiation resistance in the mutant strains, we measured the survival after exposure to both electron-beam and ultraviolet radiation, UV-A, B, and C All mutant strains showed increased resistance to electrons when compared with the parent. However, the mutant strains do not display increased UV resistance, and in one case is more sensitive than the parent strain. Thus, the protective role of increased RPA expression within a cell may be specific to the DNA damage caused by the different physical effects induced by high energy electron-beam radiation.« less

Conservation of chromosomal arrangement among three strains of the genetically unstable archaeon Halobacterium salinarium.

PubMed Central

Hackett, N R; Bobovnikova, Y; Heyrovska, N

1994-01-01

Phenotypic variants of Halobacterium salinarium NRC-1 arise at a frequency of 10(-2). These result from transpositions of halobacterial insertion sequences and rearrangements mediated by halobacterial insertion sequences. We have tested the hypothesis that such mutations are confined to only a portion of the genome by comparing the chromosomal restriction map of H. salinarium NRC-1 and that of the derivative S9, which was made in 1969. The two chromosomes were mapped by using two-dimensional pulsed-field gel electrophoresis and the restriction enzymes AflII, AseI, and DraI. A comparison of the two deduced maps showed a domain of about 210 kbp to be subject to many rearrangements, including an inversion in S9 relative to NRC-1. However, the rest of the chromosome was conserved among NRC-1, S9, and an independent Halobacterium isolate, GRB, previously mapped by St. Jean et al. (A. St. Jean, B. A. Trieselmann, and R. L. Charlebois, Nucleic Acids Res. 22:1476-1483, 1994). This concurs with data from eubacteria suggesting strong selective forces maintaining gene order even in the face of rearrangement events occurring at a high frequency. Images PMID:8002597

Conservation of chromosomal arrangement among three strains of the genetically unstable archaeon Halobacterium salinarium.

PubMed

Hackett, N R; Bobovnikova, Y; Heyrovska, N

1994-12-01

Phenotypic variants of Halobacterium salinarium NRC-1 arise at a frequency of 10(-2). These result from transpositions of halobacterial insertion sequences and rearrangements mediated by halobacterial insertion sequences. We have tested the hypothesis that such mutations are confined to only a portion of the genome by comparing the chromosomal restriction map of H. salinarium NRC-1 and that of the derivative S9, which was made in 1969. The two chromosomes were mapped by using two-dimensional pulsed-field gel electrophoresis and the restriction enzymes AflII, AseI, and DraI. A comparison of the two deduced maps showed a domain of about 210 kbp to be subject to many rearrangements, including an inversion in S9 relative to NRC-1. However, the rest of the chromosome was conserved among NRC-1, S9, and an independent Halobacterium isolate, GRB, previously mapped by St. Jean et al. (A. St. Jean, B. A. Trieselmann, and R. L. Charlebois, Nucleic Acids Res. 22:1476-1483, 1994). This concurs with data from eubacteria suggesting strong selective forces maintaining gene order even in the face of rearrangement events occurring at a high frequency.

Prototype Protein-Based Three-Dimensional Memory

DTIC Science & Technology

2003-01-01

9 Figure 3.2: Hypothetical mutational landscape ...to explore the genetic mutational landscape of a protein without any a priori knowledge of structure- function relationships. As such, it explores...native organism, Halobacterium salinarum, the protein acts as a photosynthetic sunlight to chemical energy transducer. Through several billion years of

Halobacterium saccharovorum sp. nov., a carbohydrate-metabolizing, extremely halophilic bacterium

NASA Technical Reports Server (NTRS)

Tomlinson, G. A.; Hochstein, L. I.

1976-01-01

The previously described extremely halophilic bacterium, strain M6, metabolizes a variety of carbohydrates with the production of acid. In addition, the organism produces nitrite (but no gas) from nitrate, is motile, and grows most rapidly at about 50 C. These characteristics distinguish it from all previously described halophilic bacteria in the genus Halobacterium. It is suggested that it be designated as a new species, Halobacterium saccharovorum.

Bacterioruberin and salinixanthin carotenoids of extremely halophilic Archaea and Bacteria: A Raman spectroscopic study

NASA Astrophysics Data System (ADS)

Jehlička, J.; Edwards, H. G. M.; Oren, A.

2013-04-01

Laboratory cultures of a number of red extremely halophilic Archaea (Halobacterium salinarum strains NRC-1 and R1, Halorubrum sodomense, Haloarcula valismortis) and of Salinibacter ruber, a red extremely halophilic member of the Bacteria, have been investigated by Raman spectroscopy using 514.5 nm excitation to characterize their carotenoids. The 50-carbon carotenoid α-bacterioruberin was detected as the major carotenoid in all archaeal strains. Raman spectroscopy also detected bacterioruberin as the main pigment in a red pellet of cells collected from a saltern crystallizer pond. Salinibacter contains the C40-carotenoid acyl glycoside salinixanthin (all-E, 2'S)-2'-hydroxy-1'-[6-O-(methyltetradecanoyl)-β-D-glycopyranosyloxy]-3',4'-didehydro-1',2'-dihydro-β,ψ-carotene-4-one), for which the Raman bands assignments of are given here for the first time.

On archaebacterial ATPase from Halobacterium saccharovorum

NASA Technical Reports Server (NTRS)

Kristjansson, H.; Ponnamperuma, C.; Hochstein, L.; Altekar, W.

1984-01-01

The energy transducing ATPase from Halobacterium saccharovorum was studied in order to define the origin of energy transducing systems. The ATPase required high salt concentration (4M NaCl) for activity; activity was rapidly lost when NaCl was below 1 Molar. At low salt concentration, the membrane bound ATPase activity could be stabilized in presence of spermine. However, following solubilization spermine was ineffective. Furthermore, F1 ATPase activity was stabilized by ammonium sulfate even when the NaCl concentration was less than 1 Molar. These studies suggest that stabilization by hydrophobic interactions preceded ionic ones in the evolution of the energy transducing ATPases.

Inference of Expanded Lrp-Like Feast/Famine Transcription Factor Targets in a Non-Model Organism Using Protein Structure-Based Prediction

PubMed Central

Ashworth, Justin; Plaisier, Christopher L.; Lo, Fang Yin; Reiss, David J.; Baliga, Nitin S.

2014-01-01

Widespread microbial genome sequencing presents an opportunity to understand the gene regulatory networks of non-model organisms. This requires knowledge of the binding sites for transcription factors whose DNA-binding properties are unknown or difficult to infer. We adapted a protein structure-based method to predict the specificities and putative regulons of homologous transcription factors across diverse species. As a proof-of-concept we predicted the specificities and transcriptional target genes of divergent archaeal feast/famine regulatory proteins, several of which are encoded in the genome of Halobacterium salinarum. This was validated by comparison to experimentally determined specificities for transcription factors in distantly related extremophiles, chromatin immunoprecipitation experiments, and cis-regulatory sequence conservation across eighteen related species of halobacteria. Through this analysis we were able to infer that Halobacterium salinarum employs a divergent local trans-regulatory strategy to regulate genes (carA and carB) involved in arginine and pyrimidine metabolism, whereas Escherichia coli employs an operon. The prediction of gene regulatory binding sites using structure-based methods is useful for the inference of gene regulatory relationships in new species that are otherwise difficult to infer. PMID:25255272

Inference of expanded Lrp-like feast/famine transcription factor targets in a non-model organism using protein structure-based prediction.

PubMed

Ashworth, Justin; Plaisier, Christopher L; Lo, Fang Yin; Reiss, David J; Baliga, Nitin S

2014-01-01

Widespread microbial genome sequencing presents an opportunity to understand the gene regulatory networks of non-model organisms. This requires knowledge of the binding sites for transcription factors whose DNA-binding properties are unknown or difficult to infer. We adapted a protein structure-based method to predict the specificities and putative regulons of homologous transcription factors across diverse species. As a proof-of-concept we predicted the specificities and transcriptional target genes of divergent archaeal feast/famine regulatory proteins, several of which are encoded in the genome of Halobacterium salinarum. This was validated by comparison to experimentally determined specificities for transcription factors in distantly related extremophiles, chromatin immunoprecipitation experiments, and cis-regulatory sequence conservation across eighteen related species of halobacteria. Through this analysis we were able to infer that Halobacterium salinarum employs a divergent local trans-regulatory strategy to regulate genes (carA and carB) involved in arginine and pyrimidine metabolism, whereas Escherichia coli employs an operon. The prediction of gene regulatory binding sites using structure-based methods is useful for the inference of gene regulatory relationships in new species that are otherwise difficult to infer.

Growth-Phase-Specific Modulation of Cell Morphology and Gene Expression by an Archaeal Histone Protein.

PubMed

Dulmage, Keely A; Todor, Horia; Schmid, Amy K

2015-09-08

In all three domains of life, organisms use nonspecific DNA-binding proteins to compact and organize the genome as well as to regulate transcription on a global scale. Histone is the primary eukaryotic nucleoprotein, and its evolutionary roots can be traced to the archaea. However, not all archaea use this protein as the primary DNA-packaging component, raising questions regarding the role of histones in archaeal chromatin function. Here, quantitative phenotyping, transcriptomic, and proteomic assays were performed on deletion and overexpression mutants of the sole histone protein of the hypersaline-adapted haloarchaeal model organism Halobacterium salinarum. This protein is highly conserved among all sequenced haloarchaeal species and maintains hallmark residues required for eukaryotic histone functions. Surprisingly, despite this conservation at the sequence level, unlike in other archaea or eukaryotes, H. salinarum histone is required to regulate cell shape but is not necessary for survival. Genome-wide expression changes in histone deletion strains were global, significant but subtle in terms of fold change, bidirectional, and growth phase dependent. Mass spectrometric proteomic identification of proteins from chromatin enrichments yielded levels of histone and putative nucleoid-associated proteins similar to those of transcription factors, consistent with an open and transcriptionally active genome. Taken together, these data suggest that histone in H. salinarum plays a minor role in DNA compaction but important roles in growth-phase-dependent gene expression and regulation of cell shape. Histone function in haloarchaea more closely resembles a regulator of gene expression than a chromatin-organizing protein like canonical eukaryotic histone. Histones comprise the major protein component of eukaryotic chromatin and are required for both genome packaging and global regulation of expression. The current paradigm maintains that archaea whose genes encode

Mechanism of Dissolution of Envelopes of the Extreme Halophile Halobacterium cutirubrum1

PubMed Central

Onishi, H.; Kushner, D. J.

1966-01-01

Onishi, H. (National Research Council, Ottawa, Ontario, Canada), and D. J. Kushner. Mechanism of dissolution of envelopes of the extreme halophile Halobacterium cutirubrum. J. Bacteriol. 91:646–652. 1966.—Envelopes of Halobacterium cutirubrum dissolved rapidly in media of low ionic strength. Heating partially inhibited breakdown, probably because of nonspecific protein coagulation rather than inactivation of a lytic enzyme(s). Dissolution of envelopes in water did not involve splitting of peptide bonds or protein-lipid bonds, or any extensive breakdown of carbohydrate polymers. Dissolution was increased by alcohols and urea, even at high salt concentrations, but was not affected by metabolic inhibitors. Thus, no evidence was found for a dilution-activated lytic enzyme that contributes to envelope breakdown. Cells of H. cutirubrum were stable in 2 m NaCl, but lysis occurred in 2 m KCl or NH4Cl. This lysis did not involve an extensive breakdown of the envelope. No evidence for different sites of Na+, K+, and NH4+ action was obtained from the pattern of release of envelope constituents in different concentrations of these salts. Ultracentrifugation studies showed that adding salts to envelopes that had been dissolved in water led to a nonspecific reaggregation of envelope material. No difference was seen between the effects of KCl and NaCl, except at 3 to 4 m concentrations where KCl caused more aggregation. The preferential effect of Na+ on intact cells is probably due to its ability specifically to prevent leakage rather than to an overall effect on envelope integrity. Images PMID:5883109

Bacterio-opsin mutants of Halobacterium halobium

PubMed Central

Betlach, Mary; Pfeifer, Felicitas; Friedman, James; Boyer, Herbert W.

1983-01-01

The bacterio-opsin (bop) gene of Halobacterium halobium R1 has been cloned with about 40 kilobases of flanking genomic sequence. The 40-kilobase segment is derived from the (G+C)-rich fraction of the chromosome and is not homologous to the major (pHH1) or minor endogenous covalently closed circular DNA species of H. halobium. A 5.1-kilobase Pst I fragment containing the bop gene was subcloned in pBR322 and a partial restriction map was determined. Defined restriction fragments of this clone were used as probes to analyze the defects associated with the bop gene in 12 bacterio-opsin mutants. Eleven out of 12 of the mutants examined had inserts ranging from 350 to 3,000 base pairs either in the bop gene or up to 1,400 base pairs upstream. The positions of the inserts were localized to four regions in the 5.1-kilobase genomic fragment: within the gene (one mutant), in a region that overlaps the 5′ end of the gene (seven mutants), and in two different upstream regions (three mutants). Two revertants of the mutant with the most distal insert had an additional insert in the same region. The polar effects of these inserts are discussed in terms of inactivation of a regulatory gene or disruption of part of a coordinately expressed operon. Given the defined nature of the bop mRNA—i.e., it has a 5′ leader sequence of three ribonucleotides—these observations indicate that the bop mRNA might be processed from a large mRNA transcript. Images PMID:16593291

Haloplanus salinarum sp. nov., an extremely halophilic archaeon isolated from a solar saltern.

PubMed

Hwang, Han-Bit; Kim, Ye-Eun; Koh, Hyeon-Woo; Song, Hye Seon; Roh, Seong Woon; Kim, So-Jeong; Nam, Seung Won; Park, Soo-Je

2017-11-01

An extremely halophilic archaeal strain SP28 T was isolated from the Gomso solar saltern, Republic of Korea. Cells of the new strain SP28 T were pleomorphic and Gram stain negative, and produced red-pigmented colonies. These grew in medium with 2.5-4.5 M NaCl (optimum 3.1 M) and 0.05-0.5 M MgCl2 (optimum 0.1 M), at 25-50 °C (optimum 37 °C) and at a pH of 6.5-8.5 (optimum pH 8.0). Mg 2+ was required for growth. A concentration of at least 2 M NaCl was required to prevent cell lysis. Polar lipids included phosphatidylglycerol, phosphatidylglycerol phosphate methyl ester, phosphatidylglycerol sulfate and one glycolipid chromatographically identical to sulfated mannosyl glucosyl diether. 16S rRNA and rpoB' gene sequence analyses showed that strain SP28 T is closely related to Haloplanus ruber R35 T (97.3 and 94.1 %, 16S rRNA and rpoB' gene sequence similarity, respectively), Haloplanus litoreus GX21 T (97.0 and 92.1 %), Haloplanus salinus YGH66 T (96.0 and 91.9 %), Haloplanus vescus RO5-8 T (95.9 and 90.9 %), Haloplanus aerogenes TBN37 T (95.6 and 90.3 %) and Haloplanus natans RE-101 T (95.3 and 89.8 %). The DNA G+C content of the novel strain SP28 T was 66.2 mol%, which is slightly higher than that of Hpn.litoreus GX21 T (65.8 mol%) and Hpn.ruber R35 T (66.0 mol%). DNA-DNA hybridization values betweenHpn.ruber R35 T and strain SP28 T and between Hpn.litoreus GX21 T and strain SP28 T were about 24.8 and 20.7 %, respectively. We conclude that strain SP28 T represents a novel species of the genus Haloplanus and propose the name Haloplanus salinarum sp. nov. The type strain is SP28 T (=JCM 31424 T =KCCM 43210 T ).

A DIETHER ANALOG OF PHOSPHATIDYL GLYCEROPHOSPHATE IN HALOBACTERIUM CUTIRUBRUM,

DTIC Science & Technology

The major phosphatide in the extremely halophilic bacterium, Halobacterium cutirubrum, was isolated by a combination of solvent fractionation...precipitation through the barium salt, and final purification as the sodium salt. Analytical and degradative data showed the phosphatide to be a...phosphatidyl glycerophosphate with two long-chain ether groups instead of fatty acid ester groups. Both long-chain groups were found to be identical and were

Acidic Ribosomal Proteins from the Extreme ’Halobacterium cutirubrum’,

DTIC Science & Technology

the extreme halophilic bacterium, Halobacterium cutirubrum. The identification of the protein moieties involved in these and other interactions in...the halophile ribosome requires a rapid and reproducible screening method for the separation, enumeration and identification of these acidic...polypeptides in the complex ribosomal protein mixtures. In this paper the authors present the results of analyses of the halophile ribosomal proteins using a

Approach toward enhancement of halophilic protease production by Halobacterium sp. strain LBU50301 using statistical design response surface methodology.

PubMed

Chuprom, Julalak; Bovornreungroj, Preeyanuch; Ahmad, Mehraj; Kantachote, Duangporn; Dueramae, Sawitree

2016-06-01

A new potent halophilic protease producer, Halobacterium sp. strain LBU50301 was isolated from salt-fermented fish samples ( budu ) and identified by phenotypic analysis, and 16S rDNA gene sequencing. Thereafter, sequential statistical strategy was used to optimize halophilic protease production from Halobacterium sp. strain LBU50301 by shake-flask fermentation. The classical one-factor-at-a-time (OFAT) approach determined gelatin was the best nitrogen source. Based on Plackett - Burman (PB) experimental design; gelatin, MgSO 4 ·7H 2 O, NaCl and pH significantly influenced the halophilic protease production. Central composite design (CCD) determined the optimum level of medium components. Subsequently, an 8.78-fold increase in corresponding halophilic protease yield (156.22 U/mL) was obtained, compared with that produced in the original medium (17.80 U/mL). Validation experiments proved the adequacy and accuracy of model, and the results showed the predicted value agreed well with the experimental values. An overall 13-fold increase in halophilic protease yield was achieved using a 3 L laboratory fermenter and optimized medium (231.33 U/mL).

Comparative genomic analysis of the Haloferax volcanii DS2 and Halobacterium salinarium GRB contig maps reveals extensive rearrangement.

PubMed Central

St Jean, A; Charlebois, R L

1996-01-01

Anonymous probes from the genome of Halobacterium salinarium GRB and 12 gene probes were hybridized to the cosmid clones representing the chromosome and plasmids of Halobacterium salinarium GRB and Haloferax volcanii DS2. The order of and pairwise distances between 35 loci uniquely cross-hybridizing to both chromosomes were analyzed in a search for conservation. No conservation between the genomes could be detected at the 15-kbp resolution used in this study. We found distinct sets of low-copy-number repeated sequences in the chromosome and plasmids of Halobacterium salinarium GRB, indicating some degree of partitioning between these replicons. We propose alternative courses for the evolution of the haloarchaeal genome: (i) that the majority of genomic differences that exist between genera came about at the inception of this group or (ii) that the differences have accumulated over the lifetime of the lineage. The strengths and limitations of investigating these models through comparative genomic studies are discussed. PMID:8682791

A light-driven proton pump from Haloterrigena turkmenica: Functional expression in Escherichia coli membrane and coupling with a H{sup +} co-transporter

DOE Office of Scientific and Technical Information (OSTI.GOV)

Kamo, Naoki; Hashiba, Tsuyoshi; Kikukawa, Takashi

2006-03-10

A gene encoding putative retinal protein was cloned from Haloterrigena turkmenica (JCM9743). The deduced amino acid sequence was most closely related to that of deltarhodopsin, which functions as a light-driven H{sup +} pump and was identified in a novel strain Haloterrigena sp. arg-4 (K. Ihara, T. Uemura, I. Katagiri, T. Kitajima-Ihara, Y. Sugiyama, Y. Kimura, Y. Mukohata, Evolution of the archaeal rhodopsins: Evolution rate changes by gene duplication and functional differentiation, J. Mol. Biol. 285 (1999) 163-174. GenBank Accession No. AB009620). Thus, we called the present protein H. turkmenica deltarhodopsin (HtdR) in this report. Differing from the Halobacterium salinarum bacteriorhodopsinmore » (bR), functional expression of HtdR was achieved in Escherichia coli membrane with a high yield of 10-15mg protein/L culture. The photocycle of purified HtdR was similar to that of bR. The photo-induced electrogenic proton pumping activity of HtdR was verified. We co-expressed both HtdR and EmrE, a proton-coupled multi-drug efflux transporter in E. coli, and the cells successfully extruded ethidium, a substrate of EmrE, on illumination.« less

Mapping flexible protein domains at subnanometer resolution with the atomic force microscope.

PubMed

Müller, D J; Fotiadis, D; Engel, A

1998-06-23

The mapping of flexible protein domains with the atomic force microscope is reviewed. Examples discussed are the bacteriorhodopsin from Halobacterium salinarum, the head-tail-connector from phage phi29, and the hexagonally packed intermediate layer from Deinococcus radiodurans which all were recorded in physiological buffer solution. All three proteins undergo reversible structural changes that are reflected in standard deviation maps calculated from aligned topographs of individual protein complexes. Depending on the lateral resolution (up to 0.8 nm) flexible surface regions can ultimately be correlated with individual polypeptide loops. In addition, multivariate statistical classification revealed the major conformations of the protein surface.

Lipid-protein stoichiometries in a crystalline biological membrane: NMR quantitative analysis of the lipid extract of the purple membrane.

PubMed

Corcelli, Angela; Lattanzio, Veronica M T; Mascolo, Giuseppe; Papadia, Paride; Fanizzi, Francesco

2002-01-01

The lipid/protein stoichiometries of a naturally crystalline biological membrane, the purple membrane (PM) of Halobacterium salinarum, have been obtained by a combination of (31)P- and (1)H-NMR analyses of the lipid extract. In total, 10 lipid molecules per retinal were found to be present in the PM lipid extract: 2-3 molecules of phosphatidylglycerophosphate methyl ester (PGP-Me), 3 of glycolipid sulfate, 1 of phosphatidylglycerol, 1 of archaeal glycocardiolipin (GlyC), 2 of squalene plus minor amounts of phosphatidylglycerosulfate (PGS) and bisphosphatidylglycerol (archaeal cardiolipin) (BPG) and a negligible amount of vitamin MK8. The novel data of the present study are necessary to identify the lipids in the electron density map, and to shed light on the structural relationships of the lipid and protein components of the PM.

Halobacterium denitrificans sp. nov. - An extremely halophilic denitrifying bacterium

NASA Technical Reports Server (NTRS)

Tomlinson, G. A.; Jahnke, L. L.; Hochstein, L. I.

1986-01-01

Halobacterium denitrificans was one of several carbohydrate-utilizing, denitrifying, extremely halophilic bacteria isolated by anaerobic enrichment in the presence of nitrate. Anaerobic growth took place only when nitrate (or nitrite) was present and was accompanied by the production of dinitrogen. In the presence of high concentrations of nitrate (i.e., 0.5 percent), nitrous oxide and nitrite were also detected. When grown aerobically in a mineral-salts medium containing 0.005 percent yeast extract, H. denitrificans utilized a variety of carbohydrates as sources of carbon and energy. In every case, carbohydrate utilization was accompanied by acid production.

Halobacterium denitrificans sp. nov., an extremely halophilic denitrifying bacterium

NASA Technical Reports Server (NTRS)

Tomlinson, G. A.; Jahnke, L. L.; Hochstein, L. I.

1986-01-01

Halobacterium denitrificans was one of several carbohydrate-utilizing, denitrifying, extremely halophilic bacteria isolated by anaerobic enrichment in the presence of nitrate. Anaerobic growth took place only when nitrate (or nitrite) was present and was accompanied by the production of dinitrogen. In the presence of high concentrations of nitrate (i.e., 0.5 percent), nitrous oxide and nitrite were also detected. When grown aerobically in a mineral-salts medium containing 0.005 percent yeast extract, H. denitrificans utilized a variety of carbohydrates as sources of carbon and energy. In every case, carbohydrate utilization was accompanied by acid production.

ATP synthesis in Halobacterium saccharovorum: evidence that synthesis may be catalysed by an F0F1-ATP synthase

NASA Technical Reports Server (NTRS)

Hochstein, L. I.

1992-01-01

Halobacterium saccharovorum synthesized ATP in response to a pH shift from 8 to 6.2. Synthesis was inhibited by carbonyl cyanide m-chloro-phenylhydrazone, dicyclohexylcarbodiimide, and azide. Nitrate, an inhibitor of the membrane-bound ATPase previously isolated from this organism, did not inhibit ATP synthesis. N-Ethymaleimide, which also inhibited this ATPase, stimulated the production of ATP. These observations suggested that H. saccharovorum synthesized and hydrolysed ATP using different enzymes and that the vacuolar-like ATPase activity previously described in H. saccharovorum was an ATPase whose function is yet to be identified.

Photonic Potential of Haloarchaeal Pigment Bacteriorhodopsin for Future Electronics: A Review.

PubMed

Ashwini, Ravi; Vijayanand, S; Hemapriya, J

2017-08-01

Haloarchaea are known for its adaptation in extreme saline environment. Halophilic archaea produces carotenoid pigments and proton pumps to protect them from extremes of salinity. Bacteriorhodopsin (bR) is a light-driven proton pump that resides in the membrane of haloarchaea Halobacterium salinarum. The photocycle of Bacteriorhodopsin passes through several states from K to O, finally liberating ATP for host's survival. Extensive studies on Bacteriorhodopsin photocycle has provided in depth knowledge on their sequential mechanism of converting solar energy into chemical energy inside the cell. This ability of Bacteriorhodopsin to harvest sunlight has now been experimented to exploit the unexplored and extensively available solar energy in various biotechnological applications. Currently, bacteriorhodopsin finds its importance in dye-sensitized solar cell (DSSC), logic gates (integrated circuits, IC's), optical switching, optical memories, storage devices (random access memory, RAM), biosensors, electronic sensors and optical microcavities. This review deals with the optical and electrical applications of the purple pigment Bacteriorhodopsin.

Flagellar filament bio-templated inorganic oxide materials - towards an efficient lithium battery anode

NASA Astrophysics Data System (ADS)

Beznosov, Sergei N.; Veluri, Pavan S.; Pyatibratov, Mikhail G.; Chatterjee, Abhijit; Macfarlane, Douglas R.; Fedorov, Oleg V.; Mitra, Sagar

2015-01-01

Designing a new generation of energy-intensive and sustainable electrode materials for batteries to power a variety of applications is an imperative task. The use of biomaterials as a nanosized structural template for these materials has the potential to produce hitherto unachievable structures. In this report, we have used genetically modified flagellar filaments of the extremely halophilic archaea species Halobacterium salinarum to synthesize nanostructured iron oxide composites for use as a lithium-ion battery anode. The electrode demonstrated a superior electrochemical performance compared to existing literature results, with good capacity retention of 1032 mAh g-1 after 50 cycles and with high rate capability, delivering 770 mAh g-1 at 5 A g-1 (~5 C) discharge rate. This unique flagellar filament based template has the potential to provide access to other highly structured advanced energy materials in the future.

Flagellar filament bio-templated inorganic oxide materials - towards an efficient lithium battery anode.

PubMed

Beznosov, Sergei N; Veluri, Pavan S; Pyatibratov, Mikhail G; Chatterjee, Abhijit; MacFarlane, Douglas R; Fedorov, Oleg V; Mitra, Sagar

2015-01-13

Designing a new generation of energy-intensive and sustainable electrode materials for batteries to power a variety of applications is an imperative task. The use of biomaterials as a nanosized structural template for these materials has the potential to produce hitherto unachievable structures. In this report, we have used genetically modified flagellar filaments of the extremely halophilic archaea species Halobacterium salinarum to synthesize nanostructured iron oxide composites for use as a lithium-ion battery anode. The electrode demonstrated a superior electrochemical performance compared to existing literature results, with good capacity retention of 1032 mAh g(-1) after 50 cycles and with high rate capability, delivering 770 mAh g(-1) at 5 A g(-1) (~5 C) discharge rate. This unique flagellar filament based template has the potential to provide access to other highly structured advanced energy materials in the future.

Flagellar filament bio-templated inorganic oxide materials – towards an efficient lithium battery anode

PubMed Central

Beznosov, Sergei N.; Veluri, Pavan S.; Pyatibratov, Mikhail G.; Chatterjee, Abhijit; MacFarlane, Douglas R.; Fedorov, Oleg V.; Mitra, Sagar

2015-01-01

Designing a new generation of energy-intensive and sustainable electrode materials for batteries to power a variety of applications is an imperative task. The use of biomaterials as a nanosized structural template for these materials has the potential to produce hitherto unachievable structures. In this report, we have used genetically modified flagellar filaments of the extremely halophilic archaea species Halobacterium salinarum to synthesize nanostructured iron oxide composites for use as a lithium-ion battery anode. The electrode demonstrated a superior electrochemical performance compared to existing literature results, with good capacity retention of 1032 mAh g−1 after 50 cycles and with high rate capability, delivering 770 mAh g−1 at 5 A g−1 (~5 C) discharge rate. This unique flagellar filament based template has the potential to provide access to other highly structured advanced energy materials in the future. PMID:25583370

Light-driven solute transport in Halobacterium halobium

NASA Technical Reports Server (NTRS)

Lanyi, J. K.

1979-01-01

The cell membrane of Halobacterium halobium exhibits differential regions which contain crystalline arrays of a single kind of protein, termed bacteriorhodopsin. This bacterial retinal-protein complex resembles the visual pigment and, after the absorption of protons, translocates H(+) across the cell membrane, leading to an electrochemical gradient for protons between the inside and the outside of the cell. Thus, light is an alternate source of energy in these bacteria, in addition to terminal oxidation. The paper deals with work on light-driven transport in H. halobium with cell envelope vesicles. The discussion covers light-driven movements of H(+), Na(+), and K(+); light-driven amino acid transport; and apparent allosteric control of amino acid transport. The scheme of energy coupling in H. halobium vesicles appears simple, its quantitative details are quite complex and reveal regulatory phenomena. More knowledge is required of the way the coupling components are regulated by the ion gradients present.

Resonance energy transfer improves the biological function of bacteriorhodopsin within a hybrid material built from purple membranes and semiconductor quantum dots.

PubMed

Rakovich, Aliaksandra; Sukhanova, Alyona; Bouchonville, Nicolas; Lukashev, Evgeniy; Oleinikov, Vladimir; Artemyev, Mikhail; Lesnyak, Vladimir; Gaponik, Nikolai; Molinari, Michael; Troyon, Michel; Rakovich, Yury P; Donegan, John F; Nabiev, Igor

2010-07-14

Purple membrane (PM) from bacteria Halobacterium salinarum contains a photochromic protein bacteriorhodopsin (bR) arranged in a 2D hexagonal nanocrystalline lattice (Figure 1 ). Absorption of light by the protein-bound chromophore retinal results in pumping the protons through the PM creating an electrochemical gradient which is then used by the ATPases to energize the cellular processes. Energy conversion, photochromism, and photoelectrism are the inherent effects which are employed in many bR technical applications. bR, along with the other photosensitive proteins, is not able to deal with the excess energy of photons in UV and blue spectral region and utilizes less than 0.5% of the energy from the available incident solar light for its biological function. Here, we proceed with optimization of bR functions through the engineering of a "nanoconverter" of solar energy based on semiconductor quantum dots (QDs) tagged with the PM. These nanoconverters are able to harvest light from deep-UV to the visible region and to transfer this additionally collected energy to bR via Förster resonance energy transfer (FRET). We show that specific nanobio-optical and spatial coupling of QDs (donor) and bR retinal (acceptor) provide a means to achieve FRET with efficiency approaching 100%. We have finally demonstrated that the integration of QDs within PM significantly increases the efficiency of light-driven transmembrane proton pumping, which is the main bR biological function. This new QD-PM hybrid material will have numerous optoelectronic, photonic, and photovoltaic applications based on its energy conversion, photochromism, and photoelectrism properties.

Nucleotide-Protectable Labeling of Sulfhydryl Groups in Subunit I of the ATPhase from Halobacterium Saccharovorum

NASA Technical Reports Server (NTRS)

Sulzner, Michael; Stan-Lotter, Helga; Hochstein, Lawrence I.

1992-01-01

A membrane-bound ATPase from the archaebacterium Halobacterium saccharovorum is inhibited by N-ethyl-maleimide in a nucleotide-protectable manner. When the enzyme was incubated with N-[C-14]jethylmaleimide, the bulk of radioactivity was as- sociated with the 87,000-Da subunit (subunit 1). ATP, ADP, or AMP reduced incorporation of the inhibitor. No charge shift of subunit I was detected following labeling with N-ethylmaleimide, indicating an electroneutral reaction. The results are consistent with the selective modification of sulfhydryl groups in subunit I at or near the catalytic site and are further evidence of a resemblance between this archaebacterial ATPase and the vacuolar-type ATPases.

Irregular bilayer structure in vesicles prepared from Halobacterium cutirubrum lipids

NASA Technical Reports Server (NTRS)

Lanyi, J. K.

1974-01-01

Fluorescent probes were used to study the structure of the cell envelope of Halobacterium cutirubrum, and, in particular, to explore the effect of the heterogeneity of the lipids in this organism on the structure of the bilayers. The fluorescence polarization of perylene was followed in vesicles of unfractionated lipids and polar lipids as a function of temperature in 3.4 M solutions of NaCl, NaNO3, and KSCN, and it was found that vesicles of unfractionated lipids were more perturbed by chaotropic agents than polar lipids. The dependence of the relaxation times of perylene on temperature was studied in cell envelopes and in vesicles prepared from polar lipids, unfractionated lipids, and mixtures of polar and neutral lipids.

Light energy conservation processes in Halobacterium halobium cells

NASA Technical Reports Server (NTRS)

Bogomolni, R. A.

1977-01-01

Proton pumping driven by light or by respiration generates an electrochemical potential difference across the membrane in Halobacterium halobium. The pH changes induced by light or by respiration in cell suspensions are complicated by proton flows associated with the functioning of the cellular energy transducers. A proton-per-ATP ratio of about 3 is calculated from simultaneous measurements of phosphorylation and the proton inflow. This value is compatible with the chemiosmotic coupling hypothesis. The time course of the light-induced changes in membrane potential indicates that light-driven pumping increases a dark pre-existing potential of about 130 mV only by a small amount (20 to 30 mV). The complex kinetic features of the membrane potential changes do not closely follow those of the pH changes, which suggests that flows of ions other than protons are involved. A qualitative model consistent with the available data is presented.

Relationship of the Membrane ATPase from Halobacterium saccharovorum to Vacuolar ATPases

NASA Technical Reports Server (NTRS)

Stan-Lotter, Helga; Bowman, Emma J.; Hochstein, Lawrence I.

1991-01-01

Polyclonal antiserum against subunit A (67 kDa) of the vacuolar ATPase from Neurospora crassa reacted with subunit I (87 kDa) from a membrane ATPase of the extremely halophilic archaebacterium Halobacterium saccharovorum. The halobacterial ATPase was inhibited by nitrate and N-ethylmaleimide; the extent of the latter inhibition was diminished in the presence of adenosine di- or triphosphates. 4-Chloro-7-nitrobenzofurazan in- hibited the hatobacterial ATPase also in a nucleotide- protectable manner; the bulk of inhibitor was associated with subunit II (60 kDa). The data suggested that this halobacterial ATPase may have conserved structural features from both the vacuotar and the F-type ATPases.

A comparison of an ATPase from the archaebacterium Halobacterium saccharovorum with the F1 moiety from the Escherichia coli ATP Synthase

NASA Technical Reports Server (NTRS)

Stan-Lotter, Helga; Hochstein, Lawrence I.

1989-01-01

A purified ATPase associated with membranes from Halobacterium saccharovorum was compared with the F sub 1 moiety from the Escherichia coli ATP Synthase. The halobacterial enzyme was composed of two major (I and II) and two minor subunits (III and IV), whose molecular masses were 87 kDa, 60 kDa, 29 kDa, and 20 kDa, respectively. The isoelectric points of these subunits ranged from 4.1 to 4.8, which in the case of the subunits I and II was consistent with the presence of an excess of acidic amino acids (20 to 22 Mol percent). Peptide mapping of sodium dodecylsulfate-denatured subunits I and II showed no relationship between the primary structures of the individual halobacterial subunits or similarities to the subunits of the F sub 1 ATPase (EC 3.6.1.34) from E. coli. Trypsin inactivation of the halobacterial ATPase was accompanied by the partial degradation of the major subunits. This observation, taken in conjunction with molecular masses of the subunits and the native enzyme, was consistent with the previously proposed stoichiometry of 2:2:1:1. These results suggest that H. saccharovorum, and possibly, Halobacteria in general, possess an ATPase which is unlike the ubiquitous F sub o F sub 1 - ATP Synthase.

Light-activated amino acid transport in Halobacterium halobium envelope vesicles

NASA Technical Reports Server (NTRS)

Macdonald, R. E.; Lanyi, J. K.

1977-01-01

Vesicles prepared from Halobacterium halobium cell envelopes accumulate amino acids in response to light-induced electrical and chemical gradients. Nineteen of 20 commonly occurring amino acids have been shown to be actively accumulated by these vesicles in response to illumination or in response to an artificially created Na+ gradient. On the basis of shared common carriers the transport systems can be divided into eight classes, each responsible for the transport of one or several amino acids: arginine, lysine, histidine; asparagine, glutamine; alanine, glycine, threonine, serine; leucine, valine, isoleucine, methionine; phenylalanine, tyrosine, tryptophan; aspartate; glutamate; proline. Available evidence suggests that these carriers are symmetrical in that amino acids can be transported equally well in both directions across the vesicle membranes. A tentative working model to account for these observations is presented.

The Function of Gas Vesicles in Halophilic Archaeaand Bacteria: Theories and Experimental Evidence

PubMed Central

Oren, Aharon

2012-01-01

A few extremely halophilic Archaea (Halobacterium salinarum, Haloquadratum walsbyi, Haloferax mediterranei, Halorubrum vacuolatum, Halogeometricum borinquense, Haloplanus spp.) possess gas vesicles that bestow buoyancy on the cells. Gas vesicles are also produced by the anaerobic endospore-forming halophilic Bacteria Sporohalobacter lortetii and Orenia sivashensis. We have extensive information on the properties of gas vesicles in Hbt. salinarum and Hfx. mediterranei and the regulation of their formation. Different functions were suggested for gas vesicle synthesis: buoying cells towards oxygen-rich surface layers in hypersaline water bodies to prevent oxygen limitation, reaching higher light intensities for the light-driven proton pump bacteriorhodopsin, positioning the cells optimally for light absorption, light shielding, reducing the cytoplasmic volume leading to a higher surface-area-to-volume ratio (for the Archaea) and dispersal of endospores (for the anaerobic spore-forming Bacteria). Except for Hqr. walsbyi which abounds in saltern crystallizer brines, gas-vacuolate halophiles are not among the dominant life forms in hypersaline environments. There only has been little research on gas vesicles in natural communities of halophilic microorganisms, and the few existing studies failed to provide clear evidence for their possible function. This paper summarizes the current status of the different theories why gas vesicles may provide a selective advantage to some halophilic microorganisms. PMID:25371329

Improved purification, crystallization and primary structure of pyruvate:ferredoxin oxidoreductase from Halobacterium halobium.

PubMed

Plaga, W; Lottspeich, F; Oesterhelt, D

1992-04-01

An improved purification procedure, including nickel chelate affinity chromatography, is reported which resulted in a crystallizable pyruvate:ferredoxin oxidoreductase preparation from Halobacterium halobium. Crystals of the enzyme were obtained using potassium citrate as the precipitant. The genes coding for pyruvate:ferredoxin oxidoreductase were cloned and their nucleotide sequences determined. The genes of both subunits were adjacent to one another on the halobacterial genome. The derived amino acid sequences were confirmed by partial primary structure analysis of the purified protein. The structural motif of thiamin-diphosphate-binding enzymes was unequivocally located in the deduced amino acid sequence of the small subunit.

Light-dependent cation gradients and electrical potential in Halobacterium halobium cell envelope vesicles

NASA Technical Reports Server (NTRS)

Lanyi, J. K.; Macdonald, R. E.

1977-01-01

Vesicles can be prepared from Halobacterium halobium cell envelopes, which contain properly oriented bacteriorhodopsin and which extrude H(+) during illumination. The pH difference that is generated across the membranes is accompanied by an electrical potential of 90 to 100 mV (interior negative) and the movements of other cations. Among these is the efflux of Na(+), which proceeds against its electrochemical potential. The relationship between the size and direction of the light-induced pH gradient and the rate of depletion of Na(+) from the vesicles, as well as other evidence, suggest that the active Na(+) extrusion is facilitated by a membrane component that exchanges H(+) for Na(+) with a stoichiometry greater than 1. The gradients of H(+) and Na(+) are thus coupled to one another. The Na(+) gradient (efflux much larger than influx), which arises during illumination, plays a major role in energizing the active transport of amino acids.

Metabolism of halophilic archaea

PubMed Central

Falb, Michaela; Müller, Kerstin; Königsmaier, Lisa; Oberwinkler, Tanja; Horn, Patrick; von Gronau, Susanne; Gonzalez, Orland; Pfeiffer, Friedhelm; Bornberg-Bauer, Erich

2008-01-01

In spite of their common hypersaline environment, halophilic archaea are surprisingly different in their nutritional demands and metabolic pathways. The metabolic diversity of halophilic archaea was investigated at the genomic level through systematic metabolic reconstruction and comparative analysis of four completely sequenced species: Halobacterium salinarum, Haloarcula marismortui, Haloquadratum walsbyi, and the haloalkaliphile Natronomonas pharaonis. The comparative study reveals different sets of enzyme genes amongst halophilic archaea, e.g. in glycerol degradation, pentose metabolism, and folate synthesis. The carefully assessed metabolic data represent a reliable resource for future system biology approaches as it also links to current experimental data on (halo)archaea from the literature. Electronic supplementary material The online version of this article (doi:10.1007/s00792-008-0138-x) contains supplementary material, which is available to authorized users. PMID:18278431

Controlled In Meso Phase Crystallization – A Method for the Structural Investigation of Membrane Proteins

PubMed Central

Kubicek, Jan; Schlesinger, Ramona; Baeken, Christian; Büldt, Georg; Schäfer, Frank; Labahn, Jörg

2012-01-01

We investigated in meso crystallization of membrane proteins to develop a fast screening technology which combines features of the well established classical vapor diffusion experiment with the batch meso phase crystallization, but without premixing of protein and monoolein. It inherits the advantages of both methods, namely (i) the stabilization of membrane proteins in the meso phase, (ii) the control of hydration level and additive concentration by vapor diffusion. The new technology (iii) significantly simplifies in meso crystallization experiments and allows the use of standard liquid handling robots suitable for 96 well formats. CIMP crystallization furthermore allows (iv) direct monitoring of phase transformation and crystallization events. Bacteriorhodopsin (BR) crystals of high quality and diffraction up to 1.3 Å resolution have been obtained in this approach. CIMP and the developed consumables and protocols have been successfully applied to obtain crystals of sensory rhodopsin II (SRII) from Halobacterium salinarum for the first time. PMID:22536388

A predictive computational model of the kinetic mechanism of stimulus-induced transducer methylation and feedback regulation through CheY in archaeal phototaxis and chemotaxis.

PubMed

Streif, Stefan; Oesterhelt, Dieter; Marwan, Wolfgang

2010-03-18

Photo- and chemotaxis of the archaeon Halobacterium salinarum is based on the control of flagellar motor switching through stimulus-specific methyl-accepting transducer proteins that relay the sensory input signal to a two-component system. Certain members of the transducer family function as receptor proteins by directly sensing specific chemical or physical stimuli. Others interact with specific receptor proteins like the phototaxis photoreceptors sensory rhodopsin I and II, or require specific binding proteins as for example some chemotaxis transducers. Receptor activation by light or a change in receptor occupancy by chemical stimuli results in reversible methylation of glutamate residues of the transducer proteins. Both, methylation and demethylation reactions are involved in sensory adaptation and are modulated by the response regulator CheY. By mathematical modeling we infer the kinetic mechanisms of stimulus-induced transducer methylation and adaptation. The model (deterministic and in the form of ordinary differential equations) correctly predicts experimentally observed transducer demethylation (as detected by released methanol) in response to attractant and repellent stimuli of wildtype cells, a cheY deletion mutant, and a mutant in which the stimulated transducer species is methylation-deficient. We provide a kinetic model for signal processing in photo- and chemotaxis in the archaeon H. salinarum suggesting an essential role of receptor cooperativity, antagonistic reversible methylation, and a CheY-dependent feedback on transducer demethylation.

Nucleotide-protectable labeling of sulfhydryl groups in subunit I of the ATPase from Halobacterium saccharovorum

NASA Technical Reports Server (NTRS)

Sulzner, Michael; Stan-Lotter, Helga; Hochstein, Lawrence I.

1992-01-01

The membrane ATPase from Halobacterium saccharovorum was purified as described by Hochstein et al. (1987) and was incubated with C-14 labeled N-ethylmaleimide (NEM), with and without adenine nucleotides, to determine the effect of nucleotides on the enzyme labeling. It was found that NEM incorporates into the 87,000-Da subunit (subunit I) of the enzyme and that the conditions for enzyme modification are similar to those which result in the inhibition of the enzyme activity. The presence of ATP, ADP, and AMP was found to reduce both the inhibitor incorporation and enzyme inhibition. It was shown that the reaction involves a modification of thiol groups.

The N-terminal sequence of ribosomal protein L10 from the archaebacterium Halobacterium marismortui and its relationship to eubacterial protein L6 and other ribosomal proteins.

PubMed

Dijk, J; van den Broek, R; Nasiulas, G; Beck, A; Reinhardt, R; Wittmann-Liebold, B

1987-08-01

The amino-terminal sequence of ribosomal protein L10 from Halobacterium marismortui has been determined up to residue 54, using both a liquid- and a gas-phase sequenator. The two sequences are in good agreement. The protein is clearly homologous to protein HcuL10 from the related strain Halobacterium cutirubrum. Furthermore, a weaker but distinct homology to ribosomal protein L6 from Escherichia coli and Bacillus stearothermophilus can be detected. In addition to 7 identical amino acids in the first 36 residues in all four sequences a number of conservative replacements occurs, of mainly hydrophobic amino acids. In this common region the pattern of conserved amino acids suggests the presence of a beta-alpha fold as it occurs in ribosomal proteins L12 and L30. Furthermore, several potential cases of homology to other ribosomal components of the three ur-kingdoms have been found.

Amino acid sequences of the ribosomal proteins HL30 and HmaL5 from the archaebacterium Halobacterium marismortui.

PubMed

Hatakeyama, T; Hatakeyama, T

1990-07-06

The complete amino acid sequences of the ribosomal proteins HL30 and HmaL5 from the archaebacterium Halobacterium marismortui were determined. Protein HL30 was found to be acetylated at its N-terminal amino acid and shows homology to the eukaryotic ribosomal proteins YL34 from yeast and RL31 from rat. Protein HmaL5 was homologous to the protein L5 from Escherichia coli and Bacillus stearothermophilus as well as to YL16 from yeast. HmaL5 shows more similarities to its eukaryotic counterpart than to eubacterial ones.

Prevalence of transcription promoters within archaeal operons and coding sequences

PubMed Central

Koide, Tie; Reiss, David J; Bare, J Christopher; Pang, Wyming Lee; Facciotti, Marc T; Schmid, Amy K; Pan, Min; Marzolf, Bruz; Van, Phu T; Lo, Fang-Yin; Pratap, Abhishek; Deutsch, Eric W; Peterson, Amelia; Martin, Dan; Baliga, Nitin S

2009-01-01

Despite the knowledge of complex prokaryotic-transcription mechanisms, generalized rules, such as the simplified organization of genes into operons with well-defined promoters and terminators, have had a significant role in systems analysis of regulatory logic in both bacteria and archaea. Here, we have investigated the prevalence of alternate regulatory mechanisms through genome-wide characterization of transcript structures of ∼64% of all genes, including putative non-coding RNAs in Halobacterium salinarum NRC-1. Our integrative analysis of transcriptome dynamics and protein–DNA interaction data sets showed widespread environment-dependent modulation of operon architectures, transcription initiation and termination inside coding sequences, and extensive overlap in 3′ ends of transcripts for many convergently transcribed genes. A significant fraction of these alternate transcriptional events correlate to binding locations of 11 transcription factors and regulators (TFs) inside operons and annotated genes—events usually considered spurious or non-functional. Using experimental validation, we illustrate the prevalence of overlapping genomic signals in archaeal transcription, casting doubt on the general perception of rigid boundaries between coding sequences and regulatory elements. PMID:19536208

Prevalence of transcription promoters within archaeal operons and coding sequences.

PubMed

Koide, Tie; Reiss, David J; Bare, J Christopher; Pang, Wyming Lee; Facciotti, Marc T; Schmid, Amy K; Pan, Min; Marzolf, Bruz; Van, Phu T; Lo, Fang-Yin; Pratap, Abhishek; Deutsch, Eric W; Peterson, Amelia; Martin, Dan; Baliga, Nitin S

2009-01-01

Despite the knowledge of complex prokaryotic-transcription mechanisms, generalized rules, such as the simplified organization of genes into operons with well-defined promoters and terminators, have had a significant role in systems analysis of regulatory logic in both bacteria and archaea. Here, we have investigated the prevalence of alternate regulatory mechanisms through genome-wide characterization of transcript structures of approximately 64% of all genes, including putative non-coding RNAs in Halobacterium salinarum NRC-1. Our integrative analysis of transcriptome dynamics and protein-DNA interaction data sets showed widespread environment-dependent modulation of operon architectures, transcription initiation and termination inside coding sequences, and extensive overlap in 3' ends of transcripts for many convergently transcribed genes. A significant fraction of these alternate transcriptional events correlate to binding locations of 11 transcription factors and regulators (TFs) inside operons and annotated genes-events usually considered spurious or non-functional. Using experimental validation, we illustrate the prevalence of overlapping genomic signals in archaeal transcription, casting doubt on the general perception of rigid boundaries between coding sequences and regulatory elements.

Constitutive signaling by the phototaxis receptor sensory rhodopsin II from disruption of its protonated Schiff base–Asp-73 interhelical salt bridge

PubMed Central

Spudich, Elena N.; Zhang, Weisheng; Alam, Maqsudul; Spudich, John L.

1997-01-01

Sensory rhodopsin II (SRII) is a repellent phototaxis receptor in the archaeon Halobacterium salinarum, similar to visual pigments in its seven-helix structure and linkage of retinal to the protein by a protonated Schiff base in helix G. Asp-73 in helix C is shown by spectroscopic analysis to be a counterion to the protonated Schiff base in the unphotolyzed SRII and to be the proton acceptor from the Schiff base during photoconversion to the receptor signaling state. Coexpression of the genes encoding mutated SRII with Asn substituted for Asp-73 (D73N) and the SRII transducer HtrII in H. salinarum cells results in a 3-fold higher swimming reversal frequency accompanied by demethylation of HtrII in the dark, showing that D73N SRII produces repellent signals in its unphotostimulated state. Analogous constitutive signaling has been shown to be produced by the similar neutral residue substitution of the Schiff base counterion and proton acceptor Glu-113 in human rod rhodopsin. The interpretation for both seven-helix receptors is that light activation of the wild-type protein is caused primarily by photoisomerization-induced transfer of the Schiff base proton on helix G to its primary carboxylate counterion on helix C. Therefore receptor activation by helix C–G salt-bridge disruption in the photoactive site is a general mechanism in retinylidene proteins spanning the vast evolutionary distance between archaea and humans. PMID:9144172

Apparent cooperativity of amino acid transport in Halobacterium halobium - Effect of electrical potential

NASA Technical Reports Server (NTRS)

Lanyi, J. K.

1978-01-01

Active serine accumulation in cell envelope vesicles from Halobacterium halobium proceeds by co-transport with Na(+) and can be induced by either transmembrane electrical potential or transmembrane Na(+) concentration difference. It was shown earlier that in the former case the initial transport rate is a fourth-power function of the magnitude of the electrochemical potential difference of sodium ions, and in the latter, a second-power function. A possible interpretation of this finding is cooperativity of sodium-transporting sites in the transport carrier. When both kinds of driving force are imposed simultaneously on the vesicles, fourth-power dependence on the total potential difference of sodium ions is obtained, suggesting that the transport carrier is regulated by the electrical potential. Heat treatment of the vesicles at 48 C partially inactivates transport and abolishes this effect of the electrical potential.

Transcriptome changes and cAMP oscillations in an archaeal cell cycle.

PubMed

Baumann, Anke; Lange, Christian; Soppa, Jörg

2007-06-11

The cell cycle of all organisms includes mass increase by a factor of two, replication of the genetic material, segregation of the genome to different parts of the cell, and cell division into two daughter cells. It is tightly regulated and typically includes cell cycle-specific oscillations of the levels of transcripts, proteins, protein modifications, and signaling molecules. Until now cell cycle-specific transcriptome changes have been described for four eukaryotic species ranging from yeast to human, but only for two prokaryotic species. Similarly, oscillations of small signaling molecules have been identified in very few eukaryotic species, but not in any prokaryote. A synchronization procedure for the archaeon Halobacterium salinarum was optimized, so that nearly 100% of all cells divide in a time interval that is 1/4th of the generation time of exponentially growing cells. The method was used to characterize cell cycle-dependent transcriptome changes using a genome-wide DNA microarray. The transcript levels of 87 genes were found to be cell cycle-regulated, corresponding to 3% of all genes. They could be clustered into seven groups with different transcript level profiles. Cluster-specific sequence motifs were detected around the start of the genes that are predicted to be involved in cell cycle-specific transcriptional regulation. Notably, many cell cycle genes that have oscillating transcript levels in eukaryotes are not regulated on the transcriptional level in H. salinarum. Synchronized cultures were also used to identify putative small signaling molecules. H. salinarum was found to contain a basal cAMP concentration of 200 microM, considerably higher than that of yeast. The cAMP concentration is shortly induced directly prior to and after cell division, and thus cAMP probably is an important signal for cell cycle progression. The analysis of cell cycle-specific transcriptome changes of H. salinarum allowed to identify a strategy of transcript level

Extremely halophilic archaea from ancient salt sediments and their possible survival in halite fluid inclusions

NASA Astrophysics Data System (ADS)

Stan-Lotter, H.; Fendrihan, S.; Gerbl, F. W.; Dornmayr-Pfaffenhuemer, M.; Frethem, C.

2008-09-01

Halophilic archaebacteria (haloarchaea) thrive in environments with salt concentrations approaching saturation, such as natural brines, marine solar salterns and alkaline salt lakes; they have also been isolated from ancient subsurface salt sediments of great geological age (195-280 million years) and some of those strains were described as novel species (1). The cells survived perhaps while being enclosed within small fluid inclusions in the halite. The characterization of subsurface microbial life is of astrobiological relevance since extraterrestrial halite has been detected and since microbial life on Mars, if existent, may have retreated into the subsurface. We attempted to simulate the embedding process of extremely halophilic archaea and to analyse any cellular changes which might occur. When enclosing haloarchaea in laboratory grown halite, cells accumulated preferentially in fluid inclusions, as could be demonstrated by pre-staining with fluorescent dyes. With increased time of embedding, rod-shaped cells of Halobacterium salinarum strains were found to assume roundish morphologies. Upon dissolution of the salt crystals, these spheres were stable and viable for months when kept in buffers containing 4 M NaCl. Scanning electron microscopy (SEM) following fixation with glutaraldehyde suggested a potentially gradual transformation from rods to spheres. This notion was supported by fluorescence microscopy of Halobacterium cells, following embedding in halite and staining with SYTO 9. One-dimensional protein patterns of rods and spheres, following SDS polyacrylamide gel electrophoresis, were similar except that the S-layer protein appeared reduced by about 15 - 20 % in spheres. The reddish-orange pigmentation of spheres was much lighter compared to that of rod-shaped cells, suggesting lowered concentrations of carotenoids; this was confirmed by extraction and spectrometry of pigments. The data suggested that Halobacterium cells are capable of forming specific

Inhibition of the ATPase from Halobacterium Saccharovorum by Thiol Inhibitors: Evidence for the Presence of More Than One Essential Cysteinyl Residue

NASA Technical Reports Server (NTRS)

Hochstein, Lawrence I.; Emrich, Errol; Stan-Lotter, Helga; DeVincenzi, Donald L. (Technical Monitor)

1995-01-01

The vacuolar-like ATPase from Halobacterium saccha vorum is inhibited by N-ethylmaleimide and p-chloromercudphenylsulfonate. The failure of adenine nucleotides to protect against p-chloromercuriphenyisulfonate inhibition, of p-chloromercuriphenylsulfonate to protect against N-ethylmaleimide inhibition, and the difference in the temperature dependence of inactivation infers that the enzyme contains at least two thiols that are essential for enzyme activity. CNBr cleavage of C-14-N-ethylmaleimide labeled subunit results in two radioactive peptides that locates the N-ethylmaleimide-reactive cysteinyl residue as cysteine-262 in the H. salinarium sequence.

Using a portable Raman spectrometer to detect carotenoids of halophilic prokaryotes in synthetic inclusions in NaCl, KCl, and sulfates.

PubMed

Jehlička, Jan; Culka, Adam; Mana, Lilly; Oren, Aharon

2018-05-03

Cell suspensions of the haloarchaea Halorubrum sodomense and Halobacterium salinarum and the extremely halophilic bacterium Salinibacter ruber (Bacteroidetes) in saturated solutions of chlorides and sulfates (NaCl, KCl, MgSO 4 ·7H 2 O, K 2 SO 4 , and (NH 4 )Al(SO 4 ) 2 ·12H 2 O) were left to evaporate to produce micrometric inclusions in laboratory-grown crystals. Raman spectra of these pinkish inclusions were obtained using a handheld Raman spectrometer with green excitation (532 nm). This portable instrument does not include any microscopic tool. Acceptable Raman spectra of carotenoids were obtained in the range of 200-4000 cm -1 . This detection achievement was related to the mode of illumination and collection of scattered light as well as due to resonance Raman enhancement of carotenoid signals under green excitation. The position of diagnostic Raman carotenoid bands corresponds well to those specific carotenoids produced by a given halophile. To our best knowledge, this is the first study of carotenoids included in the laboratory in crystalline chlorides and sulfates, using a miniature portable Raman spectrometer. Graphical abstract ᅟ.

Isolation, characterization and exploring biotechnological potential of halophilic archaea from salterns of western India.

PubMed

Singh, Aparna; Singh, Anil Kumar

2018-01-01

Thirteen halophilic archaea were isolated from Kandla and Bhayander salt pans. These isolates were grouped into three different genera Halobacterium, Haloferax and Haloarcula based on morphological and biochemical characterization, polar lipid analysis, Amplified 16S rDNA restriction analysis (ARDRA) and 16S rDNA sequence analysis. Biochemical characterization suggested the ability of isolates to produce protease, amylase and poly-hydroxybutyrate (PHB) indicating their biotechnological potential. The isolates were further screened for the amount of extracellular protease produced. Halobacterium sp. SP1(1) showed significant protease production compared to other isolates. Protease producing ability of the isolate was influenced by several factors such as NaCl concentration, type of protein source, metal ions and surfactants, and presence of amino acid supplements in the production medium. Soybean flour, FeCl 3 and dicotylsulfosuccinate were found to increase protease production by 2.36, 1.54 and 1.26 folds, respectively compared to production in basal medium. Effect of organic solvents used in paints (n-decane, n-undecane and n-dodecane) was also investigated on protease production by the isolate. Protease production by Halobacterium sp. SP1(1) was enhanced by 1.2 folds in presence of n-decane compared to control. Furthermore, the ability of isolate to hydrolyse fish protein was investigated using three different edible fishes (Pomfret, Flat fish and Seer fish) as sole protein source. Pomfret was found to be a good protein source for protease production by the isolate. These results revealed that Halobacterium sp. SP1(1) may have potential for paint-based antifouling coating preparations and fish sauce preparation by virtue of its extracellular protease.

Primary structures of ribosomal proteins from the archaebacterium Halobacterium marismortui and the eubacterium Bacillus stearothermophilus.

PubMed

Arndt, E; Scholzen, T; Krömer, W; Hatakeyama, T; Kimura, M

1991-06-01

Approximately 40 ribosomal proteins from each Halobacterium marismortui and Bacillus stearothermophilus have been sequenced either by direct protein sequence analysis or by DNA sequence analysis of the appropriate genes. The comparison of the amino acid sequences from the archaebacterium H marismortui with the available ribosomal proteins from the eubacterial and eukaryotic kingdoms revealed four different groups of proteins: 24 proteins are related to both eubacterial as well as eukaryotic proteins. Eleven proteins are exclusively related to eukaryotic counterparts. For three proteins only eubacterial relatives-and for another three proteins no counterpart-could be found. The similarities of the halobacterial ribosomal proteins are in general somewhat higher to their eukaryotic than to their eubacterial counterparts. The comparison of B stearothermophilus proteins with their E coli homologues showed that the proteins evolved at different rates. Some proteins are highly conserved with 64-76% identity, others are poorly conserved with only 25-34% identical amino acid residues.

The primary structures of ribosomal proteins L16, L23 and L33 from the archaebacterium Halobacterium marismortui.

PubMed

Hatakeyama, T; Hatakeyama, T; Kimura, M

1988-11-21

The complete amino acid sequences of ribosomal proteins L16, L23 and L33 from the archaebacterium Halobacterium marismortui were determined. The sequences were established by manual sequencing of peptides produced with several proteases as well as by cleavage with dilute HCl. Proteins L16, L23 and L33 consist of 119, 154 and 69 amino acid residues, and their molecular masses are 13,538, 16,812 and 7620 Da, respectively. The comparison of their sequences with those of ribosomal proteins from other organisms revealed that L23 and L33 are related to eubacterial ribosomal proteins from Escherichia coli and Bacillus stearothermophilus, while protein L16 was found to be homologous to a eukaryotic ribosomal protein from yeast. These results provide information about the special phylogenetic position of archaebacteria.

Prospects for robust biocatalysis: engineering of novel specificity in a halophilic amino acid dehydrogenase.

PubMed

Munawar, Nayla; Engel, Paul C

2013-01-01

Heat- and solvent-tolerant enzymes from halophiles, potentially important industrially, offer a robust framework for protein engineering, but few solved halophilic structures exist to guide this. Homology modelling has guided mutations in glutamate dehydrogenase (GDH) from Halobacterium salinarum to emulate conversion of a mesophilic GDH to a methionine dehydrogenase. Replacement of K89, A163 and S367 by leucine, glycine and alanine converted halophilic GDH into a dehydrogenase accepting L-methionine, L-norleucine and L-norvaline as substrates. Over-expression in the halophilic expression host Haloferax volcanii and three-step purification gave ~98 % pure protein exhibiting maximum activity at pH 10. This enzyme also showed enhanced thermostability and organic solvent tolerance even at 70 °C, offering a biocatalyst resistant to harsh industrial environments. To our knowledge, this is the first reported amino acid specificity change engineered in a halophilic enzyme, encouraging use of mesophilic models to guide engineering of novel halophilic biocatalysts for industrial application. Calibrated gel filtration experiments show that both the mutant and the wild-type enzyme are stable hexamers.

Coordination of frontline defense mechanisms under severe oxidative stress.

PubMed

Kaur, Amardeep; Van, Phu T; Busch, Courtney R; Robinson, Courtney K; Pan, Min; Pang, Wyming Lee; Reiss, David J; DiRuggiero, Jocelyne; Baliga, Nitin S

2010-07-01

Complexity of cellular response to oxidative stress (OS) stems from its wide-ranging damage to nucleic acids, proteins, carbohydrates, and lipids. We have constructed a systems model of OS response (OSR) for Halobacterium salinarum NRC-1 in an attempt to understand the architecture of its regulatory network that coordinates this complex response. This has revealed a multi-tiered OS-management program to transcriptionally coordinate three peroxidase/catalase enzymes, two superoxide dismutases, production of rhodopsins, carotenoids and gas vesicles, metal trafficking, and various other aspects of metabolism. Through experimental validation of interactions within the OSR regulatory network, we show that despite their inability to directly sense reactive oxygen species, general transcription factors have an important function in coordinating this response. Remarkably, a significant fraction of this OSR was accurately recapitulated by a model that was earlier constructed from cellular responses to diverse environmental perturbations--this constitutes the general stress response component. Notwithstanding this observation, comparison of the two models has identified the coordination of frontline defense and repair systems by regulatory mechanisms that are triggered uniquely by severe OS and not by other environmental stressors, including sub-inhibitory levels of redox-active metals, extreme changes in oxygen tension, and a sub-lethal dose of gamma rays.

Coordination of frontline defense mechanisms under severe oxidative stress

PubMed Central

Kaur, Amardeep; Van, Phu T; Busch, Courtney R; Robinson, Courtney K; Pan, Min; Pang, Wyming Lee; Reiss, David J; DiRuggiero, Jocelyne; Baliga, Nitin S

2010-01-01

Complexity of cellular response to oxidative stress (OS) stems from its wide-ranging damage to nucleic acids, proteins, carbohydrates, and lipids. We have constructed a systems model of OS response (OSR) for Halobacterium salinarum NRC-1 in an attempt to understand the architecture of its regulatory network that coordinates this complex response. This has revealed a multi-tiered OS-management program to transcriptionally coordinate three peroxidase/catalase enzymes, two superoxide dismutases, production of rhodopsins, carotenoids and gas vesicles, metal trafficking, and various other aspects of metabolism. Through experimental validation of interactions within the OSR regulatory network, we show that despite their inability to directly sense reactive oxygen species, general transcription factors have an important function in coordinating this response. Remarkably, a significant fraction of this OSR was accurately recapitulated by a model that was earlier constructed from cellular responses to diverse environmental perturbations—this constitutes the general stress response component. Notwithstanding this observation, comparison of the two models has identified the coordination of frontline defense and repair systems by regulatory mechanisms that are triggered uniquely by severe OS and not by other environmental stressors, including sub-inhibitory levels of redox-active metals, extreme changes in oxygen tension, and a sub-lethal dose of γ rays. PMID:20664639

Protein-DNA binding dynamics predict transcriptional response to nutrients in archaea.

PubMed

Todor, Horia; Sharma, Kriti; Pittman, Adrianne M C; Schmid, Amy K

2013-10-01

Organisms across all three domains of life use gene regulatory networks (GRNs) to integrate varied stimuli into coherent transcriptional responses to environmental pressures. However, inferring GRN topology and regulatory causality remains a central challenge in systems biology. Previous work characterized TrmB as a global metabolic transcription factor in archaeal extremophiles. However, it remains unclear how TrmB dynamically regulates its ∼100 metabolic enzyme-coding gene targets. Using a dynamic perturbation approach, we elucidate the topology of the TrmB metabolic GRN in the model archaeon Halobacterium salinarum. Clustering of dynamic gene expression patterns reveals that TrmB functions alone to regulate central metabolic enzyme-coding genes but cooperates with various regulators to control peripheral metabolic pathways. Using a dynamical model, we predict gene expression patterns for some TrmB-dependent promoters and infer secondary regulators for others. Our data suggest feed-forward gene regulatory topology for cobalamin biosynthesis. In contrast, purine biosynthesis appears to require TrmB-independent regulators. We conclude that TrmB is an important component for mediating metabolic modularity, integrating nutrient status and regulating gene expression dynamics alone and in concert with secondary regulators.

Haloarchaea and the Formation of Gas Vesicles

PubMed Central

Pfeifer, Felicitas

2015-01-01

Halophilic Archaea (Haloarchaea) thrive in salterns containing sodium chloride concentrations up to saturation. Many Haloarchaea possess genes encoding gas vesicles, but only a few species, such as Halobacterium salinarum and Haloferax mediterranei, produce these gas-filled, proteinaceous nanocompartments. Gas vesicles increase the buoyancy of cells and enable them to migrate vertically in the water body to regions with optimal conditions. Their synthesis depends on environmental factors, such as light, oxygen supply, temperature and salt concentration. Fourteen gas vesicle protein (gvp) genes are involved in their formation, and regulation of gvp gene expression occurs at the level of transcription, including the two regulatory proteins, GvpD and GvpE, but also at the level of translation. The gas vesicle wall is solely formed of proteins with the two major components, GvpA and GvpC, and seven additional accessory proteins are also involved. Except for GvpI and GvpH, all of these are required to form the gas permeable wall. The applications of gas vesicles include their use as an antigen presenter for viral or pathogen proteins, but also as a stable ultrasonic reporter for biomedical purposes. PMID:25648404

Perchlorate and halophilic prokaryotes: implications for possible halophilic life on Mars.

PubMed

Oren, Aharon; Elevi Bardavid, Rahel; Mana, Lily

2014-01-01

In view of the finding of perchlorate among the salts detected by the Phoenix Lander on Mars, we investigated the relationships of halophilic heterotrophic microorganisms (archaea of the family Halobacteriaceae and the bacterium Halomonas elongata) toward perchlorate. All strains tested grew well in NaCl-based media containing 0.4 M perchlorate, but at the highest perchlorate concentrations, tested cells were swollen or distorted. Some species (Haloferax mediterranei, Haloferax denitrificans, Haloferax gibbonsii, Haloarcula marismortui, Haloarcula vallismortis) could use perchlorate as an electron acceptor for anaerobic growth. Although perchlorate is highly oxidizing, its presence at a concentration of 0.2 M for up to 2 weeks did not negatively affect the ability of a yeast extract-based medium to support growth of the archaeon Halobacterium salinarum. These findings show that presence of perchlorate among the salts on Mars does not preclude the possibility of halophilic life. If indeed the liquid brines that may exist on Mars are inhabited by salt-requiring or salt-tolerant microorganisms similar to the halophiles on Earth, presence of perchlorate may even be stimulatory when it can serve as an electron acceptor for respiratory activity in the anaerobic Martian environment.

A system-level model for the microbial regulatory genome.

PubMed

Brooks, Aaron N; Reiss, David J; Allard, Antoine; Wu, Wei-Ju; Salvanha, Diego M; Plaisier, Christopher L; Chandrasekaran, Sriram; Pan, Min; Kaur, Amardeep; Baliga, Nitin S

2014-07-15

Microbes can tailor transcriptional responses to diverse environmental challenges despite having streamlined genomes and a limited number of regulators. Here, we present data-driven models that capture the dynamic interplay of the environment and genome-encoded regulatory programs of two types of prokaryotes: Escherichia coli (a bacterium) and Halobacterium salinarum (an archaeon). The models reveal how the genome-wide distributions of cis-acting gene regulatory elements and the conditional influences of transcription factors at each of those elements encode programs for eliciting a wide array of environment-specific responses. We demonstrate how these programs partition transcriptional regulation of genes within regulons and operons to re-organize gene-gene functional associations in each environment. The models capture fitness-relevant co-regulation by different transcriptional control mechanisms acting across the entire genome, to define a generalized, system-level organizing principle for prokaryotic gene regulatory networks that goes well beyond existing paradigms of gene regulation. An online resource (http://egrin2.systemsbiology.net) has been developed to facilitate multiscale exploration of conditional gene regulation in the two prokaryotes. © 2014 The Authors. Published under the terms of the CC BY 4.0 license.

Suppressor Mutation Analysis of the Sensory Rhodopsin I-Transducer Complex: Insights into the Color-Sensing Mechanism

PubMed Central

Jung, Kwang-Hwan; Spudich, John L.

1998-01-01

The molecular complex containing the phototaxis receptor sensory rhodopsin I (SRI) and transducer protein HtrI (halobacterial transducer for SRI) mediates color-sensitive phototaxis responses in the archaeon Halobacterium salinarum. One-photon excitation of the complex by orange light elicits attractant responses, while two-photon excitation (orange followed by near-UV light) elicits repellent responses in swimming cells. Several mutations in SRI and HtrI cause an unusual mutant phenotype, called orange-light-inverted signaling, in which the cell produces a repellent response to normally attractant light. We applied a selection procedure for intragenic and extragenic suppressors of orange-light-inverted mutants and identified 15 distinct second-site mutations that restore the attractant response. Two of the 3 suppressor mutations in SRI are positioned at the cytoplasmic ends of helices F and G, and 12 suppressor mutations in HtrI cluster at the cytoplasmic end of the second HtrI transmembrane helix (TM2). Nearly all suppressors invert the normally repellent response to two-photon stimulation to an attractant response when they are expressed with their suppressible mutant alleles or in an otherwise wild-type strain. The results lead to a model for control of flagellar reversal by the SRI-HtrI complex. The model invokes an equilibrium between the A (reversal-inhibiting) and R (reversal-stimulating) conformers of the signaling complex. Attractant light and repellent light shift the equilibrium toward the A and R conformers, respectively, and mutations are proposed to cause intrinsic shifts in the equilibrium in the dark form of the complex. Differences in the strength of the two-photon signal inversion and in the allele specificity of suppression are correlated, and this correlation can be explained in terms of different values of the equilibrium constant (Keq) for the conformational transition in different mutants and mutant-suppressor pairs. PMID:9555883

Evolution of context dependent regulation by expansion of feast/famine regulatory proteins.

PubMed

Plaisier, Christopher L; Lo, Fang-Yin; Ashworth, Justin; Brooks, Aaron N; Beer, Karlyn D; Kaur, Amardeep; Pan, Min; Reiss, David J; Facciotti, Marc T; Baliga, Nitin S

2014-11-14

Expansion of transcription factors is believed to have played a crucial role in evolution of all organisms by enabling them to deal with dynamic environments and colonize new environments. We investigated how the expansion of the Feast/Famine Regulatory Protein (FFRP) or Lrp-like proteins into an eight-member family in Halobacterium salinarum NRC-1 has aided in niche-adaptation of this archaeon to a complex and dynamically changing hypersaline environment. We mapped genome-wide binding locations for all eight FFRPs, investigated their preference for binding different effector molecules, and identified the contexts in which they act by analyzing transcriptional responses across 35 growth conditions that mimic different environmental and nutritional conditions this organism is likely to encounter in the wild. Integrative analysis of these data constructed an FFRP regulatory network with conditionally active states that reveal how interrelated variations in DNA-binding domains, effector-molecule preferences, and binding sites in target gene promoters have tuned the functions of each FFRP to the environments in which they act. We demonstrate how conditional regulation of similar genes by two FFRPs, AsnC (an activator) and VNG1237C (a repressor), have striking environment-specific fitness consequences for oxidative stress management and growth, respectively. This study provides a systems perspective into the evolutionary process by which gene duplication within a transcription factor family contributes to environment-specific adaptation of an organism.

Subatmospheric vapor pressures for fluoromethane (R41), 1,1-difluoroethane (R152a), and 1,1,1-trifluoroethane (R143a) evaluated from internal-energy measurements

DOE Office of Scientific and Technical Information (OSTI.GOV)

Duarte-Garza, H.A.; Magee, J.W.

1999-09-01

Vapor pressures were evaluated from measured internal-energy changes {Delta}U{sup (2)} in the vapor + liquid two-phase region. The method employed a thermodynamic relationship between the derivative quantity ({partial_derivative}U{sup (2)}/{partial_derivative}V){sub T}, the vapor pressure p{sub {sigma}}, and its temperature derivative ({partial_derivative}p/{partial_derivative}T){sub {sigma}}. This method was applied at temperatures between the triple point and the normal boiling point of three substances: fluoromethane (R41), 1,1-difluoroethane (R152a), and 1,1,1-trifluoroethane (R143a). In the case of R41, vapor pressures up to 1 MPa were calculated to validate the technique at higher pressures. For R152a, the calculated vapor pressure at the triple-point temperature differed from a directmore » experimental measurement by less than the claimed uncertainty (5 Pa) of the measurement. The calculated vapor pressures for R41 helped to resolve discrepancies in several published vapor pressure sources. Agreement with experimentally measured vapor pressures for R152a and for R143a near the normal boiling point (101.325 kPa) was within the experimental uncertainty of approximately 0.04 kPa (0.04%) for the published measurements.« less

Effects of halophilic peptide fusion on solubility, stability, and catalytic performance of D-phenylglycine aminotransferase.

PubMed

Javid, Hossein; Jomrit, Juntratip; Chantarasiri, Aiya; Isarangkul, Duangnate; Meevootisom, Vithaya; Wiyakrutta, Suthep

2014-05-01

D-Phenylglycine aminotransferase (D-PhgAT) from Pseudomonas stutzeri ST-201 is useful for enzymatic synthesis of enantiomerically pure D-phenylglycine. However, its low protein solubility prevents its application at high substrate concentration. With an aim to increase the protein solubility, the N-terminus of D-PhgAT was genetically fused with short peptides (A1 α- helix, A2 α-helix, and ALAL, which is a hybrid of A1 and A2) from a ferredoxin enzyme of a halophilic archaeon, Halobacterium salinarum. The fused enzymes A1-D-PhgAT, A2-D-PhgAT, and ALAL-D-PhgAT displayed a reduced pI and increased in solubility by 6.1-, 5.3-, and 8.1- fold in TEMP (pH 7.6) storage, respectively, and 5-, 4.5-, and 5.9-fold in CAPSO (pH 9.5) reaction buffers, respectively, compared with the wild-type enzyme (WT-D-PhgAT). In addition, all the fused D-PhgAT displayed higher enzymatic reaction rates than the WT-DPhgAT at all concentrations of L-glutamate monosodium salt used. The highest rate, 23.82 ± 1.47 mM/h, was that obtained from having ALAL-D-PhgAT reacted with 1,500 mM of the substrate. Moreover, the halophilic fusion significantly increased the tolerance of D-PhgAT in the presence of NaCl and KCl, being slightly in favor of KCl, where under the same condition at 3.5 M NaCl or KCl all halophilic-fused variants showed higher activity than WT-D-PhgAT.

Analogies between respiration and a light-driven proton pump as sources of energy for active glutamate transport in Halobacterium halobium

NASA Technical Reports Server (NTRS)

Belliveau, J. W.; Lanyi, J. K.

1977-01-01

Halobacterium halobium is known to contain sheets of bacteriorhodopsin, a pigment which upon exposure to light undergoes cyclic protonation and deprotonation, resulting in net H(+) translocation. In this paper, experiments were conducted to test H. halobium cell envelope vesicles for respiration-induced glutamate uptake. It is shown that glutamate transport in H. halobium cell envelope vesicles can occur as a result of respiration, as well as light acting on bacteriorhodopsin. Glutamate transport can be energized by the oxidation of dimethyl phenylenediamine, and the properties of the transport system are entirely analogous to those observed with illumination as the source of energy. In the case of respiration-dependent glutamate transport, the transportation is also driven by a Na(+) gradient, thereby confirming the existence of a single glutamate transport system independent of the source of energy. The analogy observed is indirect evidence that the cytochrome oxidase of H. halobium functions as a H(+) pump.

Amino acid sequences of ribosomal proteins S11 from Bacillus stearothermophilus and S19 from Halobacterium marismortui. Comparison of the ribosomal protein S11 family.

PubMed

Kimura, M; Kimura, J; Hatakeyama, T

1988-11-21

The complete amino acid sequences of ribosomal proteins S11 from the Gram-positive eubacterium Bacillus stearothermophilus and of S19 from the archaebacterium Halobacterium marismortui have been determined. A search for homologous sequences of these proteins revealed that they belong to the ribosomal protein S11 family. Homologous proteins have previously been sequenced from Escherichia coli as well as from chloroplast, yeast and mammalian ribosomes. A pairwise comparison of the amino acid sequences showed that Bacillus protein S11 shares 68% identical residues with S11 from Escherichia coli and a slightly lower homology (52%) with the homologous chloroplast protein. The halophilic protein S19 is more related to the eukaryotic (45-49%) than to the eubacterial counterparts (35%).

Volumetric properties under pressure for 1,2-dichlorofluoroethane (R141), 1-fluoro-1,1,2-trichloroethane (R131a), and 1,2-dichloro-1,1-difluoroethane (R132b)

DOE Office of Scientific and Technical Information (OSTI.GOV)

Malhotra, R.; Woolf, L.A.

1997-01-01

The effect of pressure on the volume of R141, R131, and R132b is reported as volume ratios (the volume under pressure relative to its value at atmospheric pressure) at six temperatures covering the range 278.15 to 338.13 K and pressures up to 380 MPa for R141 and R131a. For R132b the same temperature range has been used, but above its normal boiling point experimental arrangements have limited maximum pressures to below 300 MPa, with some loss of accuracy. Densities have been measured at atmospheric pressure for each liquid. The experimental data have been used to calculate isothermal compressibilities, thermal expansivities,more » and internal pressures; the change in isobaric heat capacity from its value at atmospheric pressure has also been estimated. The volume ratios for all three compounds can be represented by a version of the Tait equation based on previously reported data for 1,2-dichloroethane and 1,1,2-trichloroethane with the inclusion of allowances of the substitution in the former of chlorine or fluorine for the hydrogens on one of the carbons.« less

An evolutionarily conserved RNase-based mechanism for repression of transcriptional positive autoregulation

PubMed Central

Wurtmann, Elisabeth J.; Ratushny, Alexander V.; Pan, Min; Beer, Karlyn D.; Aitchison, John D.; Baliga, Nitin S.

2014-01-01

Summary It is known that environmental context influences the degree of regulation at the transcriptional and post-transcriptional levels. However, the principles governing the differential usage and interplay of regulation at these two levels are not clear. Here, we show that the integration of transcriptional and post-transcriptional regulatory mechanisms in a characteristic network motif drives efficient environment-dependent state transitions. Through phenotypic screening, systems analysis, and rigorous experimental validation, we discovered an RNase (VNG2099C) in Halobacterium salinarum that is transcriptionally co-regulated with genes of the aerobic physiologic state but acts on transcripts of the anaerobic state. Through modeling and experimentation we show that this arrangement generates an efficient state-transition switch, within which RNase-repression of a transcriptional positive autoregulation (RPAR) loop is critical for shutting down ATP-consuming active potassium uptake to reserve energy required for salinity adaptation under aerobic, high potassium, or dark conditions. Subsequently, we discovered that many Escherichia coli operons with energy-associated functions are also putatively controlled by RPAR indicating that this network motif may have evolved independently in phylogenetically distant organisms. Thus, our data suggest that interplay of transcriptional and post-transcriptional regulation in the RPAR motifis a generalized principle for efficient environment-dependent state transitions across prokaryotes. PMID:24612392

Ultrafast Excited-state Deactivation of Flavins Bound to Dodecin*

PubMed Central

Staudt, Heike; Oesterhelt, Dieter; Grininger, Martin; Wachtveitl, Josef

2012-01-01

Dodecins, a group of flavin-binding proteins with a dodecameric quaternary structure, are able to incorporate two flavins within each of their six identical binding pockets building an aromatic tetrade with two tryptophan residues. Dodecin from the archaeal Halobacterium salinarum is a riboflavin storage device. We demonstrate that unwanted side reactions induced by reactive riboflavin species and degradation of riboflavin are avoided by ultrafast depopulation of the reactive excited state of riboflavin. Intriguingly, in this process, the staggered riboflavin dimers do not interact in ground and photoexcited states. Rather, within the tetrade assembly, each riboflavin is kept under the control of the respective adjacent tryptophan, which suggests that the stacked arrangement is a matter of optimizing the flavin load. We further identify an electron transfer in combination with a proton transfer as a central element of the effective excited state depopulation mechanism. Structural and functional comparisons of the archaeal dodecin with bacterial homologs reveal diverging evolution. Bacterial dodecins bind the flavin FMN instead of riboflavin and exhibit a clearly different binding pocket design with inverse incorporations of flavin dimers. The different adoption of flavin changes photochemical properties, making bacterial dodecin a comparably less efficient quencher of flavins. This supports a functional role different for bacterial and archaeal dodecins. PMID:22451648

High-throughput single-molecule force spectroscopy for membrane proteins

NASA Astrophysics Data System (ADS)

Bosshart, Patrick D.; Casagrande, Fabio; Frederix, Patrick L. T. M.; Ratera, Merce; Bippes, Christian A.; Müller, Daniel J.; Palacin, Manuel; Engel, Andreas; Fotiadis, Dimitrios

2008-09-01

Atomic force microscopy-based single-molecule force spectroscopy (SMFS) is a powerful tool for studying the mechanical properties, intermolecular and intramolecular interactions, unfolding pathways, and energy landscapes of membrane proteins. One limiting factor for the large-scale applicability of SMFS on membrane proteins is its low efficiency in data acquisition. We have developed a semi-automated high-throughput SMFS (HT-SMFS) procedure for efficient data acquisition. In addition, we present a coarse filter to efficiently extract protein unfolding events from large data sets. The HT-SMFS procedure and the coarse filter were validated using the proton pump bacteriorhodopsin (BR) from Halobacterium salinarum and the L-arginine/agmatine antiporter AdiC from the bacterium Escherichia coli. To screen for molecular interactions between AdiC and its substrates, we recorded data sets in the absence and in the presence of L-arginine, D-arginine, and agmatine. Altogether ~400 000 force-distance curves were recorded. Application of coarse filtering to this wealth of data yielded six data sets with ~200 (AdiC) and ~400 (BR) force-distance spectra in each. Importantly, the raw data for most of these data sets were acquired in one to two days, opening new perspectives for HT-SMFS applications.

Role of Mn2+ and compatible solutes in the radiation resistance of thermophilic bacteria and archaea.

PubMed

Webb, Kimberly M; DiRuggiero, Jocelyne

2012-01-01

Radiation-resistant bacteria have garnered a great deal of attention from scientists seeking to expose the mechanisms underlying their incredible survival abilities. Recent analyses showed that the resistance to ionizing radiation (IR) in the archaeon Halobacterium salinarum is dependent upon Mn-antioxidant complexes responsible for the scavenging of reactive oxygen species (ROS) generated by radiation. Here we examined the role of the compatible solutes trehalose, mannosylglycerate, and di-myo-inositol phosphate in the radiation resistance of aerobic and anaerobic thermophiles. We found that the IR resistance of the thermophilic bacteria Rubrobacter xylanophilus and Rubrobacter radiotolerans was highly correlated to the accumulation of high intracellular concentration of trehalose in association with Mn, supporting the model of Mn(2+)-dependent ROS scavenging in the aerobes. In contrast, the hyperthermophilic archaea Thermococcus gammatolerans and Pyrococcus furiosus did not contain significant amounts of intracellular Mn, and we found no significant antioxidant activity from mannosylglycerate and di-myo-inositol phosphate in vitro. We therefore propose that the low levels of IR-generated ROS under anaerobic conditions combined with highly constitutively expressed detoxification systems in these anaerobes are key to their radiation resistance and circumvent the need for the accumulation of Mn-antioxidant complexes in the cell.

Crystal structure of Escherichia coli-expressed Haloarcula marismortui bacteriorhodopsin I in the trimeric form.

PubMed

Shevchenko, Vitaly; Gushchin, Ivan; Polovinkin, Vitaly; Round, Ekaterina; Borshchevskiy, Valentin; Utrobin, Petr; Popov, Alexander; Balandin, Taras; Büldt, Georg; Gordeliy, Valentin

2014-01-01

Bacteriorhodopsins are a large family of seven-helical transmembrane proteins that function as light-driven proton pumps. Here, we present the crystal structure of a new member of the family, Haloarcula marismortui bacteriorhodopsin I (HmBRI) D94N mutant, at the resolution of 2.5 Å. While the HmBRI retinal-binding pocket and proton donor site are similar to those of other archaeal proton pumps, its proton release region is extended and contains additional water molecules. The protein's fold is reinforced by three novel inter-helical hydrogen bonds, two of which result from double substitutions relative to Halobacterium salinarum bacteriorhodopsin and other similar proteins. Despite the expression in Escherichia coli and consequent absence of native lipids, the protein assembles as a trimer in crystals. The unique extended loop between the helices D and E of HmBRI makes contacts with the adjacent protomer and appears to stabilize the interface. Many lipidic hydrophobic tail groups are discernible in the membrane region, and their positions are similar to those of archaeal isoprenoid lipids in the crystals of other proton pumps, isolated from native or native-like sources. All these features might explain the HmBRI properties and establish the protein as a novel model for the microbial rhodopsin proton pumping studies.

Evolution of context dependent regulation by expansion of feast/famine regulatory proteins

DOE PAGES

Plaisier, Christopher L.; Lo, Fang -Yin; Ashworth, Justin; ...

2014-11-14

Expansion of transcription factors is believed to have played a crucial role in evolution of all organisms by enabling them to deal with dynamic environments and colonize new environments. We investigated how the expansion of the Feast/Famine Regulatory Protein (FFRP) or Lrp-like proteins into an eight-member family in Halobacterium salinarum NRC-1 has aided in niche-adaptation of this archaeon to a complex and dynamically changing hypersaline environment. We mapped genome-wide binding locations for all eight FFRPs, investigated their preference for binding different effector molecules, and identified the contexts in which they act by analyzing transcriptional responses across 35 growth conditions thatmore » mimic different environmental and nutritional conditions this organism is likely to encounter in the wild. Integrative analysis of these data constructed an FFRP regulatory network with conditionally active states that reveal how interrelated variations in DNA-binding domains, effector-molecule preferences, and binding sites in target gene promoters have tuned the functions of each FFRP to the environments in which they act. We demonstrate how conditional regulation of similar genes by two FFRPs, AsnC (an activator) and VNG1237C (a repressor), have striking environment-specific fitness consequences for oxidative stress management and growth, respectively. This study provides a systems perspective into the evolutionary process by which gene duplication within a transcription factor family contributes to environment-specific adaptation of an organism.« less

Isolation, characterization and phylogenetic analysis of halophilic archaea from a salt mine in central Anatolia (Turkey).

PubMed

Yildiz, Evrim; Ozcan, Birgul; Caliskan, Mahmut

2012-01-01

The haloarchaeal diversity of a salt mine, a natural cave in central Anatolia, was investigated using convential microbiological and molecular biology methods. Eight halophilic archaeal isolates selected based on their colony morphology and whole cell protein profiles were taxonomically classified on the basis of their morphological, physiological, biochemical properties, polar lipid and protein profiles and 16S rDNA sequences. From the 16S rDNA sequences comparisons it was established that the isolates CH2, CH3 and CHC resembled Halorubrum saccharovorum by 98.8%, 98.9% and 99.5%, respectively. There was a 99.7% similarity between the isolate CH11 and Halobacterium noricense and 99.2% between the isolate CHA1 and Haloarcula argentinensis. The isolate CH8K and CH8B revealed a similarity rate of 99.8% and 99.3% to Halococcus dombrowskii, respectively. It was concluded that the isolates named CH2, CH3 and CHC were clustered in the genus Halorubrum and that CHA1 and CH7 in the genus Haloarcula, CH8K and CH8B in the genus Halococcus and CH11 in the genus Halobacterium.

Halocin C8: an antimicrobial peptide distributed among four halophilic archaeal genera: Natrinema, Haloterrigena, Haloferax, and Halobacterium.

PubMed

Besse, Alison; Vandervennet, Manon; Goulard, Christophe; Peduzzi, Jean; Isaac, Stéphanie; Rebuffat, Sylvie; Carré-Mlouka, Alyssa

2017-05-01

Halophilic archaea thrive in hypersaline ecosystems and produce antimicrobial peptides (AMPs) named halocins. AMPs are essential effectors of microbial interactions in natural ecosystems. Halocin C8 is a 7.4 kDa peptide produced by Natrinema sp. AS7092. Surrounded by genes involved in regulation and transport, the halC8 gene encodes a precursor, processed into the mature halocin and an immunity protein, protecting the producing strain against its halocin. This feature constitutes a unique property of halocin C8, as known AMPs and their immunity proteins are generally encoded on distinct ORFs in an operon. By complementary in silico and PCR-based approaches, the presence of halC8 in halophilic archaea collected from various parts of the world was evidenced. The full-length halC8 gene is restricted and consistently found in the genomes of strains belonging to the phylogenetically related genera Natrinema and Haloterrigena, along with transport and regulation genes. Functional expression of halC8 was demonstrated by RT-PCR and antimicrobial assays. Active halocin C8 was shown to contain five disulphide bridges, presumably conferring a compact structure resistant to harsh environmental conditions. In other archaeal genera, Haloferax and Halobacterium, genes encoding halocin C8 with diverging immunity protein moiety were evidenced. A phylogenetic analysis of halocin C8 sequences was conducted.

Distinct human and mouse membrane trafficking systems for sweet taste receptors T1r2 and T1r3.

PubMed

Shimizu, Madoka; Goto, Masao; Kawai, Takayuki; Yamashita, Atsuko; Kusakabe, Yuko

2014-01-01

The sweet taste receptors T1r2 and T1r3 are included in the T1r taste receptor family that belongs to class C of the G protein-coupled receptors. Heterodimerization of T1r2 and T1r3 is required for the perception of sweet substances, but little is known about the mechanisms underlying this heterodimerization, including membrane trafficking. We developed tagged mouse T1r2 and T1r3, and human T1R2 and T1R3 and evaluated membrane trafficking in human embryonic kidney 293 (HEK293) cells. We found that human T1R3 surface expression was only observed when human T1R3 was coexpressed with human T1R2, whereas mouse T1r3 was expressed without mouse T1r2 expression. A domain-swapped chimera and truncated human T1R3 mutant showed that the Venus flytrap module and cysteine-rich domain (CRD) of human T1R3 contain a region related to the inhibition of human T1R3 membrane trafficking and coordinated regulation of human T1R3 membrane trafficking. We also found that the Venus flytrap module of both human T1R2 and T1R3 are needed for membrane trafficking, suggesting that the coexpression of human T1R2 and T1R3 is required for this event. These results suggest that the Venus flytrap module and CRD receive taste substances and play roles in membrane trafficking of human T1R2 and T1R3. These features are different from those of mouse receptors, indicating that human T1R2 and T1R3 are likely to have a novel membrane trafficking system.

Complete amino acid sequences of the ribosomal proteins L25, L29 and L31 from the archaebacterium Halobacterium marismortui.

PubMed

Hatakeyama, T; Kimura, M

1988-03-15

Ribosomal proteins were extracted from 50S ribosomal subunits of the archaebacterium Halobacterium marismortui by decreasing the concentration of Mg2+ and K+, and the proteins were separated and purified by ion-exchange column chromatography on DEAE-cellulose. Ten proteins were purified to homogeneity and three of these proteins were subjected to sequence analysis. The complete amino acid sequences of the ribosomal proteins L25, L29 and L31 were established by analyses of the peptides obtained by enzymatic digestion with trypsin, Staphylococcus aureus protease, chymotrypsin and lysylendopeptidase. Proteins L25, L29 and L31 consist of 84, 115 and 95 amino acid residues with the molecular masses of 9472 Da, 12293 Da and 10418 Da respectively. A comparison of their sequences with those of other large-ribosomal-subunit proteins from other organisms revealed that protein L25 from H. marismortui is homologous to protein L23 from Escherichia coli (34.6%), Bacillus stearothermophilus (41.8%), and tobacco chloroplasts (16.3%) as well as to protein L25 from yeast (38.0%). Proteins L29 and L31 do not appear to be homologous to any other ribosomal proteins whose structures are so far known.

The Firegoose: two-way integration of diverse data from different bioinformatics web resources with desktop applications

PubMed Central

Bare, J Christopher; Shannon, Paul T; Schmid, Amy K; Baliga, Nitin S

2007-01-01

Background Information resources on the World Wide Web play an indispensable role in modern biology. But integrating data from multiple sources is often encumbered by the need to reformat data files, convert between naming systems, or perform ongoing maintenance of local copies of public databases. Opportunities for new ways of combining and re-using data are arising as a result of the increasing use of web protocols to transmit structured data. Results The Firegoose, an extension to the Mozilla Firefox web browser, enables data transfer between web sites and desktop tools. As a component of the Gaggle integration framework, Firegoose can also exchange data with Cytoscape, the R statistical package, Multiexperiment Viewer (MeV), and several other popular desktop software tools. Firegoose adds the capability to easily use local data to query KEGG, EMBL STRING, DAVID, and other widely-used bioinformatics web sites. Query results from these web sites can be transferred to desktop tools for further analysis with a few clicks. Firegoose acquires data from the web by screen scraping, microformats, embedded XML, or web services. We define a microformat, which allows structured information compatible with the Gaggle to be embedded in HTML documents. We demonstrate the capabilities of this software by performing an analysis of the genes activated in the microbe Halobacterium salinarum NRC-1 in response to anaerobic environments. Starting with microarray data, we explore functions of differentially expressed genes by combining data from several public web resources and construct an integrated view of the cellular processes involved. Conclusion The Firegoose incorporates Mozilla Firefox into the Gaggle environment and enables interactive sharing of data between diverse web resources and desktop software tools without maintaining local copies. Additional web sites can be incorporated easily into the framework using the scripting platform of the Firefox browser. Performing data

The Firegoose: two-way integration of diverse data from different bioinformatics web resources with desktop applications.

PubMed

Bare, J Christopher; Shannon, Paul T; Schmid, Amy K; Baliga, Nitin S

2007-11-19

Information resources on the World Wide Web play an indispensable role in modern biology. But integrating data from multiple sources is often encumbered by the need to reformat data files, convert between naming systems, or perform ongoing maintenance of local copies of public databases. Opportunities for new ways of combining and re-using data are arising as a result of the increasing use of web protocols to transmit structured data. The Firegoose, an extension to the Mozilla Firefox web browser, enables data transfer between web sites and desktop tools. As a component of the Gaggle integration framework, Firegoose can also exchange data with Cytoscape, the R statistical package, Multiexperiment Viewer (MeV), and several other popular desktop software tools. Firegoose adds the capability to easily use local data to query KEGG, EMBL STRING, DAVID, and other widely-used bioinformatics web sites. Query results from these web sites can be transferred to desktop tools for further analysis with a few clicks. Firegoose acquires data from the web by screen scraping, microformats, embedded XML, or web services. We define a microformat, which allows structured information compatible with the Gaggle to be embedded in HTML documents. We demonstrate the capabilities of this software by performing an analysis of the genes activated in the microbe Halobacterium salinarum NRC-1 in response to anaerobic environments. Starting with microarray data, we explore functions of differentially expressed genes by combining data from several public web resources and construct an integrated view of the cellular processes involved. The Firegoose incorporates Mozilla Firefox into the Gaggle environment and enables interactive sharing of data between diverse web resources and desktop software tools without maintaining local copies. Additional web sites can be incorporated easily into the framework using the scripting platform of the Firefox browser. Performing data integration in the browser

40 CFR Table 1 to Subpart R of... - General Provisions Applicability to Subpart R

Code of Federal Regulations, 2012 CFR

2012-07-01

... Subpart R 1 Table 1 to Subpart R of Part 63 Protection of Environment ENVIRONMENTAL PROTECTION AGENCY... Pipeline Breakout Stations) Pt. 63, Subpt. R, Table 1 Table 1 to Subpart R of Part 63—General Provisions Applicability to Subpart R Reference Applies to subpart R Comment 63.1(a)(1) Yes 63.1(a)(2) Yes 63.1(a)(3) Yes...

40 CFR Table 1 to Subpart R of... - General Provisions Applicability to Subpart R

Code of Federal Regulations, 2014 CFR

2014-07-01

... Subpart R 1 Table 1 to Subpart R of Part 63 Protection of Environment ENVIRONMENTAL PROTECTION AGENCY... Pipeline Breakout Stations) Pt. 63, Subpt. R, Table 1 Table 1 to Subpart R of Part 63—General Provisions Applicability to Subpart R Reference Applies to subpart R Comment 63.1(a)(1) Yes 63.1(a)(2) Yes 63.1(a)(3) Yes...

40 CFR Table 1 to Subpart R of... - General Provisions Applicability to Subpart R

Code of Federal Regulations, 2011 CFR

2011-07-01

... Subpart R 1 Table 1 to Subpart R of Part 63 Protection of Environment ENVIRONMENTAL PROTECTION AGENCY... Pipeline Breakout Stations) Pt. 63, Subpt. R, Table 1 Table 1 to Subpart R of Part 63—General Provisions Applicability to Subpart R Reference Applies to subpart R Comment 63.1(a)(1) Yes 63.1(a)(2) Yes 63.1(a)(3) Yes...

40 CFR Table 1 to Subpart R of... - General Provisions Applicability to Subpart R

Code of Federal Regulations, 2010 CFR

2010-07-01

... Subpart R 1 Table 1 to Subpart R of Part 63 Protection of Environment ENVIRONMENTAL PROTECTION AGENCY... Pipeline Breakout Stations) Pt. 63, Subpt. R, Table 1 Table 1 to Subpart R of Part 63—General Provisions Applicability to Subpart R Reference Applies to subpart R Comment 63.1(a)(1) Yes 63.1(a)(2) Yes 63.1(a)(3) Yes...

40 CFR Table 1 to Subpart R of... - General Provisions Applicability to Subpart R

Code of Federal Regulations, 2013 CFR

2013-07-01

... Subpart R 1 Table 1 to Subpart R of Part 63 Protection of Environment ENVIRONMENTAL PROTECTION AGENCY... Pipeline Breakout Stations) Pt. 63, Subpt. R, Table 1 Table 1 to Subpart R of Part 63—General Provisions Applicability to Subpart R Reference Applies to subpart R Comment 63.1(a)(1) Yes 63.1(a)(2) Yes 63.1(a)(3) Yes...

Taste responses in mice lacking taste receptor subunit T1R1

PubMed Central

Kusuhara, Yoko; Yoshida, Ryusuke; Ohkuri, Tadahiro; Yasumatsu, Keiko; Voigt, Anja; Hübner, Sandra; Maeda, Katsumasa; Boehm, Ulrich; Meyerhof, Wolfgang; Ninomiya, Yuzo

2013-01-01

The T1R1 receptor subunit acts as an umami taste receptor in combination with its partner, T1R3. In addition, metabotropic glutamate receptors (brain and taste variants of mGluR1 and mGluR4) are thought to function as umami taste receptors. To elucidate the function of T1R1 and the contribution of mGluRs to umami taste detection in vivo, we used newly developed knock-out (T1R1−/−) mice, which lack the entire coding region of the Tas1r1 gene and express mCherry in T1R1-expressing cells. Gustatory nerve recordings demonstrated that T1R1−/− mice exhibited a serious deficit in inosine monophosphate-elicited synergy but substantial residual responses to glutamate alone in both chorda tympani and glossopharyngeal nerves. Interestingly, chorda tympani nerve responses to sweeteners were smaller in T1R1−/− mice. Taste cell recordings demonstrated that many mCherry-expressing taste cells in T1R1+/− mice responded to sweet and umami compounds, whereas those in T1R1−/− mice responded to sweet stimuli. The proportion of sweet-responsive cells was smaller in T1R1−/− than in T1R1+/− mice. Single-cell RT-PCR demonstrated that some single mCherry-expressing cells expressed all three T1R subunits. Chorda tympani and glossopharyngeal nerve responses to glutamate were significantly inhibited by addition of mGluR antagonists in both T1R1−/− and T1R1+/− mice. Conditioned taste aversion tests demonstrated that both T1R1−/− and T1R1+/− mice were equally capable of discriminating glutamate from other basic taste stimuli. Avoidance conditioned to glutamate was significantly reduced by addition of mGluR antagonists. These results suggest that T1R1-expressing cells mainly contribute to umami taste synergism and partly to sweet sensitivity and that mGluRs are involved in the detection of umami compounds. PMID:23339178

Differentiation of Forebrain and Hippocampal Dopamine 1-Class Receptors, D1R and D5R, in Spatial Learning and Memory

PubMed Central

Sariñana, Joshua; Tonegawa, Susumu

2017-01-01

Activation of prefrontal cortical (PFC), striatal, and hippocampal dopamine 1-class receptors (D1R and D5R) is necessary for normal spatial information processing. Yet the precise role of the D1R versus the D5R in the aforementioned structures, and their specific contribution to the water-maze spatial learning task remains unknown. D1R- and D5R- specific in situ hybridization probes showed that forebrain restricted D1R and D5R KO mice (F-D1R/D5R KO) displayed D1R mRNA deletion in the medial (m)PFC, dorsal and ventral striatum, and the dentate gyrus (DG) of the hippocampus. D5R mRNA deletion was limited to the mPFC, the CA1 and DG hippocampal subregions. F-D1R/D5R KO mice were given water-maze training and displayed subtle spatial latency differences between genotypes and spatial memory deficits during both regular and reversal training. To differentiate forebrain D1R from D5R activation, forebrain restricted D1R KO (F-D1R KO) and D5R KO (F-D5R KO) mice were trained on the water-maze task. F-D1R KO animals exhibited escape latency deficits throughout regular and reversal training as well as spatial memory deficits during reversal training. F-D1R KO mice also showed perseverative behavior during the reversal spatial memory probe test. In contrast, F-D5R KO animals did not present observable deficits on the water-maze task. Because F-D1R KO mice showed water-maze deficits we tested the necessity of hippocampal D1R activation for spatial learning and memory. We trained DG restricted D1R KO (DG-D1R KO) mice on the water-maze task. DG-D1R KO mice did not present detectable spatial memory deficit, but did show subtle deficits during specific days of training. Our data provides evidence that forebrain D5R activation plays a unique role in spatial learning and memory in conjunction with D1R activation. Moreover, these data suggest that mPFC and striatal, but not DG D1R activation are essential for spatial learning and memory. PMID:26174222

Methionine Regulates mTORC1 via the T1R1/T1R3-PLCβ-Ca2+-ERK1/2 Signal Transduction Process in C2C12 Cells.

PubMed

Zhou, Yuanfei; Ren, Jiao; Song, Tongxing; Peng, Jian; Wei, Hongkui

2016-10-11

The mammalian target of rapamycin complex 1 (mTORC1) integrates amino acid (AA) availability to support protein synthesis and cell growth. Taste receptor type 1 member (T1R) is a G protein-coupled receptor that functions as a direct sensor of extracellular AA availability to regulate mTORC1 through Ca 2+ stimulation and extracellular signal-regulated kinases 1 and 2 (ERK1/2) activation. However, the roles of specific AAs in T1R1/T1R3-regulated mTORC1 are poorly defined. In this study, T1R1 and T1R3 subunits were expressed in C2C12 myotubes, and l-AA sensing was accomplished by T1R1/T1R3 to activate mTORC1. In response to l-AAs, such as serine (Ser), arginine (Arg), threonine (Thr), alanine (Ala), methionine (Met), glutamine (Gln), and glycine (Gly), Met induced mTORC1 activation and promoted protein synthesis. Met also regulated mTORC1 via T1R1/T1R3-PLCβ-Ca 2+ -ERK1/2 signal transduction. Results revealed a new role for Met-regulated mTORC1 via an AA receptor. Further studies should be performed to determine the role of T1R1/T1R3 in mediating extracellular AA to regulate mTOR signaling and to reveal its mechanism.

Screening of allergic components mediated by H(1)R in homoharringtonine injection through H(1)R/CMC-HPLC/MS.

PubMed

Guo, Ying; Han, Shengli; Cao, Jingjing; Liu, Qi; Zhang, Tao

2014-12-01

It has been reported that the histamine H1 receptor (H(1)R) gene is up-regulated in patients with allergic rhinitis and H(1)R expression level strongly correlates with the severity of allergy symptoms. Drugs for therapy should avoid allergy symptoms, especially for patients with over-expressed H(1)R. Therefore, screening of the components which could induce H(1)R activation is urgently needed for drug safety evaluation. Homoharringtonine injection is a preparation for acute nonlymphocytic leukemia, which is approved by China Food and Drug Administration (CFDA) and US Food and Drug Administration. However, severely adverse reactions often occur with intravenous injection of the preparation. In present study, an H(1)R/CMC model was applied for capturing membrane retained components which could induce H(1)R activation. Retention components were enriched and analyzed by H(1)R/CMC-HPLC/MS. Homoharringtonine was recognized, separated and identified in homoharringtonine injection. Ca(2+) flux assay and p-IP3R expression founded that homoharringtonine retained by the H1 R/CMC model increased phosphorylation of IP3R and promoted cytosolic free Ca(2+) elevation in a dose-dependent manner which further verified the activity of homoharringtonine in activating the H1 R. In conclusion, homoharringtonine was screened and identified as a potential allergic factor. This provides an indication that a patient with over-expressed H1 R should be aware of possible allergic reaction when applying homoharringtonine injection. Copyright © 2014 John Wiley & Sons, Ltd.

Interleukin-1 receptor (IL-1R) mediates epilepsy-induced sleep disruption.

PubMed

Huang, Tzu-Rung; Jou, Shuo-Bin; Chou, Yu-Ju; Yi, Pei-Lu; Chen, Chun-Jen; Chang, Fang-Chia

2016-11-22

Sleep disruptions are common in epilepsy patients. Our previous study demonstrates that homeostatic factors and circadian rhythm may mediate epilepsy-induced sleep disturbances when epilepsy occurs at different zeitgeber hours. The proinflammatory cytokine, interleukin-1 (IL-1), is a somnogenic cytokine and may also be involved in epileptogenesis; however, few studies emphasize the effect of IL-1 in epilepsy-induced sleep disruption. We herein hypothesized that IL-1 receptor type 1 (IL-1R1) mediates the pathogenesis of epilepsy and epilepsy-induced sleep disturbances. We determined the role of IL-1R1 by using IL-1R1 knockout (IL-1R1 -/- KO) mice. Our results elucidated the decrease of non-rapid eye movement (NREM) sleep during the light period in IL-1R -/- mice and confirmed the somnogenic role of IL-1R1. Rapid electrical amygdala kindling was performed to induce epilepsy at the particular zeitgeber time (ZT) point, ZT13. Our results demonstrated that seizure thresholds induced by kindling stimuli, such as the after-discharge threshold and successful kindling rates, were not altered in IL-1R -/- mice when compared to those obtained from the wildtype mice (IL-1R +/+ mice). This result suggests that IL-1R1 is not involved in kindling-induced epileptogenesis. During sleep, ZT13 kindling stimulation significantly enhanced NREM sleep during the subsequent 6 h (ZT13-18) in wildtype mice, and sleep returned to the baseline the following day. However, the kindling-induced sleep alteration was absent in the IL-1R -/- KO mice. These results indicate that the IL-1 signal mediates epilepsy-induced sleep disturbance, but dose not participate in kindling-induced epileptogenesis.

FTIR Studies of Internal Water Molecules of Bacteriorhodopsin: Structural Analysis of Halide-bound D85S and D212N Mutants in the Schiff Base Region

NASA Astrophysics Data System (ADS)

Shibata, Mikihiro; Kandori, Hideki

2007-12-01

Bacteriorhodopsin (BR), a membrane protein found in Halobacterium salinarum, functions as a light-driven proton pump. The Schiff base region has a quadropolar structure with positive charges located at the protonated Schiff base and Arg82, and counterbalancing negative charges located at Asp85 and Asp212 (Figure 1A). It is known that BR lacks a proton-pumping activity if Asp85 or Asp212 is neutralized by mutation. On the other hand, binding of C1- brings different effects for pumping functions in mutants at D85 and D212 position. While C1--bound D85T and D85S pump C1-, photovoltage measurements suggested that C1--bound D212N pumps protons at low pH. In this study, we measured low-temperature FTIR spectra of D85S and D212N containing various halides to compare the halide binding site of both proteins. In the case of D85S, the N-D stretching vibrations of the Schiff base were halide-dependent. This result suggests that the halide is a hydrogen-bond acceptor of the Schiff base, being consistent with the X-ray crystal structure. On the other hand, no halide dependence was observed for vibrational bands of the retinal skeleton and the Schiff base in the D212N mutant. This result suggests that the halide does not form a hydrogen bond with the Schiff base directly, unlike the mutation at D85 position. Halide-dependent water bands in the Schiff base region also differ between D85S and D212N. From these results, halide binding site of both proteins and role of two negative charges in BR will be discussed.

InsR/IGF1R pathway mediates resistance to EGFR inhibitors in glioblastoma

PubMed Central

Ma, Yufang; Tang, Nan; Thompson, Reid; Mobley, Bret C.; Clark, Steven W.; Sarkaria, Jann N.; Wang, Jialiang

2015-01-01

Purpose Aberrant activation of epidermal growth factor receptor (EGFR) is a hallmark of glioblastoma. However, EGFR inhibitors exhibit at best modest efficacy in glioblastoma. This is in sharp contrast to the observations in EGFR-mutant lung cancer. We examined whether activation of functionally redundant receptor tyrosine kinases (RTKs) conferred resistance to EGFR inhibitors in glioblastoma. Experimental Design We collected a panel of patient-derived glioblastoma xenograft (PDX) lines that maintained expression of wild type or mutant EGFR in serial xenotransplantation and tissue cultures. Using this physiologically relevant platform, we tested the abilities of several RTK ligands to protect glioblastoma cells against an EGFR inhibitor, gefitinib. Based on the screening results, we further developed a combination therapy co-targeting EGFR and insulin receptor (InsR)/insulin-like growth factor 1 receptor (IGF1R). Results Insulin and IGF1 induced significant protection against gefitinib in the majority of EGFR-dependent PDX lines with one exception that did not expression InsR or IGF1R. Blockade of the InsR/IGF1R pathway synergistically improved sensitivity to gefitinib or dacomitinib. Gefitinib alone effectively attenuated EGFR activities and the downstream MEK/ERK pathway. However, repression of AKT and induction of apoptosis required concurrent inhibition of both EGFR and InsR/IGF1R. A combination of gefitinib and OSI-906, a dual InsR/IGF1R inhibitor, was more effective than either agent alone to treat subcutaneous glioblastoma xenograft tumors. Conclusions Our results suggest that activation of the InsR/IGF1R pathway confers resistance to EGFR inhibitors in EGFR-dependent glioblastoma through AKT regulation. Concurrent blockade of these two pathways holds promise to treat EGFR-dependent glioblastoma. PMID:26561558

The combinatorial PP1-binding consensus Motif (R/K)x( (0,1))V/IxFxx(R/K)x(R/K) is a new apoptotic signature.

PubMed

Godet, Angélique N; Guergnon, Julien; Maire, Virginie; Croset, Amélie; Garcia, Alphonse

2010-04-01

Previous studies established that PP1 is a target for Bcl-2 proteins and an important regulator of apoptosis. The two distinct functional PP1 consensus docking motifs, R/Kx((0,1))V/IxF and FxxR/KxR/K, involved in PP1 binding and cell death were previously characterized in the BH1 and BH3 domains of some Bcl-2 proteins. In this study, we demonstrate that DPT-AIF(1), a peptide containing the AIF(562-571) sequence located in a c-terminal domain of AIF, is a new PP1 interacting and cell penetrating molecule. We also showed that DPT-AIF(1) provoked apoptosis in several human cell lines. Furthermore, DPT-APAF(1) a bi-partite cell penetrating peptide containing APAF-1(122-131), a non penetrating sequence from APAF-1 protein, linked to our previously described DPT-sh1 peptide shuttle, is also a PP1-interacting death molecule. Both AIF(562-571) and APAF-1(122-131) sequences contain a common R/Kx((0,1))V/IxFxxR/KxR/K motif, shared by several proteins involved in control of cell survival pathways. This motif combines the two distinct PP1c consensus docking motifs initially identified in some Bcl-2 proteins. Interestingly DPT-AIF(2) and DPT-APAF(2) that carry a F to A mutation within this combinatorial motif, no longer exhibited any PP1c binding or apoptotic effects. Moreover the F to A mutation in DPT-AIF(2) also suppressed cell penetration. These results indicate that the combinatorial PP1c docking motif R/Kx((0,1))V/IxFxxR/KxR/K, deduced from AIF(562-571) and APAF-1(122-131) sequences, is a new PP1c-dependent Apoptotic Signature. This motif is also a new tool for drug design that could be used to characterize potential anti-tumour molecules.

Biotransformation of (-)-(1R,4S)-Menthone and (+)-(1S,4R)-Menthone by the Common Cutworm Spodoptera litura Larvae.

PubMed

Marumoto, Shinsuke; Okuno, Yoshiharu; Hagiwara, Yuki; Miyazawa, Mitsuo

2017-08-01

Using biotransformation as a biocatalytic process has the advantage of being able to proceed under mild conditions and with high regio- and enantioselectivity. This study investigated the biotransformation of (-)-(1R,4S)-menthone (1) and (+)-(1S,4R)-menthone (2) by Spodoptera litura larvae. Compound 1 was converted to (-)-(1R,4S)-7-hydroxymenthone (1-1), (+)-(1R,3S,4S)-7-hydroxyneomenthol (1-2) and (-)-(1R,4S,8R)-p-menth-3-one-9-oic acid (1-3). The metabolism of substrate 2 generated three enantiomers of the above metabolites, designated as 2-1 to 2-3, respectively. The C-9 position of (-)-menthone and (+)-menthone was oxidized to carboxylic acid by S. litura, which is a metabolic pathway not observed in any other example of biocatalysis.

1/ r potential in higher dimensions

NASA Astrophysics Data System (ADS)

Chakraborty, Sumanta; Dadhich, Naresh

2018-01-01

In Einstein gravity, gravitational potential goes as 1/r^{d-3} in d non-compactified spacetime dimensions, which assumes the familiar 1 / r form in four dimensions. On the other hand, it goes as 1/r^{α }, with α =(d-2m-1)/m, in pure Lovelock gravity involving only one mth order term of the Lovelock polynomial in the gravitational action. The latter offers a novel possibility of having 1 / r potential for the non-compactified dimension spectrum given by d=3m+1. Thus it turns out that in the two prototype gravitational settings of isolated objects, like black holes and the universe as a whole - cosmological models, the Einstein gravity in four and mth order pure Lovelock gravity in 3m+1 dimensions behave in a similar fashion as far as gravitational interactions are considered. However propagation of gravitational waves (or the number of degrees of freedom) does indeed serve as a discriminator because it has two polarizations only in four dimensions.

The Adiponectin Homolog Osmotin Enhances Neurite Outgrowth and Synaptic Complexity via AdipoR1/NgR1 Signaling in Alzheimer's Disease.

PubMed

Yoon, Gwangho; Shah, Shahid Ali; Ali, Tahir; Kim, Myeong Ok

2018-01-15

Alzheimer's disease is a major neurodegenerative disease characterized by memory loss and cognitive deficits. Recently, we reported that osmotin, which is a homolog of adiponectin, improved long-term potentiation and cognitive functions in Alzheimer's disease mice. Several lines of evidence have suggested that Nogo-A and the Nogo-66 receptor 1 (NgR1), which form a complex that inhibits long-term potentiation and cognitive function, might be associated with the adiponectin receptor 1 (AdipoR1), which is a receptor for osmotin. Here, we explore whether osmotin's effects on long-term potentiation and memory function are associated with NgR1 signaling via AdipoR1 in Alzheimer's disease. Osmotin reduced the expression of NgR1 without affecting Nogo-A expression. Furthermore, osmotin inhibited NgR1 signaling by prohibiting the formation of the Nogo-A and NgR1 ligand-receptor complex, resulting in enhanced neurite outgrowth; these effects disappeared in the presence of AdipoR1 interference. In addition, osmotin increased the expression of the pre- and postsynaptic markers synaptophysin and PSD-95, as well as the activation of the memory-associated markers AMPA receptor and CREB; these effects occurred in an AdipoR1- and NgR1-dependent manner. Osmotin was also found to enhance dendritic complexity and spine density in the hippocampal region of Alzheimer's disease mouse brains. These results suggest that osmotin can enhance neurite outgrowth and synaptic complexity through AdipoR1 and NgR1 signaling, implying that osmotin might be an effective therapeutic agent for Alzheimer's disease and that AdipoR1 might be a crucial therapeutic target for neurodegenerative diseases such as Alzheimer's.

(2R,1'S,2'R)- and (2S,1'S,2'R)-3-[2-Mono(di,tri)fluoromethylcyclopropyl]alanines and their incorporation into hormaomycin analogues

PubMed Central

Kozhushkov, Sergei I; Yufit, Dmitrii S; Grosse, Christian; Kaiser, Marcel

2014-01-01

Summary Efficient and scalable syntheses of enantiomerically pure (2R,1'S,2'R)- and (2S,1'S,2'R)-3-[2-mono(di,tri)fluoromethylcyclopropyl]alanines 9a–c, as well as allo-D-threonine (4) and (2S,3R)-β-methylphenylalanine (3), using the Belokon' approach with (S)- and (R)-2-[(N-benzylprolyl)amino]benzophenone [(S)- and (R)-10] as reusable chiral auxiliaries have been developed. Three new fluoromethyl analogues of the naturally occurring octadepsipeptide hormaomycin (1) with (fluoromethylcyclopropyl)alanine moieties have been synthesized and subjected to preliminary tests of their antibiotic activity. PMID:25550751

Interleukin 1 Receptor (IL-1R1) Activation Exacerbates Toxin-Induced Acute Kidney Injury.

PubMed

Privratsky, Jamie R; Zhang, Jiandong; Lu, Xiaohan; Rudemiller, Nathan; Wei, Qingqing; Yu, Yen-Rei; Gunn, Michael Dee; Crowley, Steven D

2018-05-23

Acute kidney injury (AKI) is a leading cause of morbidity and mortality. Cisplatin is an effective chemotherapeutic agent whose administration is limited by nephrotoxicity. Therapies to prevent cisplatin-induced AKI are lacking. While tumor necrosis factor-α (TNF) plays a key role in the pathogenesis of cisplatin nephrotoxicity, the immune signaling pathways that trigger TNF generation in this context require elucidation. Sterile injury triggers the release and activation of both isoforms of interleukin(IL)-1, IL-1α and IL-1β, and stimulation of the interleukin-1 receptor (IL-1R1) by these ligands engages a pro-inflammatory signaling cascade that induces TNF induction. We therefore hypothesized that IL-1R1 activation exacerbates cisplatin-induced AKI by inducing TNF production thereby augmenting inflammatory signals between kidney parenchymal cells and infiltrating myeloid cells. IL-1R1+/+ (WT) and IL-1R1-/- (KO) mice were subjected to cisplatin-induced AKI. Compared to WT mice, IL-1R1 KO mice had attenuated AKI as measured by serum creatinine and BUN; renal NGAL mRNA levels; and blinded histological analysis of kidney pathology. In the cisplatin-injured kidney, IL-1R1 KO mice had diminished levels of whole kidney TNF and fewer Ly6G-expressing neutrophils. In addition, an unbiased machine learning analysis of intra-renal immune cells revealed a diminished number of CD11bint/CD11cint myeloid cells in IL-1R1 KO injured kidneys compared to IL-1R1 WT kidneys. Following cisplatin, IL-1R1 KO kidneys, compared to WTs, had fewer TNF-producing macrophages, CD11bint/CD11cint cells, and neutrophils, consistent with an effect of IL-1R1 to polarize intra-renal myeloid cells toward a pro-inflammatory phenotype. Interruption of IL-1-dependent signaling pathways warrants further evaluation to decrease nephrotoxicity during cisplatin therapy.

Activation of the umami taste receptor (T1R1/T1R3) initiates the peristaltic reflex and pellet propulsion in the distal colon.

PubMed

Kendig, Derek M; Hurst, Norman R; Bradley, Zachary L; Mahavadi, Sunila; Kuemmerle, John F; Lyall, Vijay; DeSimone, John; Murthy, Karnam S; Grider, John R

2014-12-01

Intraluminal nutrients in the gut affect the peristaltic reflex, although the mechanism is not well defined. Recent evidence supports the presence of taste receptors and their signaling components in enteroendocrine cells, although their function is unclear. This study aimed to determine if nutrients modify colonic motility through activation of taste receptors. Colonic sections were immunostained for the umami taste receptor T1R1/T1R3, which mediates the response to umami ligands, such as monosodium glutamate (MSG), in taste cells. Ascending contraction, descending relaxation, and calcitonin gene-related peptide release were measured in three-chamber flat-sheet preparations of rat colon in response to MSG alone or with inosine 5'-monophosphate (IMP). Velocity of artificial fecal pellet propulsion was measured by video recording in guinea pig distal colon. T1R1/T1R3 receptors were present in enteroendocrine cells of colonic sections from human, rat, mouse, and guinea pig. MSG initiated ascending contraction and descending relaxation components of the peristaltic reflex and calcitonin gene-related peptide release in flat-sheet preparations. IMP augmented the MSG-induced effects, suggesting activation of T1R1/T1R3 receptors. In T1R1(-/-) mice, mucosal stroking, but not MSG, elicited a peristaltic reflex. Intraluminal perfusion of MSG enhanced the velocity of artificial fecal pellet propulsion, which was also augmented by IMP. Propulsion was also increased by l-cysteine, but not l-tryptophan, supporting a role of T1R1/T1R3 receptors. We conclude that T1R1/T1R3 activation by luminal MSG or l-cysteine elicits a peristaltic reflex and CGRP release and increases the velocity of pellet propulsion in distal colon. This mechanism may explain how nutrients regulate colonic propulsion. Copyright © 2014 the American Physiological Society.

Activation of the umami taste receptor (T1R1/T1R3) initiates the peristaltic reflex and pellet propulsion in the distal colon

PubMed Central

Kendig, Derek M.; Hurst, Norman R.; Bradley, Zachary L.; Mahavadi, Sunila; Kuemmerle, John F.; Lyall, Vijay; DeSimone, John; Murthy, Karnam S.

2014-01-01

Intraluminal nutrients in the gut affect the peristaltic reflex, although the mechanism is not well defined. Recent evidence supports the presence of taste receptors and their signaling components in enteroendocrine cells, although their function is unclear. This study aimed to determine if nutrients modify colonic motility through activation of taste receptors. Colonic sections were immunostained for the umami taste receptor T1R1/T1R3, which mediates the response to umami ligands, such as monosodium glutamate (MSG), in taste cells. Ascending contraction, descending relaxation, and calcitonin gene-related peptide release were measured in three-chamber flat-sheet preparations of rat colon in response to MSG alone or with inosine 5′-monophosphate (IMP). Velocity of artificial fecal pellet propulsion was measured by video recording in guinea pig distal colon. T1R1/T1R3 receptors were present in enteroendocrine cells of colonic sections from human, rat, mouse, and guinea pig. MSG initiated ascending contraction and descending relaxation components of the peristaltic reflex and calcitonin gene-related peptide release in flat-sheet preparations. IMP augmented the MSG-induced effects, suggesting activation of T1R1/T1R3 receptors. In T1R1−/− mice, mucosal stroking, but not MSG, elicited a peristaltic reflex. Intraluminal perfusion of MSG enhanced the velocity of artificial fecal pellet propulsion, which was also augmented by IMP. Propulsion was also increased by l-cysteine, but not l-tryptophan, supporting a role of T1R1/T1R3 receptors. We conclude that T1R1/T1R3 activation by luminal MSG or l-cysteine elicits a peristaltic reflex and CGRP release and increases the velocity of pellet propulsion in distal colon. This mechanism may explain how nutrients regulate colonic propulsion. PMID:25324508

Standardized Procedures for Use of Nucleic Acid-Based Tools

DTIC Science & Technology

2008-08-01

Dhc size or cell wall characteristics including Brevundimonas diminuta (small), Micrococcus sp. (small coccoid) and Halobacterium sp. (Dhc-like cell...Halobacterium species as a Dhc surrogate. Another potential surrogate based on size and shape is Micrococcus species including Micrococcus luteus... Micrococcus luteus (ATCC-4442) are relatively small (1,000 nm) spherical bacteria (Madigan et al., 2006). Due to the fact that introduction and

Predicting IGF-1R therapy response in bone sarcomas: immuno-SPECT imaging with radiolabeled R1507

PubMed Central

Fleuren, Emmy D.G.; Versleijen-Jonkers, Yvonne M.H.; van de Luijtgaarden, Addy C.M.; Molkenboer-Kuenen, Janneke D.M.; Heskamp, Sandra; Roeffen, Melissa H.S.; van Laarhoven, Hanneke W.M.; Houghton, Peter J.; Oyen, Wim J.G.; Boerman, Otto C.; van der Graaf, Winette T.A.

2011-01-01

Purpose To investigate whether 111In-R1507 immuno-SPECT, a novel non-invasive, in vivo screening method to visualize membranous Insulin-like Growth Factor 1 Receptor (IGF-1R) expression and accessibility, can be used to predict IGF-1R treatment (R1507) responsein bone sarcomas. Experimental design BALB/c nude mice were subcutaneously implanted with IGF-1R-expressing human bone sarcoma xenografts (OS-1, EW-5 and EW-8) which demonstrated high, modest or no response, respectively, to R1507, a monoclonal antibody targeting the extracellular domain of IGF-1R. An IGF-1R-negative tumor (OS-33), unresponsive to IGF-1R inhibitors, was examined as well. Mice were injected with indium-111 labeled R1507 (111In-R1507). Biodistribution and immuno-SPECT/CT imaging studies were performed 1, 3 and 7 days p.i. in mice with OS-1 and EW-5 xenografts and 3 days p.i. in mice with EW-8 and OS-33 xenografts. Results Biodistribution studies showed specific accumulation of 111In-R1507 in OS-1 and EW-5 xenografts (27.5±6.5%ID/g and 14.0±2.8%ID/g, 3 days p.i., respectively). Most importantly, 111In-R1507 uptake in IGF-1R-positive, but unresponsive, EW-8 xenografts (6.5±1.5%ID/g, 3 days p.i.) was similar to that of the IGF-1R-negative OS-33 tumor (5.5±0.6%ID/g, 3 days p.i.). Uptake in normal tissues was low and non-specific. Corresponding immuno-SPECT images clearly discriminated between high, modest and non-responding tumors by demonstrating a homogeneous (OS-1), heterogeneous (EW-5) or non-specific (EW-8 and OS-33)tumor uptake of 111In-R1507. Conclusions 111In-R1507 immuno-SPECT is an excellent method to visualize membranous IGF-1R expression and target accessibility in vivo in human bone sarcoma xenografts and may serve as an independent marker to predict IGF-1R therapy (R1507) responsein bone sarcoma patients. PMID:22038993

Thermopower of CexR1-xB6 (R=La, Pr and Nd)

NASA Astrophysics Data System (ADS)

Kim, Moo‑Sung; Nakai, Yuki; Tou, Hideki; Sera, Masafumi; Iga, Fumitoshi; Takabatake, Toshiro; Kunii, Satoru

2006-06-01

The thermopower, S, of CexR1-xB6 (R=La, Pr, Nd) was investigated. S with a positive sign shows a typical behavior observed in the Ce Kondo system, an increase with decreasing temperature at high temperatures and a maximum at low temperatures. The S values of all the systems at high temperatures are roughly linearly dependent on the Ce concentration, indicating the conservation of the single-impurity character of the Kondo effect in a wide x range. However, the maximum value of S, Smax, and the temperature, Tmax, at which Smax is observed exhibit different x dependences between CexLa1-xB6 and CexR1-xB6 (R=Pr, Nd). In CexLa1-xB6, Tmax, which is ˜8 K in CeB6, decreases with decreasing x and converges to ˜1 K in a very dilute alloy and Smax shows an increase below x ˜ 0.1 after decreasing with decreasing x. In CexR1-xB6 (R=Pr, Nd), Tmax shows a weak x dependence but Smax shows a roughly linear decrease in x. These results are discussed from the standpoint of the chemical pressure effect and the Ce-Ce interaction. S in the long-range ordered phase shows very different behaviors between CexPr1-xB6 and CexNd1-xB6.

miR-598 acts as a tumor suppressor in human gastric cancer by targeting IGF-1R.

PubMed

Liu, Na; Yang, Hua; Wang, Hong

2018-01-01

In recent years, the aberrant expression of miR-598 in tumorigenesis has been demonstrated, as well as the fact that the IGF-1R pathway is also involved in the development of human gastric cancer (GC). The present study aimed to investigate the molecular mechanisms underlying miR-598-regulated IGF-1R expression in human GC. We analyzed the expression of miR-598 and IGF-1R in GC samples and cells, and evaluated the clinical significance of miR-598 and IGF-1R in GC patients. Furthermore, in vitro and in vivo assays were used to investigate the molecular mechanisms of miR-598 and IGF-1R. miR-598 expression was frequently downregulated in GC tissues and cells, and significantly correlated with poor prognosis, vascular invasion, TNM stage, and lymph node metastases as well as IGF-1R expression. The overexpression of miR-598 obviously inhibited cell proliferation, migration, invasion, and induced cell cycle arrest in the G1/S phase, and increased the apoptosis of GC cells. The overexpression of miR-598 also significantly inhibited ERK1/2 and Akt phosphorylation level. In vivo assay validated the inhibitory effect of miR-598 on tumor growth. Further studies showed that miR-598 inhibited IGF-1R protein expression by directly targeting its 3'-UTR. Besides, over-expression of IGF-1R reversed the inhibitory effects of miR-598, while suppression of IGF-1R expression showed inverse effects. miR-598 suppresses GC cell proliferation, migration and invasion by directly targeting IGF-1R expression. Thus, miR-598 may be a useful target for GC patients.

26 CFR 1.414(r)-0 - Table of contents.

Code of Federal Regulations, 2011 CFR

2011-04-01

... 26 Internal Revenue 5 2011-04-01 2011-04-01 false Table of contents. 1.414(r)-0 Section 1.414(r)-0...) INCOME TAXES (CONTINUED) Pension, Profit-Sharing, Stock Bonus Plans, Etc. § 1.414(r)-0 Table of contents. (a) In general. Sections 1.414(r)-1 through 1.414(r)-11 provide rules for determining whether an...

26 CFR 1.414(r)-0 - Table of contents.

Code of Federal Regulations, 2013 CFR

2013-04-01

... 26 Internal Revenue 5 2013-04-01 2013-04-01 false Table of contents. 1.414(r)-0 Section 1.414(r)-0...) INCOME TAXES (CONTINUED) Pension, Profit-Sharing, Stock Bonus Plans, Etc. § 1.414(r)-0 Table of contents. (a) In general. Sections 1.414(r)-1 through 1.414(r)-11 provide rules for determining whether an...

26 CFR 1.414(r)-0 - Table of contents.

Code of Federal Regulations, 2012 CFR

2012-04-01

... 26 Internal Revenue 5 2012-04-01 2011-04-01 true Table of contents. 1.414(r)-0 Section 1.414(r)-0...) INCOME TAXES (CONTINUED) Pension, Profit-Sharing, Stock Bonus Plans, Etc. § 1.414(r)-0 Table of contents. (a) In general. Sections 1.414(r)-1 through 1.414(r)-11 provide rules for determining whether an...

26 CFR 1.414(r)-0 - Table of contents.

Code of Federal Regulations, 2014 CFR

2014-04-01

... 26 Internal Revenue 5 2014-04-01 2014-04-01 false Table of contents. 1.414(r)-0 Section 1.414(r)-0...) INCOME TAXES (CONTINUED) Pension, Profit-Sharing, Stock Bonus Plans, Etc. § 1.414(r)-0 Table of contents. (a) In general. Sections 1.414(r)-1 through 1.414(r)-11 provide rules for determining whether an...

Dynamic Metabolite Profiling in an Archaeon Connects Transcriptional Regulation to Metabolic Consequences.

PubMed

Todor, Horia; Gooding, Jessica; Ilkayeva, Olga R; Schmid, Amy K

2015-01-01

Previous work demonstrated that the TrmB transcription factor is responsible for regulating the expression of many enzyme-coding genes in the hypersaline-adapted archaeon Halobacterium salinarum via a direct interaction with a cis-regulatory sequence in their promoters. This interaction is abolished in the presence of glucose. Although much is known about the effects of TrmB at the transcriptional level, it remains unclear whether and to what extent changes in mRNA levels directly affect metabolite levels. In order to address this question, here we performed a high-resolution metabolite profiling time course during a change in nutrients using a combination of targeted and untargeted methods in wild-type and ΔtrmB strain backgrounds. We found that TrmB-mediated transcriptional changes resulted in widespread and significant changes to metabolite levels across the metabolic network. Additionally, the pattern of growth complementation using various purines suggests that the mis-regulation of gluconeogenesis in the ΔtrmB mutant strain in the absence of glucose results in low phosphoribosylpyrophosphate (PRPP) levels. We confirmed these low PRPP levels using a quantitative mass spectrometric technique and found that they are associated with a metabolic block in de novo purine synthesis, which is partially responsible for the growth defect of the ΔtrmB mutant strain in the absence of glucose. In conclusion, we show how transcriptional regulation of metabolism affects metabolite levels and ultimately, phenotypes.

Electron-impact excitation of the 1{sup 1}{ital S}{r_arrow}3{sup 1}{ital P} and 1{sup 1}{ital S}{r_arrow}4{sup 1}{ital P} transitions in helium

DOE Office of Scientific and Technical Information (OSTI.GOV)

Khakoo, M.A.; Roundy, D.; Rugamas, F.

1996-11-01

The method of electron-photon coincidence is used to {open_quote}{open_quote}resolve{close_quote}{close_quote} the electron-impact excitation of the {ital n}{sup 1}{ital P} levels ({ital n}=3 and 4) from nearby levels. Experimentally determined ratios of the differential cross sections for the electron-impact excitation of 1{sup 1}{ital S}{r_arrow}2{sup 1}{ital P}, to 1{sup 1}{ital S}{r_arrow}3{sup 1}{ital P}, and 4{sup 1}{ital P} transitions are presented at 30-, 40-, and 80-eV incident electron energies. Differential cross sections for the 1{sup 1}{ital S}{r_arrow}3{sup 1}{ital P} and 1{sup 1}{ital S}{r_arrow}4{sup 1}{ital P} transitions are derived by normalizing these ratios to available experimental differential cross sections for the 1{sup 1}{ital S}{r_arrow}2{sup 1}{italmore » P} transition. The ratios and differential cross sections are compared to available theoretical and semiempirical data. {copyright} {ital 1996 The American Physical Society.}« less

Palmitoylation-dependent activation of MC1R prevents melanomagenesis

PubMed Central

Chen, Shuyang; Zhu, Bo; Yin, Chengqian; Liu, Wei; Han, Changpeng; Chen, Baoen; Liu, Tongzheng; Li, Xin; Chen, Xiang; Li, Chunying; Hu, Limin; Zhou, Jun; Xu, Zhi-Xiang; Gao, Xiumei; Wu, Xu; Goding, Colin R.; Cui, Rutao

2017-01-01

The melanocortin-1 receptor (MC1R), a G protein-coupled receptor, plays a crucial role in human and mouse pigmentation1–8. Activation of MC1R in melanocytes by α-melanocyte-stimulating hormone (α-MSH)9 stimulates cAMP signaling and melanin production and enhances DNA repair after UV irradiation (UVR)10–16. Individuals carrying MC1R variants, especially those associated with red hair color, fair skin and poor tanning ability (RHC-variants), are associated with higher risk of melanoma5,17,18,19,20. However, how MC1R activity might be modulated by UV irradiation, why redheads are more prone to developing melanoma, and whether the activity of RHC variants might be restored for therapeutic benefit remain unresolved questions. Here we demonstrate a potential MC1R-targeted intervention strategy to rescue loss-of-function MC1R in MC1R RHC-variants for therapeutic benefit based on activating MC1R protein palmitoylation. Specifically, MC1R palmitoylation, primarily mediated by the protein-acyl transferase (PAT) ZDHHC13, is essential for activating MC1R signaling that triggers increased pigmentation, UVB-induced G1-like cell cycle arrest and control of senescence and melanomagenesis in vitro and in vivo. Using C57BL/6J-MC1Re/eJ mice expressing MC1R RHC-variants we show that pharmacological activation of palmitoylation rescues the defects of MC1R RHC-variants and prevents melanomagenesis. The results highlight a central role for MC1R palmitoylation in pigmentation and protection against melanoma. PMID:28869973

Rapid dynamic R1 /R2 */temperature assessment: a method with potential for monitoring drug delivery.

PubMed

Lorenzato, Cyril; Oerlemans, Chris; Cernicanu, Alexandru; Ries, Mario; Denis de Senneville, Baudouin; Moonen, Chrit; Bos, Clemens

2014-11-01

Local drug delivery by hyperthermia-induced drug release from thermosensitive liposomes (TSLs) may reduce the systemic toxicity of chemotherapy, whilst maintaining or increasing its efficacy. Relaxivity contrast agents can be co-encapsulated with the drug to allow the visualization of the presence of liposomes, by means of R2 *, as well as the co-release of the contrast agent and the drug, by means of R1, on heating. Here, the mathematical method used to extract both R2 * and R1 from a fast dynamic multi-echo spoiled gradient echo (ME-SPGR) is presented and analyzed. Finally, this method is used to monitor such release events. R2 * was obtained from a fit to the ME-SPGR data. Absolute R1 was calculated from the signal magnitude changes corrected for the apparent proton density changes and a baseline Look-Locker R1 map. The method was used to monitor nearly homogeneous water bath heating and local focused ultrasound heating of muscle tissue, and to visualize the release of a gadolinium chelate from TSLs in vitro. R2 *, R1 and temperature maps were measured with a 5-s temporal resolution. Both R2 *and R1 measured were found to change with temperature. The dynamic R1 measurements after heating agreed with the Look-Locker R1 values if changes in equilibrium magnetization with temperature were considered. Release of gadolinium from TSLs was detected by an R1 increase near the phase transition temperature, as well as a shallow R2 * increase. Simultaneous temperature, R2 * and R1 mapping is feasible in real time and has the potential for use in image-guided drug delivery studies. Copyright © 2014 John Wiley & Sons, Ltd.

ADCYAP1R1 and asthma in Puerto Rican children.

PubMed

Chen, Wei; Boutaoui, Nadia; Brehm, John M; Han, Yueh-Ying; Schmitz, Cassandra; Cressley, Alex; Acosta-Pérez, Edna; Alvarez, María; Colón-Semidey, Angel; Baccarelli, Andrea A; Weeks, Daniel E; Kolls, Jay K; Canino, Glorisa; Celedón, Juan C

2013-03-15

Epigenetic and/or genetic variation in the gene encoding the receptor for adenylate-cyclase activating polypeptide 1 (ADCYAP1R1) has been linked to post-traumatic stress disorder in adults and anxiety in children. Psychosocial stress has been linked to asthma morbidity in Puerto Rican children. To examine whether epigenetic or genetic variation in ADCYAP1R1 is associated with childhood asthma in Puerto Ricans. We conducted a case-control study of 516 children ages 6-14 years living in San Juan, Puerto Rico. We assessed methylation at a CpG site in the promoter of ADCYAP1R1 (cg11218385) using a pyrosequencing assay in DNA from white blood cells. We tested whether cg11218385 methylation (range, 0.4-6.1%) is associated with asthma using logistic regression. We also examined whether exposure to violence (assessed by the Exposure to Violence [ETV] Scale in children 9 yr and older) is associated with cg11218385 methylation (using linear regression) or asthma (using logistic regression). Logistic regression was used to test for association between a single nucleotide polymorphism in ADCYAP1R1 (rs2267735) and asthma under an additive model. All multivariate models were adjusted for age, sex, household income, and principal components. EACH 1% increment in cg11218385 methylation was associated with increased odds of asthma (adjusted odds ratio, 1.3; 95% confidence interval, 1.0-1.6; P = 0.03). Among children 9 years and older, exposure to violence was associated with cg11218385 methylation. The C allele of single nucleotide polymorphism rs2267735 was significantly associated with increased odds of asthma (adjusted odds ratio, 1.3; 95% confidence interval, 1.02-1.67; P = 0.03). Epigenetic and genetic variants in ADCYAP1R1 are associated with asthma in Puerto Rican children.

Cannabinoid-1 receptor (CB1R) blockers as medicines: beyond obesity and cardiometabolic disorders to substance abuse/drug addiction with CB1R neutral antagonists.

PubMed

Janero, David R

2012-03-01

Addiction to chemical substances with abuse potential presents medical needs largely unsolved by extant therapeutic strategies. Signal transmission through the cannabinoid-1 receptor (CB1R) in the central nervous system (CNS) modulates neurotransmitters/neuronal pathways contributing to the rewarding properties and hedonic effects of certain nondrug stimuli (e.g., food) and many prototypical addictive drugs, promoting excessive intake and its pathological consequences. Typical CB1R antagonists/inverse agonists reduce the rewarding effects and normalize behavioral phenotypes associated with food and abused drugs, but carry an unacceptable adverse-event profile that may reflect, at least partly, their intrinsic ability to alter basal homeostatic CB1R signaling in the CNS and elicit a negative efficacy response. Alternatively, peripherally biased CB1R inverse agonists with limited CNS permeability and putative CB1R neutral antagonists expressing modest (if any) inverse-agonist efficacy are garnering attention for treating obesity and related cardiometabolic complications with a potentially enhanced benefit-to-risk profile. This mini-review calls attention to the proposition that CB1R neutral antagonists offer attractive opportunities for pharmacotherapeutic exploitation in the substance abuse/drug addiction space, whereas the restricted CNS accessibility of peripherally biased CB1R inverse agonists circumscribes their therapeutic utility for this indication. The unique preclinical pharmacology, efficacy profiles, and reduced adverse-event risk of CB1R neutral antagonists make them worthy of translational study for their potential therapeutic application beyond obesity/cardiometabolic disease to include substance-abuse/drug-addiction disorders.

BCoR-L1 variation and breast cancer.

PubMed

Lose, Felicity; Arnold, Jeremy; Young, David B; Brown, Carolyn J; Mann, Graham J; Pupo, Gulietta M; Khanna, Kum Kum; Chenevix-Trench, Georgia; Spurdle, Amanda B

2007-01-01

BRCA1 is involved in numerous essential processes in the cell, and the effects of BRCA1 dysfunction in breast cancer carcinogenesis are well described. Many of the breast cancer susceptibility genes such as BRCA2, p53, ATM, CHEK2, and BRIP1 encode proteins that interact with BRCA1. BCL6 corepressor-like 1 (BCoR-L1) is a newly described BRCA1-interacting protein that displays high homology to several proteins known to be involved in the fundamental processes of DNA damage repair and transcription regulation. BCoR-L1 has been shown to play a role in transcription corepression, and expression of the X-linked BCoR-L1 gene has been reported to be dysregulated in breast cancer subjects. BCoR-L1 is located on the X chromosome and is subject to X inactivation. We performed mutation analysis of 38 BRCA1/2 mutation-negative breast cancer families with male breast cancer, prostate cancer, and/or haplotype sharing around BCoR-L1 to determine whether there is a role for BCoR-L1 as a high-risk breast cancer predisposition gene. In addition, we conducted quantitative real-time PCR (qRT-PCR) on lymphoblastoid cell lines (LCLs) from the index cases from these families and a number of cancer cell lines to assess the role of BCoR-L1 dysregulation in cancer and cancer families. Very little variation was detected in the coding region, and qRT-PCR analysis revealed that BCoR-L1 expression is highly variable in cancer-free subjects, high-risk breast cancer patients, and cancer cell lines. We also report the investigation of a new expression control, DIDO1 (death inducer-obliterator 1), that is superior to GAPDH (glyceraldehyde-3-phosphate dehydrogenase) and UBC (ubiquitin C) for analysis of expression in LCLs. Our results suggest that BCoR-L1 expression does not play a large role in predisposition to familial breast cancer.

Starvation-Survival in Haloarchaea.

PubMed

Winters, Yaicha D; Lowenstein, Tim K; Timofeeff, Michael N

2015-11-12

Recent studies claiming to revive ancient microorganisms trapped in fluid inclusions in halite have warranted an investigation of long-term microbial persistence. While starvation-survival is widely reported for bacteria, it is less well known for halophilic archaea-microorganisms likely to be trapped in ancient salt crystals. To better understand microbial survival in fluid inclusions in ancient evaporites, laboratory experiments were designed to simulate growth of halophilic archaea under media-rich conditions, complete nutrient deprivation, and a controlled substrate condition (glycerol-rich) and record their responses. Haloarchaea used for this work included Hbt. salinarum and isolate DV582A-1 (genus Haloterrigena) sub-cultured from 34 kyear Death Valley salt. Hbt. salinarum and DV582A-1 reacted to nutrient limitation with morphological and population changes. Starved populations increased and most cells converted from rods to small cocci within 56 days of nutrient deprivation. The exact timing of starvation adaptations and the physical transformations differed between species, populations of the same species, and cells of the same population. This is the first study to report the timing of starvation strategies for Hbt. salinarum and DV582A-1. The morphological states in these experiments may allow differentiation between cells trapped with adequate nutrients (represented here by early stages in nutrient-rich media) from cells trapped without nutrients (represented here by experimental starvation) in ancient salt. The hypothesis that glycerol, leaked from Dunaliella, provides nutrients for the survival of haloarchaea trapped in fluid inclusions in ancient halite, is also tested. Hbt. salinarum and DV582A-1 were exposed to a mixture of lysed and intact Dunaliella for 56 days. The ability of these organisms to utilize glycerol from Dunaliella cells was assessed by documenting population growth, cell length, and cell morphology. Hbt. salinarum and DV582A-1

CSF-1R Inhibitor Development: Current Clinical Status.

PubMed

Peyraud, Florent; Cousin, Sophie; Italiano, Antoine

2017-09-05

Colony-stimulating factor 1 receptor (CSF-1R) and its ligands, CSF-1 and interleukin 34 (IL-34), regulate the function and survival of tumor-associated macrophages, which are involved in tumorigenesis and in the suppression of antitumor immunity. Moreover, the CSF-1R/CSF-1 axis has been implicated in the pathogenesis of pigmented villonodular synovitis (PVNS), a benign tumor of the synovium. As advanced or metastatic malignant solid tumors and relapsed/refractory PVNS remain unresolved therapeutic problems, new approaches are needed to improve the outcome of patients with these conditions. In solid tumors, targeting CSF-1R via either small molecules or antibodies has shown interesting results in vitro but limited antitumor activity in vivo. Concerning PVNS, clinical trials assessing CSF-1R inhibitors have revealed promising initial outcomes. Blocking CSF-1/CSF-1R signaling represents a promising immunotherapy approach and several new potential combination therapies for future clinical testing.

The primary structures of ribosomal proteins S14 and S16 from the archaebacterium Halobacterium marismortui. Comparison with eubacterial and eukaryotic ribosomal proteins.

PubMed

Kimura, J; Kimura, M

1987-09-05

The amino acid sequences of two ribosomal proteins, S14 and S16, from the archaebacterium Halobacterium marismortui have been determined. Sequence data were obtained by the manual and solid-phase sequencing of peptides derived from enzymatic digestions with trypsin, chymotrypsin, pepsin, and Staphylococcus aureus protease as well as by chemical cleavage with cyanogen bromide. Proteins S14 and S16 contain 109 and 126 amino acid residues and have Mr values of 11,964 and 13,515, respectively. Comparison of the sequences with those of ribosomal proteins from other organisms demonstrates that S14 has a significant homology with the rat liver ribosomal protein S11 (36% identity) as well as with the Escherichia coli ribosomal protein S17 (37%), and that S16 is related to the yeast ribosomal protein YS22 (40%) and proteins S8 from E. coli (28%) and Bacillus stearothermophilus (30%). A comparison of the amino acid residues in the homologous regions of halophilic and nonhalophilic ribosomal proteins reveals that halophilic proteins have more glutamic acids, asparatic acids, prolines, and alanines, and less lysines, arginines, and isoleucines than their nonhalophilic counterparts. These amino acid substitutions probably contribute to the structural stability of halophilic ribosomal proteins.

Compensatory insulin receptor (IR) activation on inhibition of insulin-like growth factor-1 receptor (IGF-1R): rationale for cotargeting IGF-1R and IR in cancer.

PubMed

Buck, Elizabeth; Gokhale, Prafulla C; Koujak, Susan; Brown, Eric; Eyzaguirre, Alexandra; Tao, Nianjun; Rosenfeld-Franklin, Maryland; Lerner, Lorena; Chiu, M Isabel; Wild, Robert; Epstein, David; Pachter, Jonathan A; Miglarese, Mark R

2010-10-01

Insulin-like growth factor-1 receptor (IGF-1R) is a receptor tyrosine kinase (RTK) and critical activator of the phosphatidylinositol 3-kinase-AKT pathway. IGF-1R is required for oncogenic transformation and tumorigenesis. These observations have spurred anticancer drug discovery and development efforts for both biological and small-molecule IGF-1R inhibitors. The ability for one RTK to compensate for another to maintain tumor cell viability is emerging as a common resistance mechanism to antitumor agents targeting individual RTKs. As IGF-1R is structurally and functionally related to the insulin receptor (IR), we asked whether IR is tumorigenic and whether IR-AKT signaling contributes to resistance to IGF-1R inhibition. Both IGF-1R and IR(A) are tumorigenic in a mouse mammary tumor model. In human tumor cells coexpressing IGF-1R and IR, bidirectional cross talk was observed following either knockdown of IR expression or treatment with a selective anti-IGF-1R antibody, MAB391. MAB391 treatment resulted in a compensatory increase in phospho-IR, which was associated with resistance to inhibition of IRS1 and AKT. In contrast, treatment with OSI-906, a small-molecule dual inhibitor of IGF-1R/IR, resulted in enhanced reduction in phospho-IRS1/phospho-AKT relative to MAB391. Insulin or IGF-2 activated the IR-AKT pathway and decreased sensitivity to MAB391 but not to OSI-906. In tumor cells with an autocrine IGF-2 loop, both OSI-906 and an anti-IGF-2 antibody reduced phospho-IR/phospho-AKT, whereas MAB391 was ineffective. Finally, OSI-906 showed superior efficacy compared with MAB391 in human tumor xenograft models in which both IGF-1R and IR were phosphorylated. Collectively, these data indicate that cotargeting IGF-1R and IR may provide superior antitumor efficacy compared with targeting IGF-1R alone.

Rat organic solute carrier protein 1 (rOscp1) mediated the transport of organic solutes in Xenopus laevis oocytes: isolation and pharmacological characterization of rOscp1.

PubMed

Izuno, Hisanori; Kobayashi, Yasuna; Sanada, Yutaka; Nihei, Daisuke; Suzuki, Masako; Kohyama, Noriko; Ohbayashi, Masayuki; Yamamoto, Toshinori

2007-09-22

Rat organic solute carrier protein 1 (rOscp1) was isolated from a rat testis cDNA library. Isolated rOscp1 cDNA consisted of 1089 base pairs that encoded a 363-amino acid protein, and the amino acid sequence was 88% and 93% identical to that of human OSCP1 (hOSCP1) and mouse Oscp1 (mOscp1), respectively. The message for rOscp1 is highly detected in rat testis. When expressed in X. oocytes, rOscp1 mediated the high affinity transport of p-aminohippurate (PAH) with a Km value of 15.7+/-1.9 microM, and rOscp1-mediated organic solutes were exhibited in time- and Na+-independent manners. rOscp1 also transported various structurally heterogenous compounds such as testosterone, dehydroepiandrosterone sulfate (DHEA-S), and taurocholate with some differences in substrate specificity compared with hOSCP1. Immunohistochemical analysis revealed that the rOscp1 protein is localized in the basal membrane side of Sertoli cells as observed in mouse testis [Kobayashi et al., 2007; Kobayashi, Y., Tsuchiya, A., Hayashi, T., Kohyama, N., Ohbayashi, M., Yamamoto, T., 2007. Isolation and characterization of polyspecific mouse organic solute carrier protein 1 (mOscp1). Drug Metabolism and Disposition 35 (7), 1239-1245]. Thus, the present results indicate that a newly isolated cDNA clone, rOscp1, is a polyspecific organic solute carrier protein with some differences in substrate specificity compared with human and mouse OSCP1.

Role of nutrient-sensing taste 1 receptor (T1R) family members in gastrointestinal chemosensing.

PubMed

Shirazi-Beechey, Soraya P; Daly, Kristian; Al-Rammahi, Miran; Moran, Andrew W; Bravo, David

2014-06-01

Luminal nutrient sensing by G-protein-coupled receptors (GPCR) expressed on the apical domain of enteroendocrine cells activates intracellular pathways leading to secretion of gut hormones that control vital physiological processes such as digestion, absorption, food intake and glucose homeostasis. The taste 1 receptor (T1R) family of GPCR consists of three members: T1R1; T1R2; T1R3. Expression of T1R1, T1R2 and T1R3 at mRNA and protein levels has been demonstrated in the intestinal tissue of various species. It has been shown that T1R2-T1R3, in association with G-protein gustducin, is expressed in intestinal K and L endocrine cells, where it acts as the intestinal glucose (sweet) sensor. A number of studies have demonstrated that activation of T1R2-T1R3 by natural sugars and artificial sweeteners leads to secretion of glucagon-like peptides 1&2 (GLP-1 and GLP-2) and glucose dependent insulinotropic peptide (GIP). GLP-1 and GIP enhance insulin secretion; GLP-2 increases intestinal growth and glucose absorption. T1R1-T1R3 combination co-expressed on the apical domain of cholecystokinin (CCK) expressing cells is a luminal sensor for a number of L-amino acids; with amino acid-activation of the receptor eliciting CCK secretion. This article focuses on the role of the gut-expressed T1R1, T1R2 and T1R3 in intestinal sweet and L-amino acid sensing. The impact of exploiting T1R2-T1R3 as a nutritional target for enhancing intestinal glucose absorption and gut structural maturity in young animals is also highlighted.

Hydrologic Tests at Characterization Wells R-9i, R-13, R-19, R-22, and R-31, Revision 1

DOE Office of Scientific and Technical Information (OSTI.GOV)

S.G.McLin; W.J. Stone

2004-06-01

Hydrologic information is essential for environmental efforts at Los Alamos National Laboratory. Testing at new characterization wells being drilled to the regional aquifer (''R wells'') to improve the conceptual hydrogeologic model of the Pajarito Plateau is providing such information. Field tests were conducted on various zones of saturation penetrated by the R wells to collect data needed for determining hydraulic properties. This document provides details of the design and execution of testing as well as an analysis of data for five new wells: R-9i, R-13, R-19, R-22, and R-31. One well (R-13) was evaluated by a pumping test and themore » rest (R-9i, R-19, R-22, and R-31) were evaluated by injection tests. Characterization well R-9i is located in Los Alamos Canyon approximately 0.3 mi west of the Route 4/Route 502 intersection. It was completed at a depth of 322 ft below ground surface (bgs) in March 2000. This well was constructed with two screens positioned below the regional water table. Both screens were tested. Screen 1 is completed at about 189-200 ft bgs in fractured basalt, and screen 2 is completed at about 270-280 ft bgs in massive basalt. Specific capacity analysis of the screen 1 data suggests that the fractured basalt has a transmissivity (T) of 589 ft{sup 2}/day and corresponds to a hydraulic conductivity (K) of 7.1 ft/day based on a saturated thickness of 83 ft. The injection test data from the massive basalt near screen 2 were analyzed by the Bouwer-Rice slug test methodology and suggest that K is 0.11 ft/day, corresponding to a T of about 2.8 ft{sup 2}/day based on a saturated thickness of 25 ft. Characterization well R-13 is located in Mortandad Canyon just west of the eastern Laboratory boundary. It was completed at a depth of 1029 ft bgs in February 2002. This well was constructed with one 60-ft long screen positioned about 125 ft below the regional water table. This screen is completed at about 958-1019 ft bgs and straddles the geologic

Serotonin is an endogenous regulator of intestinal CYP1A1 via AhR.

PubMed

Manzella, Christopher; Singhal, Megha; Alrefai, Waddah A; Saksena, Seema; Dudeja, Pradeep K; Gill, Ravinder K

2018-04-17

Aryl hydrocarbon receptor (AhR) is a nuclear receptor that controls xenobiotic detoxification via induction of cytochrome P450 1A1 (CYP1A1) and regulates immune responses in the intestine. Metabolites of L-tryptophan activate AhR, which confers protection against intestinal inflammation. We tested the hypothesis that serotonin (5-HT) is an endogenous activator of AhR in intestinal epithelial cells. Treatment of Caco-2 monolayers with 5-HT induced CYP1A1 mRNA in a time- and concentration-dependent manner and also stimulated CYP1A1 activity. CYP1A1 induction by 5-HT was dependent upon uptake via serotonin transporter (SERT). Antagonism of AhR and knockdown of AhR and its binding partner aryl hydrocarbon receptor nuclear translocator (ARNT) attenuated CYP1A1 induction by 5-HT. Activation of AhR was evident by its nuclear translocation after 5-HT treatment and by induction of an AhR-responsive luciferase reporter. In vivo studies showed a dramatic decrease in CYP1A1 expression and other AhR target genes in SERT KO ileal mucosa by microarray analysis. These results suggest that intracellular accumulation of 5-HT via SERT induces CYP1A1 expression via AhR in intestinal epithelial cells, and SERT deficiency in vivo impairs activation of AhR. Our studies provide a novel link between the serotonergic and AhR pathways which has implications in xenobiotic metabolism and intestinal inflammation.

26 CFR 1.414(r)-1 - Requirements applicable to qualified separate lines of business.

Code of Federal Regulations, 2010 CFR

2010-04-01

... lines of business. 1.414(r)-1 Section 1.414(r)-1 Internal Revenue INTERNAL REVENUE SERVICE, DEPARTMENT... Plans, Etc. § 1.414(r)-1 Requirements applicable to qualified separate lines of business. (a) In general. Section 414(r) prescribes the conditions under which an employer is treated as operating qualified...

26 CFR 1.414(r)-1 - Requirements applicable to qualified separate lines of business.

Code of Federal Regulations, 2013 CFR

2013-04-01

... lines of business. 1.414(r)-1 Section 1.414(r)-1 Internal Revenue INTERNAL REVENUE SERVICE, DEPARTMENT..., Stock Bonus Plans, Etc. § 1.414(r)-1 Requirements applicable to qualified separate lines of business. (a) In general. Section 414(r) prescribes the conditions under which an employer is treated as operating...

26 CFR 1.414(r)-1 - Requirements applicable to qualified separate lines of business.

Code of Federal Regulations, 2011 CFR

2011-04-01

... lines of business. 1.414(r)-1 Section 1.414(r)-1 Internal Revenue INTERNAL REVENUE SERVICE, DEPARTMENT..., Stock Bonus Plans, Etc. § 1.414(r)-1 Requirements applicable to qualified separate lines of business. (a) In general. Section 414(r) prescribes the conditions under which an employer is treated as operating...

26 CFR 1.414(r)-1 - Requirements applicable to qualified separate lines of business.

Code of Federal Regulations, 2014 CFR

2014-04-01

... lines of business. 1.414(r)-1 Section 1.414(r)-1 Internal Revenue INTERNAL REVENUE SERVICE, DEPARTMENT..., Stock Bonus Plans, Etc. § 1.414(r)-1 Requirements applicable to qualified separate lines of business. (a) In general. Section 414(r) prescribes the conditions under which an employer is treated as operating...

26 CFR 1.414(r)-1 - Requirements applicable to qualified separate lines of business.

Code of Federal Regulations, 2012 CFR

2012-04-01

... lines of business. 1.414(r)-1 Section 1.414(r)-1 Internal Revenue INTERNAL REVENUE SERVICE, DEPARTMENT..., Stock Bonus Plans, Etc. § 1.414(r)-1 Requirements applicable to qualified separate lines of business. (a) In general. Section 414(r) prescribes the conditions under which an employer is treated as operating...

miR-193b Regulates Mcl-1 in Melanoma

PubMed Central

Chen, Jiamin; Zhang, Xiao; Lentz, Cindy; Abi-Daoud, Marie; Paré, Geneviève C.; Yang, Xiaolong; Feilotter, Harriet E.; Tron, Victor A.

2011-01-01

MicroRNAs play important roles in gene regulation, and their expression is frequently dysregulated in cancer cells. In a previous study, we reported that miR-193b represses cell proliferation and regulates cyclin D1 in melanoma cells, suggesting that miR-193b could act as a tumor suppressor. Herein, we demonstrate that miR-193b also down-regulates myeloid cell leukemia sequence 1 (Mcl-1) in melanoma cells. MicroRNA microarray profiling revealed that miR-193b is expressed at a significantly lower level in malignant melanoma than in benign nevi. Consistent with this, Mcl-1 is detected at a higher level in malignant melanoma than in benign nevi. In a survey of melanoma samples, the level of Mcl-1 is inversely correlated with the level of miR-193b. Overexpression of miR-193b in melanoma cells represses Mcl-1 expression. Previous studies showed that Mcl-1 knockdown cells are hypersensitive to ABT-737, a small-molecule inhibitor of Bcl-2, Bcl-XL, and Bcl-w. Similarly, overexpression of miR-193b restores ABT-737 sensitivity to ABT-737–resistant cells. Furthermore, the effect of miR-193b on the expression of Mcl-1 seems to be mediated by direct interaction between miR-193b and seed and seedless pairing sequences in the 3′ untranslated region of Mcl-1 mRNA. Thus, this study provides evidence that miR-193b directly regulates Mcl-1 and that down-regulation of miR-193b in vivo could be an early event in melanoma progression. PMID:21893020

MicroRNAs modulating angiogenesis: miR-129-1 and miR-133 act as angio-miR in HUVECs.

PubMed

Soufi-Zomorrod, Mina; Hajifathali, Abbas; Kouhkan, Fatemeh; Mehdizadeh, Mahshid; Rad, Seyed Mohammad Ali Hosseini; Soleimani, Masoud

2016-07-01

The sprouting of new blood vessels by angiogenesis is critical in vascular development and homeostasis. Aberrant angiogenesis leads to enormous pathological conditions such as ischemia and cancer. MicroRNAs (also known as miRNAs or miRs) play key roles in regulation of a range of cellular processes by posttranscriptional suppression of their target genes. Recently, new studies have indicated that miRNAs are involved in certain angiogenic settings and signaling pathways use these non-coding RNAs to promote or suppress angiogenic processes. Herein, VEGFR2 and FGFR1 were identified as miR-129-1 and miR-133 targets using bioinformatic algorithms, respectively. Afterwards, using luciferase reporter assay and gene expression analysis at both mRNA and protein levels, VEGFR2 and FGFR1 were validated as miR-129-1 and miR-133 targets. In addition, we showed that miR-129-1 and miR-133 suppress angiogenesis properties such as proliferation rate, cell viability, and migration activity of human umbilical vein endothelial cells (HUVEC) in vitro. We conclude that these miRNAs can suppress key factors of angiogenesis by directly targeting them. These results have important therapeutic implications for a variety of diseases involving deregulation of angiogenesis, including cancer.

Identification of a unique loss-of-function mutation in IGF1R and a crosstalk between IGF1R and Wnt/β-catenin signaling pathways.

PubMed

Jamwal, Gayatri; Singh, Gurjinder; Dar, Mohd Saleem; Singh, Paramjeet; Bano, Nasima; Syed, Sajad Hussain; Sandhu, Padmani; Akhter, Yusuf; Monga, Satdarshan P; Dar, Mohd Jamal

2018-06-01

IGF1R is a ubiquitous receptor tyrosine kinase that plays critical roles in cell proliferation, growth and survival. Clinical studies have demonstrated upregulation of IGF1R mediated signaling in a number of malignancies including colon, breast, and lung cancers. Overexpression of the IGF1R in these malignancies is associated with a poor prognosis and overall survival. IGF1R specific kinase inhibitors have failed in multiple clinical trials partly because of the complex nature of IGF1R signaling. Thus identifying new binding partners and allosteric sites on IGF1R are emerging areas of research. More recently, IGF1R has been shown to translocate into the nucleus and perform many functions. In this study, we generated a library of IGF1R deletion and point mutants to examine IGF1R subcellular localization and activation of downstream signaling pathways. We show that the nuclear localization of IGF1R is primarily defined by its cytoplasmic domain. We identified a cross-talk between IGF1R and Wnt/β-catenin signaling pathways and showed, for the first time, that IGF1R is associated with upregulation of TCF-mediated β-catenin transcriptional activity. Using loss-of-function mutants, deletion analysis and IGF1R specific inhibitor(s), we show that cytoplasmic and nuclear activities are two independent functions of IGF1R. Furthermore, we identified a unique loss-of-function mutation in IGF1R. This unique loss-of-function mutant retains only nuclear functions and sits in a pocket, outside ATP and substrate binding region, that is suited for designing allosteric inhibitors of IGF1R. Copyright © 2018 Elsevier B.V. All rights reserved.

miR-29a and miR-29b contribute to pancreatic beta-cell-specific silencing of monocarboxylate transporter 1 (Mct1).

PubMed

Pullen, Timothy J; da Silva Xavier, Gabriela; Kelsey, Gavin; Rutter, Guy A

2011-08-01

In pancreatic β cells, elevated glucose concentrations stimulate mitochondrial oxidative metabolism to raise intracellular ATP/ADP levels, prompting insulin secretion. Unusually low levels of expression of genes encoding the plasma membrane monocarboxylate transporter, MCT1 (SLC16A1), as well as lactate dehydrogenase A (LDHA) ensure that glucose-derived pyruvate is efficiently metabolized by mitochondria, while exogenous lactate or pyruvate is unable to stimulate metabolism and hence insulin secretion inappropriately. We show here that whereas DNA methylation at the Mct1 promoter is unlikely to be involved in cell-type-specific transcriptional repression, three microRNAs (miRNAs), miR-29a, miR-29b, and miR-124, selectively target both human and mouse MCT1 3' untranslated regions. Mutation of the cognate miR-29 or miR-124 binding sites abolishes the effects of the corresponding miRNAs, demonstrating a direct action of these miRNAs on the MCT1 message. However, despite reports of its expression in the mouse β-cell line MIN6, miR-124 was not detectably expressed in mature mouse islets. In contrast, the three isoforms of miR-29 are highly expressed and enriched in mouse islets. We show that inhibition of miR-29a in primary mouse islets increases Mct1 mRNA levels, demonstrating that miR-29 isoforms contribute to the β-cell-specific silencing of the MCT1 transporter and may thus affect insulin release.

Search for Compounds with Hypoglycemic Activity in the Series of 1-[2-(1H-Tetrazol-5-yl)-R1-phenyl]-3-R2-phenyl(ethyl)ureas and R1-Tetrazolo[1,5-c]quinazolin-5(6H)-ones

PubMed Central

Antypenko, Oleksii M.; Kovalenko, Sergiy I.; Zhernova, Galina O.

2016-01-01

Methods of 1-[2-(1H-tetrazol-5-yl)-R1-phenyl]-3-R2-phenyl(ethyl)ureas and R1-tetrazolo[1,5-c]quinazolin-5(6H)-ones synthesis were designed. IR, LC-MS, 1H NMR, and elemental analysis data evaluated the structure and purity of the obtained compounds. Different products, depending on the reaction conditions, were distinguished and discussed. The preliminary hypoglycemic activity of 36 synthesized compounds was revealed. Docking studies to 11β-hydroxysteroid dehydrogenase 1, γ-peroxisome proliferator-activated receptor, and dipeptidyl peptidase-4 were conducted. Eight of these substances were further tested on glucocorticoid-induced insulin resistance models, namely glucose tolerance, oral rapid insulin, and adrenalin tests. One of the most active compounds turned out to be tetrazolo[1,5-c]quinazolin-5(6H)-one 3.1, exceeding the reference drugs Metformin (50 and 200 mg/kg) and Gliclazide (50 mg/kg). PMID:27222601

miR-193b Regulates Mcl-1 in Melanoma.

PubMed

Chen, Jiamin; Zhang, Xiao; Lentz, Cindy; Abi-Daoud, Marie; Paré, Geneviève C; Yang, Xiaolong; Feilotter, Harriet E; Tron, Victor A

2011-11-01

MicroRNAs play important roles in gene regulation, and their expression is frequently dysregulated in cancer cells. In a previous study, we reported that miR-193b represses cell proliferation and regulates cyclin D1 in melanoma cells, suggesting that miR-193b could act as a tumor suppressor. Herein, we demonstrate that miR-193b also down-regulates myeloid cell leukemia sequence 1 (Mcl-1) in melanoma cells. MicroRNA microarray profiling revealed that miR-193b is expressed at a significantly lower level in malignant melanoma than in benign nevi. Consistent with this, Mcl-1 is detected at a higher level in malignant melanoma than in benign nevi. In a survey of melanoma samples, the level of Mcl-1 is inversely correlated with the level of miR-193b. Overexpression of miR-193b in melanoma cells represses Mcl-1 expression. Previous studies showed that Mcl-1 knockdown cells are hypersensitive to ABT-737, a small-molecule inhibitor of Bcl-2, Bcl-X(L), and Bcl-w. Similarly, overexpression of miR-193b restores ABT-737 sensitivity to ABT-737-resistant cells. Furthermore, the effect of miR-193b on the expression of Mcl-1 seems to be mediated by direct interaction between miR-193b and seed and seedless pairing sequences in the 3' untranslated region of Mcl-1 mRNA. Thus, this study provides evidence that miR-193b directly regulates Mcl-1 and that down-regulation of miR-193b in vivo could be an early event in melanoma progression. Copyright © 2011 American Society for Investigative Pathology. Published by Elsevier Inc. All rights reserved.

1. Historic American Buildings Survey, Photocopy from P.R.R. General Library, ...

Library of Congress Historic Buildings Survey, Historic Engineering Record, Historic Landscapes Survey

1. Historic American Buildings Survey, Photocopy from P.R.R. General Library, TRAIN SIDE FRONT (NORTH) c.1870 TRANSPORTATION BUILDING, 17TH STREET AND PENNSYLVANIA AVENUE, PHILADELPHIA, PENNSYLVANIA. - Pennsylvania Railroad Bryn Mawr Station, Bryn Mawr & Morris Avenues, Bryn Mawr, Montgomery County, PA

Detection of maltodextrin and its discrimination from sucrose are independent of the T1R2 + T1R3 heterodimer.

PubMed

Smith, Kimberly R; Spector, Alan C

2017-10-01

Maltodextrins, such as Maltrin and Polycose, are glucose polymer mixtures of varying chain lengths that are palatable to rodents. Although glucose and other sugars activate the T1R2 + T1R3 "sweet" taste receptor, recent evidence from T1R2- or T1R3-knockout (KO) mice suggests that maltodextrins, despite their glucose polymer composition, activate a separate receptor mechanism to generate a taste percept qualitatively distinguishable from that of sweeteners. However, explicit discrimination of maltodextrins from prototypical sweeteners has not yet been psychophysically tested in any murine model. Therefore, mice lacking T1R2 + T1R3 and wild-type controls were tested in a two-response taste discrimination task to determine whether maltodextrins are 1 ) detectable when both receptor subunits are absent and 2 ) perceptually distinct from that of sucrose irrespective of viscosity, intensity, and hedonics. Most KO mice displayed similar Polycose sensitivity as controls. However, some KO mice were only sensitive to the higher Polycose concentrations, implicating potential allelic variation in the putative polysaccharide receptor or downstream pathways unmasked by the absence of T1R2 + T1R3. Varied Maltrin and sucrose concentrations of approximately matched viscosities were then presented to render the oral somatosensory features, intensity, and hedonic value of the solutions irrelevant. Although both genotypes competently discriminated Maltrin from sucrose, performance was apparently driven by the different orosensory percepts of the two stimuli in control mice and the presence of a Maltrin but not sucrose orosensory cue in KO mice. These data support the proposed presence of an orosensory receptor mechanism that gives rise to a qualitatively distinguishable sensation from that of sucrose. Copyright © 2017 the American Physiological Society.

" L = R" - U(1) R lepton number at the LHC

NASA Astrophysics Data System (ADS)

Frugiuele, Claudia; Grégoire, Thomas; Kumar, Piyush; Pontón, Eduardo

2013-05-01

We perform a detailed study of a variety of LHC signals in supersymmetric models where lepton number is promoted to an (approximate) U(1) R symmetry. Such a symmetry has interesting implications for naturalness, as well as flavor- and CP-violation, among others. Interestingly, it makes large sneutrino vacuum expectation values phenomenologically viable, so that a slepton doublet can play the role of the down-type Higgs. As a result, (some of) the leptons and neutrinos are incorporated into the chargino and neutralino sectors. This leads to characteristic decay patterns that can be experimentally tested at the LHC. The corresponding collider phenomenology is largely determined by the new approximately conserved quantum number, which is itself closely tied to the presence of "leptonic R-parity violation". We find rather loose bounds on the first and second generation squarks, arising from a combination of suppressed production rates together with relatively small signal efficiencies of the current searches. Naturalness would indicate that such a framework should be discovered in the near future, perhaps through spectacular signals exhibiting the lepto-quark nature of the third generation squarks. The presence of fully visible decays, in addition to decay chains involving large missing energy (in the form of neutrinos) could give handles to access the details of the spectrum of new particles, if excesses over SM background were to be observed. The scale of neutrino masses is intimately tied to the source of U(1) R breaking, thus opening a window into the R-breaking sector through neutrino physics. Further theoretical aspects of the model have been presented in the companion paper [1].

Neuroprotective effects of angiotensin II type 1 receptor (AT1-R) blocker via modulating AT1-R signaling and decreased extracellular glutamate levels.

PubMed

Fujita, Tomoyoshi; Hirooka, Kazuyuki; Nakamura, Takehiro; Itano, Toshifumi; Nishiyama, Akira; Nagai, Yukiko; Shiraga, Fumio

2012-06-26

To investigate the mechanism of the neuroprotective effects of the angiotensin II type 1 receptor (AT1-R) blocker against retinal ischemia-reperfusion injury in the rat. Retinal ischemia was induced by increasing intraocular pressure. Glutamate release from the rat retina and intravitreal PO(2) (partial pressure of oxygen) profiles were monitored during and after ischemia using a microdialysis biosensor and oxygen-sensitive microelectrodes. ELISA was used to measure changes in the expression of AT1-R. Retinal mRNA expressions of p47phox and p67phox were measured by real-time polymerase chain reaction. Reactive oxygen species (ROS) were measured using dihydroethidium. Administration of candesartan, which is an AT1-R blocker (ARB), suppressed ischemia-induced increases in the extracellular glutamate. Candesartan also attenuated the increase in intravitreal PO(2) during reperfusion. AT1-R expression peaked at 12 hours after reperfusion. Although there was an increase in the retinal mRNA expression of p47phox and p64phox at 12 hours after the reperfusion, administration of candesartan suppressed these expressions. The production of ROS that was detected at 12 hours after reperfusion was also suppressed by the administration of candesartan or apocynin. NADPH oxidase-mediated ROS production increased at 12 hours after reperfusion. Candesartan may protect neurons by decreasing extracellular glutamate immediately after reperfusion and by attenuating oxidative stress via a modulation of the AT1-R signaling that occurs during ischemic insult.

The T1R2/T1R3 sweet receptor and TRPM5 ion channel taste targets with therapeutic potential.

PubMed

Sprous, Dennis; Palmer, Kyle R

2010-01-01

Taste signaling is a critical determinant of ingestive behaviors and thereby linked to obesity and related metabolic dysfunctions. Recent evidence of taste signaling pathways in the gut suggests the link to be more direct, raising the possibility that taste receptor systems could be regarded as therapeutic targets. T1R2/T1R3, the G protein coupled receptor that mediates sweet taste, and the TRPM5 ion channel have been the focus of discovery programs seeking novel compounds that could be useful in modifying taste. We review in this chapter the hypothesis of gastrointestinal taste signaling and discuss the potential for T1R2/T1R3 and TRPM5 as targets of therapeutic intervention in obesity and diabetes. Critical to the development of a drug discovery program is the creation of libraries that enhance the likelihood of identifying novel compounds that modulate the target of interest. We advocate a computer-based chemoinformatic approach for assembling natural and synthetic compound libraries as well as for supporting optimization of structure activity relationships. Strategies for discovering modulators of T1R2/T1R3 and TRPM5 using methods of chemoinformatics are presented herein. Copyright 2010 Elsevier Inc. All rights reserved.

Endoplasmic reticulum stress increases AT1R mRNA expression via TIA-1-dependent mechanism

PubMed Central

Backlund, Michael; Paukku, Kirsi; Kontula, Kimmo K.; Lehtonen, Jukka Y.A.

2016-01-01

As the formation of ribonucleoprotein complexes is a major mechanism of angiotensin II type 1 receptor (AT1R) regulation, we sought to identify novel AT1R mRNA binding proteins. By affinity purification and mass spectroscopy, we identified TIA-1. This interaction was confirmed by colocalization of AT1R mRNA and TIA-1 by FISH and immunofluorescence microscopy. In immunoprecipitates of endogenous TIA- 1, reverse transcription-PCR amplified AT1R mRNA. TIA-1 has two binding sites within AT1R 3′-UTR. The binding site proximal to the coding region is glyceraldehyde-3-phosphate dehydrogenase (GAPDH)-dependent whereas the distal binding site is not. TIA-1 functions as a part of endoplasmic reticulum (ER) stress response leading to stress granule (SG) formation and translational silencing. We and others have shown that AT1R expression is increased by ER stress-inducing factors. In unstressed cells, TIA-1 binds to AT1R mRNA and decreases AT1R protein expression. Fluorescence microscopy shows that ER stress induced by thapsigargin leads to the transfer of TIA-1 to SGs. In FISH analysis AT1R mRNA remains in the cytoplasm and no longer colocalizes with TIA-1. Thus, release of TIA-1-mediated suppression by ER stress increases AT1R protein expression. In conclusion, AT1R mRNA is regulated by TIA-1 in a ER stress-dependent manner. PMID:26681690

Synthesis, characterization, structural and biological aspects of copper(II) dithiocarbamate complexes - Part II, [Cu{S2CN(Me)(R1)}2], [Cu{S2CN(Me)(R2)}2] and [Cu{S2CN(R3)(R4)}2] {R1 = CH2CH(OMe)2, R2 = 2-methyl-1,3-dioxolane, R3 = CH2(CH2)2NCHPhOCH2Ph and R4 = CH2CH2OH}

NASA Astrophysics Data System (ADS)

Ferreira, Isabella P.; de Lima, Geraldo M.; Paniago, Eucler B.; Takahashi, Jacqueline A.; Krambrock, Klaus; Pinheiro, Carlos B.; Wardell, James L.; Visentin, Lorenzo C.

2013-09-01

Three new copper(II) dithiocarbamates (DTC), [Cu{S2CN(Me)(R1)}2] (1), [Cu{S2CN(Me)(R2)}2] (2) and [Cu{S2CN(R3)(R4)}2] (3) with R1 = CH2CH(OMe)2, R2 = 2-methyl-1,3-dioxolane, R3 = CH2(CH2)2NCHPhOCH2Ph and R4 = CH2CH2OH, have been synthesized and characterized by different spectroscopic techniques. Complexes (1) and (2) display typical EPR spectra for separated Cu(II) centers, and the spectrum of (3) is characteristic of two magnetically coupled Cu(II) ions with S = 1. The X-ray crystallographic determination has shown that complexes (1) and (2) crystallise in the triclinic and monoclinic systems. In addition both complexes are monomers in which the geometry at each Cu(II) is square planar. The in vitro antimicrobial activity of the sodium salts of ligands, and of the Cu(II)-DTC complexes have been screened against Aspergillus flavus, Aspergillus niger, Aspergillus parasiticus, Penicillium citrinum and Curvularia senegalensis, as well as Gram positive and Gram negative bacteria. Finally, the toxic effects of complexes (1)-(3) were performed using Chlorella vulgaris.

Methods for Tumor Targeting with Salmonella typhimurium A1-R.

PubMed

Hoffman, Robert M; Zhao, Ming

2016-01-01

Salmonella typhimurium A1-R (S. typhimurium A1-R) has shown great preclinical promise as a broad-based anti-cancer therapeutic (please see Chapter 1 ). The present chapter describes materials and methods for the preclinical study of S. typhimurium A1-R in clinically-relevant mouse models. Establishment of orthotopic metastatic mouse models of the major cancer types is described, as well as other useful models, for efficacy studies of S. typhimurium A1-R or other tumor-targeting bacteria, as well. Imaging methods are described to visualize GFP-labeled S. typhimurium A1-R, as well as GFP- and/or RFP-labeled cancer cells in vitro and in vivo, which S. typhimurium A1-R targets. The mouse models include metastasis to major organs that are life-threatening to cancer patients including the liver, lung, bone, and brain and how to target these metastases with S. typhimurium A1-R. Various routes of administration of S. typhimurium A1-R are described with the advantages and disadvantages of each. Basic experiments to determine toxic effects of S. typhimurium A1-R are also described. Also described are methodologies for combining S. typhimurium A1-R and chemotherapy. The testing of S. typhimurium A1-R on patient tumors in patient-derived orthotopic xenograft (PDOX) mouse models is also described. The major methodologies described in this chapter should be translatable for clinical studies.

Endoplasmic reticulum stress increases AT1R mRNA expression via TIA-1-dependent mechanism.

PubMed

Backlund, Michael; Paukku, Kirsi; Kontula, Kimmo K; Lehtonen, Jukka Y A

2016-04-20

As the formation of ribonucleoprotein complexes is a major mechanism of angiotensin II type 1 receptor (AT1R) regulation, we sought to identify novel AT1R mRNA binding proteins. By affinity purification and mass spectroscopy, we identified TIA-1. This interaction was confirmed by colocalization of AT1R mRNA and TIA-1 by FISH and immunofluorescence microscopy. In immunoprecipitates of endogenous TIA- 1, reverse transcription-PCR amplified AT1R mRNA. TIA-1 has two binding sites within AT1R 3'-UTR. The binding site proximal to the coding region is glyceraldehyde-3-phosphate dehydrogenase (GAPDH)-dependent whereas the distal binding site is not. TIA-1 functions as a part of endoplasmic reticulum (ER) stress response leading to stress granule (SG) formation and translational silencing. We and others have shown that AT1R expression is increased by ER stress-inducing factors. In unstressed cells, TIA-1 binds to AT1R mRNA and decreases AT1R protein expression. Fluorescence microscopy shows that ER stress induced by thapsigargin leads to the transfer of TIA-1 to SGs. In FISH analysis AT1R mRNA remains in the cytoplasm and no longer colocalizes with TIA-1. Thus, release of TIA-1-mediated suppression by ER stress increases AT1R protein expression. In conclusion, AT1R mRNA is regulated by TIA-1 in a ER stress-dependent manner. © The Author(s) 2015. Published by Oxford University Press on behalf of Nucleic Acids Research.

Late-Onset Inner Retinal Dysfunction in Mice Lacking Sigma Receptor 1 (σR1)

PubMed Central

Ha, Yonju; Saul, Alan; Tawfik, Amany; Williams, Cory; Bollinger, Kathryn; Smith, Robert; Tachikawa, Masanori; Zorrilla, Eric; Ganapathy, Vadivel

2011-01-01

Purpose. Sigma receptor 1 (σR1) is expressed abundantly in the eye, and several reports suggest that this putative molecular chaperone plays a role in lens cell survival, control of intraocular pressure (IOP), and retinal neuroprotection. The present study examined the consequence of the absence of σR1 on ocular development, structure, and function. Methods. Wild-type (σR1+/+), heterozygous (σR1+/−), and homozygous (σR1−/−, knockout) mice aged 5 to 59 weeks were subjected to comprehensive electrophysiological testing and IOP measurement. The eyes were examined by light and electron microscopy and subjected to morphometric examination and detection of apoptosis. Results. Cornea and lens of σR1−/− mice were similar to wild-type mice in morphologic appearance at all ages examined, and IOP was within normal limits. Comprehensive ERG and morphometric analyses initially yielded normal findings in the σR1−/− mice compared with those in the wild-type. By 12 months, however, significantly decreased ERG b-wave amplitudes and diminished negative scotopic threshold responses, consistent with inner retinal dysfunction, were detected in σR1−/− mice. Concomitant with these late-onset changes were increased TUNEL- and active caspase 3-positive cells in the inner retina and significant loss of cells in the ganglion cell layer, particularly in the central retina. Before these functional and structural abnormalities, there was ultrastructural evidence of axonal disruption in the optic nerve head of σR1−/− mice as early as 6 months of age, although there were no alterations observed in retinal vascularization in σR1−/− mice. Conclusions. These data suggest that lack of σR1 leads to development of late-onset retinal dysfunction with similarities to optic neuropathy. PMID:21862648

Late-onset inner retinal dysfunction in mice lacking sigma receptor 1 (σR1).

PubMed

Ha, Yonju; Saul, Alan; Tawfik, Amany; Williams, Cory; Bollinger, Kathryn; Smith, Robert; Tachikawa, Masanori; Zorrilla, Eric; Ganapathy, Vadivel; Smith, Sylvia B

2011-09-29

Sigma receptor 1 (σR1) is expressed abundantly in the eye, and several reports suggest that this putative molecular chaperone plays a role in lens cell survival, control of intraocular pressure (IOP), and retinal neuroprotection. The present study examined the consequence of the absence of σR1 on ocular development, structure, and function. Wild-type (σR1⁺/⁺), heterozygous (σR1⁺/⁻), and homozygous (σR1⁻/⁻, knockout) mice aged 5 to 59 weeks were subjected to comprehensive electrophysiological testing and IOP measurement. The eyes were examined by light and electron microscopy and subjected to morphometric examination and detection of apoptosis. Cornea and lens of σR1⁻/⁻ mice were similar to wild-type mice in morphologic appearance at all ages examined, and IOP was within normal limits. Comprehensive ERG and morphometric analyses initially yielded normal findings in the σR1⁻/⁻ mice compared with those in the wild-type. By 12 months, however, significantly decreased ERG b-wave amplitudes and diminished negative scotopic threshold responses, consistent with inner retinal dysfunction, were detected in σR1⁻/⁻ mice. Concomitant with these late-onset changes were increased TUNEL- and active caspase 3-positive cells in the inner retina and significant loss of cells in the ganglion cell layer, particularly in the central retina. Before these functional and structural abnormalities, there was ultrastructural evidence of axonal disruption in the optic nerve head of σR1⁻/⁻ mice as early as 6 months of age, although there were no alterations observed in retinal vascularization in σR1⁻/⁻ mice. These data suggest that lack of σR1 leads to development of late-onset retinal dysfunction with similarities to optic neuropathy.

miR-342-3p suppresses hepatocellular carcinoma proliferation through inhibition of IGF-1R-mediated Warburg effect

PubMed Central

Han, Juqiang; Wang, Yadong; Shen, Chuan; Yan, Zhifeng; Tai, Yanhong; Zhao, Caiyan

2018-01-01

Background Insulin-like growth factor-1 receptor (IGF-1R) is a well-studied oncogenic factor that promotes cell proliferation and energy metabolism and is overexpressed in numerous cancers including hepatocellular carcinoma (HCC). Aerobic glycolysis is a hallmark of cancer, and drugs targeting its regulators, including IGF-1R, are being developed. However, the mechanisms of IGF-1R inhibition and the physiological significance of the IGF-1R inhibitors in cancer cells are unclear. Materials and methods Cell proliferation was evaluated by cell counting Kit-8 and colony formation assay. Western blot and real-time PCR were accordingly used to detect the relevant proteins, miRNA and gene expression. Luciferase reporter assays were used to illustrate the interaction between miR-342-3p and IGF-1R. The effect of miR-342-3p on glycolysis was determined by glucose uptake, ATP concentration, lactate generation, extracellular acidification rate and oxygen consumption rate assays. In vivo, subcutaneous tumor formation assay and PET were performed in nude mice. Results In this study, we demonstrate that by directly targeting the 3′-UTR (3′-untranslated regions) of IGF-1R, microRNA-342-3p (miR-342-3p) suppresses IGF-1R-mediated PI3K/AKT/GLUT1 signaling pathway both in vitro and in vivo. Through suppression of IGF-1R, miR-342-3p dampens glycolysis by decreasing glucose uptake, lactate generation, ATP production, and extracellular acidification rate (ECAR), and increasing oxygen consumption rate (OCR) in hepatoma cells. Importantly, glycolysis regulated by miR-342-3p is critical for its regulating HCC growth both in vitro and in vivo. Conclusion Our findings provide clues regarding the role of miR-342-3p as a tumor suppressor in liver cancer mainly through the inhibition of IGF-1R. Targeting IGF-1R by miR-342-3p could be a potential therapeutic strategy in liver cancer. PMID:29615839

miR-342-3p suppresses hepatocellular carcinoma proliferation through inhibition of IGF-1R-mediated Warburg effect.

PubMed

Liu, Wenpeng; Kang, Lei; Han, Juqiang; Wang, Yadong; Shen, Chuan; Yan, Zhifeng; Tai, Yanhong; Zhao, Caiyan

2018-01-01

Insulin-like growth factor-1 receptor (IGF-1R) is a well-studied oncogenic factor that promotes cell proliferation and energy metabolism and is overexpressed in numerous cancers including hepatocellular carcinoma (HCC). Aerobic glycolysis is a hallmark of cancer, and drugs targeting its regulators, including IGF-1R, are being developed. However, the mechanisms of IGF-1R inhibition and the physiological significance of the IGF-1R inhibitors in cancer cells are unclear. Cell proliferation was evaluated by cell counting Kit-8 and colony formation assay. Western blot and real-time PCR were accordingly used to detect the relevant proteins, miRNA and gene expression. Luciferase reporter assays were used to illustrate the interaction between miR-342-3p and IGF-1R. The effect of miR-342-3p on glycolysis was determined by glucose uptake, ATP concentration, lactate generation, extracellular acidification rate and oxygen consumption rate assays. In vivo, subcutaneous tumor formation assay and PET were performed in nude mice. In this study, we demonstrate that by directly targeting the 3'-UTR (3'-untranslated regions) of IGF-1R, microRNA-342-3p (miR-342-3p) suppresses IGF-1R-mediated PI3K/AKT/GLUT1 signaling pathway both in vitro and in vivo. Through suppression of IGF-1R, miR-342-3p dampens glycolysis by decreasing glucose uptake, lactate generation, ATP production, and extracellular acidification rate (ECAR), and increasing oxygen consumption rate (OCR) in hepatoma cells. Importantly, glycolysis regulated by miR-342-3p is critical for its regulating HCC growth both in vitro and in vivo. Our findings provide clues regarding the role of miR-342-3p as a tumor suppressor in liver cancer mainly through the inhibition of IGF-1R. Targeting IGF-1R by miR-342-3p could be a potential therapeutic strategy in liver cancer.

Roseovarius salinarum sp. nov., isolated from a marine solar saltern.

PubMed

Wang, Nan-Nan; Liu, Zuan-Yan; Jiang, Lai-Xiang; Li, Ying-Xiu; Du, Zong-Jun

2018-06-01

A Gram-stain-negative, rod-shaped, non-motile and halophilic bacterium, designated N53 T , was isolated from a marine solar saltern in Wendeng, China. Cells of strain N53 T were 0.3-0.4 µm wide and 2.0-5.5 µm long, catalase-positive and oxidase-positive. The bacterium grew optimally at 33 °C, at pH 7.0-8.0 and in the presence of 6.0 % (w/v) NaCl. Bacteriochlorophyll a was not found. Phylogenetic analysis based on 16S rRNA gene sequences revealed that strain N53 T formed a phylogenetic lineage with members of the genus Roseovarius. Strain N53 T exhibited the highest levels of similarity to Roseovarius pacificus (94.6 %) and Roseovarius confluentis (94.6 %), with a lower level to Roseovarius tolerans was 94.0 %. The percentage of conserved proteins and average nucleotide identity values between N53 T and the type strain of the type species, Roseovarius tolerans, were 66.1 and 76.4 %, respectively. The genomic DNA G+C content was 68.1 mol%. The sole respiratory quinone was ubiquinone-10. The predominant cellular fatty acids (>10 %) were C18 : 1ω7c (54.0 %) and C16 : 0 (17.9 %). The polar lipids of strain N53 T consisted of diphosphatidylglycerol, phosphatidylglycerol, phosphatidylcholine, an unidentified aminolipid, two unidentified phospholipids and two unidentified glycolipids. The differential phenotypic properties, together with the chemotaxonomic and genomic distinctiveness, revealed that strain N53 T was separate from other recognized species of the genus Roseovarius. On the basis of the data presented here, strain N53 T represents a novel species of the genus Roseovarius, for which the name Roseovariussalinarum sp. nov. is proposed. The type strain is N53 T (=MCCC 1H00200 T =KCTC 52886 T ).

5. SWITCH TOWER AND JUNCTION OF S.A.R. #1 & S.A.R. ...

Library of Congress Historic Buildings Survey, Historic Engineering Record, Historic Landscapes Survey

5. SWITCH TOWER AND JUNCTION OF S.A.R. #1 & S.A.R. #2 TRANSMISSION LINES, MARCH 7, 1916. SCE drawing no. 4932. - Santa Ana River Hydroelectric System, Transmission Lines, Redlands, San Bernardino County, CA

Extreme variability among mammalian V1R gene families.

PubMed

Young, Janet M; Massa, Hillary F; Hsu, Li; Trask, Barbara J

2010-01-01

We report an evolutionary analysis of the V1R gene family across 37 mammalian genomes. V1Rs comprise one of three chemosensory receptor families expressed in the vomeronasal organ, and contribute to pheromone detection. We first demonstrate that Trace Archive data can be used effectively to determine V1R family sizes and to obtain sequences of most V1R family members. Analyses of V1R sequences from trace data and genome assemblies show that species-specific expansions previously observed in only eight species were prevalent throughout mammalian evolution, resulting in "semi-private" V1R repertoires for most mammals. The largest families are found in mouse and platypus, whose V1R repertoires have been published previously, followed by mouse lemur and rabbit (approximately 215 and approximately 160 intact V1Rs, respectively). In contrast, two bat species and dolphin possess no functional V1Rs, only pseudogenes, and suffered inactivating mutations in the vomeronasal signal transduction gene Trpc2. We show that primate V1R decline happened prior to acquisition of trichromatic vision, earlier during evolution than was previously thought. We also show that it is extremely unlikely that decline of the dog V1R repertoire occurred in response to selective pressures imposed by humans during domestication. Functional repertoire sizes in each species correlate roughly with anatomical observations of vomeronasal organ size and quality; however, no single ecological correlate explains the very diverse fates of this gene family in different mammalian genomes. V1Rs provide one of the most extreme examples observed to date of massive gene duplication in some genomes, with loss of all functional genes in other species.

R1507, an Anti-Insulin-Like Growth Factor-1 Receptor (IGF-1R) Antibody, and EWS/FLI-1 siRNA in Ewing's Sarcoma: Convergence at the IGF/IGFR/Akt Axis

PubMed Central

Rodon, Jordi; Sun, Michael; Kuenkele, Klaus-Peter; Parsons, Henrique A.; Trent, Jonathan C.; Kurzrock, Razelle

2011-01-01

A subset of patients with Ewing's sarcoma responds to anti-insulin-like growth factor-1 receptor (IGF-1R) antibodies. Mechanisms of sensitivity and resistance are unknown. We investigated whether an anti-IGF-1R antibody acts via a pathway that could also be suppressed by small interfering (si) RNA against the EWS/FLI-1 fusion protein, the hallmark of Ewing's sarcoma. The growth of two Ewing's sarcoma cell lines (TC-32 and TC-71) was inhibited by the fully human anti-IGF-1R antibody, R1507 (clonogenic and MTT assays). TC-32 and TC-71 cells express high levels of IGF-2, while RD-ES and A4573 Ewing's cell lines, which were less responsive to R1507 in our assays, express low or undetectable IGF-2, respectively. TC-71 cells also expressed high levels of IGF-1R, and R1507 decreased steady-state levels of this receptor by internalization/degradation, an effect which was associated with a decrease in p-IGF-1R, p-IRS-1, and p-Akt. EWS/FLI-1 siRNA also decreased p-Akt, due to its ability to increase IGF-BP3 levels and subsequently decrease IGF-1 and IGF-2 levels, thus inhibiting signaling through p-IGF-1R. This inhibition correlated with growth suppression and apoptosis. The attenuation of Akt activation was confirmed in TC-71 and HEK-293 (human embryonic kidney) cells by transfecting them with IGF-1R siRNA. We conclude that antibodies and siRNA to IGF-1R, as well as siRNA to EWS/FLI-1, act via intersecting IGF/IGF-1R signals that suppress a common point in this pathway, namely the phosphorylation of Akt. PMID:22022506

The human Kell blood group binds the erythroid 4.1R protein: new insights into the 4.1R-dependent red cell membrane complex

PubMed Central

Azouzi, Slim; Collec, Emmanuel; Mohandas, Narla; An, Xiuli; Colin, Yves; Le Van Kim, Caroline

2015-01-01

Summary Protein 4.1R plays an important role in maintaining the mechanical properties of the erythrocyte membrane. We analysed the expression of Kell blood group protein in erythrocytes from a patient with hereditary elliptocytosis associated with complete 4.1R deficiency (4.1(−) HE). Flow cytometry and Western blot analyses revealed a severe reduction of Kell. In vitro pull down and co-immunoprecipitation experiments from erythrocyte membranes showed a direct interaction between Kell and 4.1R. Using different recombinant domains of 4.1R and the cytoplasmic domain of Kell, we demonstrated that the R46R motif in the juxta-membrane region of Kell binds to lobe B of the 4.1R FERM domain. We also observed that 4.1R deficiency is associated with a reduction of XK and DARC (also termed ACKR1) proteins, the absence of the glycosylated form of the urea transporter B and a slight decrease of band 3. The functional alteration of the 4.1(−) HE erythrocyte membranes was also determined by measuring various transport activities. We documented a slower rate of HCO3−/Cl− exchange, but normal water and ammonia transport across erythrocyte membrane in the absence of 4.1. These findings provide novel insights into the structural organization of blood group antigen proteins into the 4.1R complex of the human red cell membrane. PMID:26455906

Red hair is the null phenotype of MC1R.

PubMed

Beaumont, Kimberley A; Shekar, Sri N; Cook, Anthony L; Duffy, David L; Sturm, Richard A

2008-08-01

The Melanocortin-1 Receptor (MC1R) is a G-protein coupled receptor, which is responsible for production of the darker eumelanin pigment and the tanning response. The MC1R gene has many polymorphisms, some of which have been linked to variation in pigmentation phenotypes within human populations. In particular, the p.D84E, p.R151C, p.R160W and p.D294 H alleles have been strongly associated with red hair, fair skin and increased skin cancer risk. These red hair colour (RHC) variants are relatively well described and are thought to result in altered receptor function, while still retaining varying levels of signaling ability in vitro. The mouse Mc1r null phenotype is yellow fur colour, the p.R151C, p.R160W and p.D294 H alleles were able to partially rescue this phenotype, leading to the question of what the true null phenotype of MC1R would be in humans. Due to the rarity of MC1R null alleles in human populations, they have only been found in the heterozygous state until now. We report here the first case of a homozygous MC1R null individual, phenotypic analysis indicates that red hair and fair skin is found in the absence of MC1R function.

Establishment of a novel monoclonal antibody SMab-1 specific for IDH1-R132S mutation

DOE Office of Scientific and Technical Information (OSTI.GOV)

Kaneko, Mika Kato; Tian, Wei; Takano, Shingo

2011-03-25

Research highlights: {yields} IDH1 mutations are early and frequent genetic alterations in gliomas. {yields} We newly established an anti-IDH1-R132S-specific mAb SMab-1. {yields} SMab-1 reacted with the IDH1-R132S peptide, but not with other IDH1 mutants. {yields} SMab-1 specifically stained the IDH1-R132S-expressing glioblastoma cells in immunocytochemistry and immunohistochemistry. {yields} SMab-1 should be useful in diagnosis of mutation-bearing gliomas. -- Abstract: Isocitrate dehydrogenase 1 (IDH1) mutations, which are early and frequent genetic alterations in gliomas, are specific to a single codon in the conserved and functionally important Arginine 132 (R132) in IDH1. We earlier established a monoclonal antibody (mAb), IMab-1, which is specificmore » for R132H-containing IDH1 (IDH1-R132H), the most frequent IDH1 mutation in gliomas. To establish IDH1-R132S-specific mAb, we immunized mice with R132S-containing IDH1 (IDH1-R132S) peptide. After cell fusion using Sendai virus envelope, IDH1-R132S-specific mAbs were screened in ELISA. One mAb, SMab-1, reacted with the IDH1-R132S peptide, but not with other IDH1 mutants. Western-blot analysis showed that SMab-1 reacted only with the IDH1-R132S protein, not with IDH1-WT protein or IDH1 mutants, indicating that SMab-1 is IDH1-R132S-specific. Furthermore, SMab-1 specifically stained the IDH1-R132S-expressing glioblastoma cells in immunocytochemistry and immunohistochemistry, but did not react with IDH1-WT or IDH1-R132H-containing glioblastoma cells. We newly established an anti-IDH1-R132S-specific mAb SMab-1 for use in diagnosis of mutation-bearing gliomas.« less

The HMGA1 Pseudogene 7 Induces miR-483 and miR-675 Upregulation by Activating Egr1 through a ceRNA Mechanism

PubMed Central

De Martino, Marco; Azzariti, Amalia; Arra, Claudio; Fusco, Alfredo; Esposito, Francesco

2017-01-01

Several studies have established that pseudogene mRNAs can work as competing endogenous RNAs and, when deregulated, play a key role in the onset of human neoplasias. Recently, we have isolated two HMGA1 pseudogenes, HMGA1P6 and HMGA1P7. These pseudogenes have a critical role in cancer progression, acting as micro RNA (miRNA) sponges for HMGA1 and other cancer-related genes. HMGA1 pseudogenes were found overexpressed in several human carcinomas, and their expression levels positively correlate with an advanced cancer stage and a poor prognosis. In order to investigate the molecular alterations following HMGA1 pseudogene 7 overexpression, we carried out miRNA sequencing analysis on HMGA1P7 overexpressing mouse embryonic fibroblasts. Intriguingly, the most upregulated miRNAs were miR-483 and miR-675 that have been described as key regulators in cancer progression. Here, we report that HMGA1P7 upregulates miR-483 and miR-675 through a competing endogenous RNA mechanism with Egr1, a transcriptional factor that positively regulates miR-483 and miR-675 expression. PMID:29149041

Resolution of 1-n-butyl-3-methyl-3-phospholene 1-oxide with TADDOL derivatives and calcium salts of O,O'-Dibenzoyl-(2R,3R)- or O,O'-di-p-toluoyl-(2R,3R)-tartaric acid.

PubMed

Bagi, Péter; Fekete, András; Kállay, Mihály; Hessz, Dóra; Kubinyi, Miklós; Holczbauer, Tamás; Czugler, Mátyás; Fogassy, Elemér; Keglevich, György

2014-03-01

The resolution methods applying (-)-(4R,5R)-4,5-bis(diphenylhydroxymethyl)-2,2-dimethyldioxolane ("TADDOL"), (-)-(2R,3R)-α,α,α',α'-tetraphenyl-1,4-dioxaspiro[4.5]decan-2,3-dimethanol ("spiro-TADDOL"), as well as the acidic and neutral Ca(2+) salts of (-)-O,O'-dibenzoyl- and (-)-O,O'-di-p-toluoyl-(2R,3R)-tartaric acid were extended for the preparation of 1-n-butyl-3-methyl-3-phospholene 1-oxide in optically active form. In one case, the intermediate diastereomeric complex could be identified by single-crystal X-ray analysis. The absolute P-configuration of the enantiomers of the phospholene oxide was also determined by comparing the experimentally obtained and calculated CD spectra. © 2014 Wiley Periodicals, Inc.

Induction of miR-132 and miR-212 Expression by Glucagon-Like Peptide 1 (GLP-1) in Rodent and Human Pancreatic β-Cells

PubMed Central

Li, Jing; Keller, Mark P.; Hohmeier, Hans E.; Wang, Yong; Feng, Yue; Zhou, Heather H.; Shen, Xiaolan; Rabaglia, Mary; Soni, Mufaddal; Attie, Alan D.; Newgard, Christopher B.; Thornberry, Nancy A.; Howard, Andrew D.; Zhou, Yun-Ping

2015-01-01

Better understanding how glucagon-like peptide 1 (GLP-1) promotes pancreatic β-cell function and/or mass may uncover new treatment for type 2 diabetes. In this study, we investigated the potential involvement of microRNAs (miRNAs) in the effect of GLP-1 on glucose-stimulated insulin secretion. miRNA levels in INS-1 cells and isolated rodent and human islets treated with GLP-1 in vitro and in vivo (with osmotic pumps) were measured by real-time quantitative PCR. The role of miRNAs on insulin secretion was studied by transfecting INS-1 cells with either precursors or antisense inhibitors of miRNAs. Among the 250 miRNAs surveyed, miR-132 and miR-212 were significantly up-regulated by GLP-1 by greater than 2-fold in INS-1 832/3 cells, which were subsequently reproduced in freshly isolated rat, mouse, and human islets, as well as the islets from GLP-1 infusion in vivo in mice. The inductions of miR-132 and miR-212 by GLP-1 were correlated with cAMP production and were blocked by the protein kinase A inhibitor H-89 but not affected by the exchange protein activated by cAMP activator 8-pCPT-2′-O-Me-cAMP-AM. GLP-1 failed to increase miR-132 or miR-212 expression levels in the 832/13 line of INS-1 cells, which lacks robust cAMP and insulin responses to GLP-1 treatment. Overexpression of miR-132 or miR-212 significantly enhanced glucose-stimulated insulin secretion in both 832/3 and 832/13 cells, and restored insulin responses to GLP-1 in INS-1 832/13 cells. GLP-1 increases the expression of miRNAs 132 and 212 via a cAMP/protein kinase A-dependent pathway in pancreatic β-cells. Overexpression of miR-132 or miR-212 enhances glucose and GLP-1-stimulated insulin secretion. PMID:26218441

Induction of miR-132 and miR-212 Expression by Glucagon-Like Peptide 1 (GLP-1) in Rodent and Human Pancreatic β-Cells.

PubMed

Shang, Jin; Li, Jing; Keller, Mark P; Hohmeier, Hans E; Wang, Yong; Feng, Yue; Zhou, Heather H; Shen, Xiaolan; Rabaglia, Mary; Soni, Mufaddal; Attie, Alan D; Newgard, Christopher B; Thornberry, Nancy A; Howard, Andrew D; Zhou, Yun-Ping

2015-09-01

Better understanding how glucagon-like peptide 1 (GLP-1) promotes pancreatic β-cell function and/or mass may uncover new treatment for type 2 diabetes. In this study, we investigated the potential involvement of microRNAs (miRNAs) in the effect of GLP-1 on glucose-stimulated insulin secretion. miRNA levels in INS-1 cells and isolated rodent and human islets treated with GLP-1 in vitro and in vivo (with osmotic pumps) were measured by real-time quantitative PCR. The role of miRNAs on insulin secretion was studied by transfecting INS-1 cells with either precursors or antisense inhibitors of miRNAs. Among the 250 miRNAs surveyed, miR-132 and miR-212 were significantly up-regulated by GLP-1 by greater than 2-fold in INS-1 832/3 cells, which were subsequently reproduced in freshly isolated rat, mouse, and human islets, as well as the islets from GLP-1 infusion in vivo in mice. The inductions of miR-132 and miR-212 by GLP-1 were correlated with cAMP production and were blocked by the protein kinase A inhibitor H-89 but not affected by the exchange protein activated by cAMP activator 8-pCPT-2'-O-Me-cAMP-AM. GLP-1 failed to increase miR-132 or miR-212 expression levels in the 832/13 line of INS-1 cells, which lacks robust cAMP and insulin responses to GLP-1 treatment. Overexpression of miR-132 or miR-212 significantly enhanced glucose-stimulated insulin secretion in both 832/3 and 832/13 cells, and restored insulin responses to GLP-1 in INS-1 832/13 cells. GLP-1 increases the expression of miRNAs 132 and 212 via a cAMP/protein kinase A-dependent pathway in pancreatic β-cells. Overexpression of miR-132 or miR-212 enhances glucose and GLP-1-stimulated insulin secretion.

Design and Synthesis of Cannabinoid 1 Receptor (CB1R) Allosteric Modulators: Drug Discovery Applications.

PubMed

Kulkarni, Abhijit R; Garai, Sumanta; Janero, David R; Thakur, Ganesh A

2017-01-01

Also expressed in various peripheral tissues, the type-1 cannabinoid receptor (CB1R) is the predominant G protein-coupled receptor (GPCR) in brain, where it is responsible for retrograde control of neurotransmitter release. Cellular signaling mediated by CB1R is involved in numerous physiological processes, and pharmacological CB1R modulation is considered a tenable therapeutic approach for diseases ranging from substance-use disorders and glaucoma to metabolic syndrome. Despite the design and synthesis of a variety of bioactive small molecules targeted to the CB1R orthosteric ligand-binding site, the potential of CB1R as a therapeutic GPCR has been largely unrealized due to adverse events associated with typical orthosteric CB1R agonists and antagonists/inverse agonists. Modulation of CB1R-mediated signal transmission by targeting alternative allosteric ligand-binding site(s) on the receptor has garnered interest as a potentially safer and more effective therapeutic modality. This chapter highlights the design and synthesis of novel, pharmacologically active CB1R allosteric modulators and emphasizes how their molecular properties and the positive and negative allosteric control they exert can lead to improved CB1R-targeted pharmacotherapeutics, as well as designer covalent probes that can be used to map CB1R allosteric binding domains and inform structure-based drug design. © 2017 Elsevier Inc. All rights reserved.

R34D1NG W0RD5 W1TH NUMB3R5.

PubMed

Perea, Manuel; Duñabeitia, Jon Andoni; Carreiras, Manuel

2008-02-01

Letter identities and number identities are usually thought to imply different cortical mechanisms. Specifically, the left fusiform gyrus responds more to letters than to digits (T. A. Polk et al., 2002). However, a widely circulated statement on the internet illustrates that it is possible to use numbers (leet digits) as parts of words, 4ND TH3 R35ULT1NG S3NT3NC3 C4N B3 R34D W1TH0UT GR34T 3FF0RT. Two masked priming lexical decision experiments were conducted to determine whether leet digits produce (automatic) lexical activation. Results showed that words are identified substantially faster when they are preceded by a masked leet word (M4T3R14L-MATERIAL) than when they are preceded by a control condition with other letters or digits. In addition, there was only a negligible advantage of the identity condition over the related leet condition. This leet-priming effect is not specific to numbers: A prime in which leet digits are replaced by letter-like symbols (M(Delta symbol)T(euro symbol)R!(Delta symbol)L - MATERIAL) facilitates word processing to the same degree as an identity prime. Therefore, the cognitive system regularizes the shape of the leet digits and letter-like symbols embedded in words with very little cost.

From small sweeteners to sweet proteins: anatomy of the binding sites of the human T1R2_T1R3 receptor.

PubMed

Morini, Gabriella; Bassoli, Angela; Temussi, Piero A

2005-08-25

The sweet taste receptor, a heterodimeric G protein coupled receptor (GPCR) protein, formed by the T1R2 and T1R3 subunits, recognizes several sweet compounds including carbohydrates, amino acids, peptides, proteins, and synthetic sweeteners. Its similarity with the metabotropic glutamate mGluR1 receptor allowed us to build homology models. All possible dimers formed by combinations of the human T1R2 and T1R3 subunits, modeled on the A (closed) or B (open) chains of the extracellular ligand binding domain of the mGluR1 template, yield four ligand binding sites for low-molecular-weight sweeteners. These sites were probed by docking a set of molecules representative of all classes of sweet compounds and calculating the free energy of ligand binding. These sites are not easily accessible to sweet proteins, but docking experiments in silico showed that sweet proteins can bind to a secondary site without entering the deep cleft. Our models account for many experimental observations on the tastes of sweeteners, including sweetness synergy, and can help to design new sweeteners.

Distinct Contributions of T1R2 and T1R3 Taste Receptor Subunits to the Detection of Sweet Stimuli

DOE Office of Scientific and Technical Information (OSTI.GOV)

Nie,Y.; Vigues, S.; Hobbs, J.

2005-01-01

The molecular mechanisms by which G protein-coupled receptor (GPCR)-type chemosensory receptors of animals selectively interact with their cognate ligands remain poorly understood. There is growing evidence that many chemosensory receptors exist in multimeric complexes, though little is known about the relative contributions of individual subunits to receptor functions. This study showed that each of the two subunits in the mammalian heteromeric T1R2:T1R3 sweet taste receptor binds sweet stimuli, though with distinct affinities and conformational changes. Furthermore, ligand affinities for T1R3 are drastically reduced by the introduction of a single amino acid change associated with decreased sweet taste sensitivity in mice.more » Thus, individual T1R subunits increase the receptive range of the sweet taste receptor, offering a functional mechanism for phenotypic variations in sweet taste.« less

Manumycin A Is a Potent Inhibitor of Mammalian Thioredoxin Reductase-1 (TrxR-1).

PubMed

Tuladhar, Anupama; Rein, Kathleen S

2018-04-12

The anticancer effect of manumycin A (Man A) has been attributed to the inhibition of farnesyl transferase (FTase), an enzyme that is responsible for post-translational modification of Ras proteins. However, we have discovered that Man A inhibits mammalian cytosolic thioredoxin reductase 1 (TrxR-1) in a time-dependent manner, with an IC 50 of 272 nM with preincubation and 1586 nM without preincubation. The inhibition of TrxR-1 by Man A is irreversible and is the result of a covalent interaction between Man A and TrxR-1. Evidence presented herein demonstrates that Man A forms a Michael adduct with the selenocysteine residue, which is located in the C-terminal redox center of TrxR-1. Inhibitors of TrxR-1, which act through this mechanism, convert TrxR-1 into a SecTRAP, which utilizes NADPH to reduce oxygen to superoxide radical anion (O 2 -• ).

The effect of Nullomer-derived peptides 9R, 9S1R and 124R on the NCI-60 panel and normal cell lines.

PubMed

Alileche, Abdelkrim; Hampikian, Greg

2017-08-09

Nullomer peptides are the smallest sequences absent from databases of natural proteins. We first began compiling a list of absent 5-amino acid strings in 2006 (1). We report here the effects of Nullomer-derived peptides 9R, 9S1R and 124R on the NCI-60 panel, derived from human cancers of 9 organs (kidney, ovary, skin melanoma, lung, brain, lung, colon, prostate and the hematopoietic system), and four normal cell lines (endothelial HUVEC, skin fibroblasts BJ, colon epithelial FHC and normal prostate RWPE-1). NCI-60 cancer cell panel and four normal cell lines were cultured in vitro in RPMI1640 supplemented with 10% Hyclone fetal bovine serum and exposed for 48 h to 5 μM, 25 μM and 50 μM of peptides 9R, 9S1R and 124R. Viability was assessed by CCK-8 assay. For peptide ATP depletion effects, one cell line representing each organ in the NCI-60 panel, and four normal cell lines were exposed to 50 μM of peptides 9R, 9S1R and 124R for 3 h. The ATP content was assessed in whole cells, and their supernatants. Peptides 9S1R and 9R are respectively lethal to 95 and 81.6% of the 60 cancer cell lines tested. Control peptide 124R has no effect on the growth of these cells. Especially interesting the fact that peptides 9R and 9S1R are capable of killing drug-resistant and hormone-resistant cell lines, and even cancer stem cells. Peptides 9R and 9S1R have a broader activity spectrum than many cancer drugs in current use, can completely deplete cellular ATP within 3 h, and are less toxic to 3 of the 4 normal cell lines tested than they are to several cancers. Nullomer peptides 9R and 9S1R have a large broad lethal effect on cancer cell lines derived from nine organs represented in the NCI-60 panel. This broad activity crosses many of the categorical divisions used in the general classification of cancers: solid vs liquid cancers, drug sensitive vs drug resistant, hormone sensitive vs hormone resistant, cytokine sensitive vs cytokine non sensitive, slow growing vs rapid

Lipid raft-mediated miR-3908 inhibition of migration of breast cancer cell line MCF-7 by regulating the interactions between AdipoR1 and Flotillin-1.

PubMed

Li, Yuan; Shan, Fei; Chen, Jinglong

2017-03-21

The mechanisms of lipid raft regulation by microRNAs in breast cancer are not fully understood. This work focused on the evaluation and identification of miR-3908, which may be a potential biomarker related to the migration of breast cancer cells, and elucidates lipid-raft-regulating cell migration in breast cancer. To confirm the prediction that miR-3908 is matched with AdipoR1, we used 3'-UTR luciferase activity of AdipoR1 to assess this. Then, human breast cancer cell line MCF-7 was cultured in the absence or presence of the mimics or inhibitors of miR-3908, after which the biological functions of MCF-7 cells were analyzed. The protein expression of AdipoR1, AMPK, and SIRT-1 were examined. The interaction between AdipoR1 and Flotillin-1, or its effects on lipid rafts on regulating cell migration of MCF-7, was also investigated. AdipoR1 is a direct target of miR-3908. miR-3908 suppresses the expression of AdipoR1 and its downstream pathway genes, including AMPK, p-AMPK, and SIRT-1. miR-3908 enhances the process of breast cancer cell clonogenicity. miR-3908 exerts its effects on the proliferation and migration of MCF-7 cells, which are mediated by lipid rafts regulating AdipoR1's ability to bind Flotillin-1. miR-3908 is a crucial mediator of the migration process in breast cancer cells. Lipid rafts regulate the interactions between AdipoR1 and Flotillin-1 and then the migration process associated with miR-3908 in MCF-7 cells. Our findings suggest that targeting miR-3908 and the lipid raft, may be a promising strategy for the treatment and prevention of breast cancer.

INFLUENCE OF IRON CHELATION ON R1 AND R2 CALIBRATION CURVES IN GERBIL LIVER AND HEART

PubMed Central

Wood, John C.; Aguilar, Michelle; Otto-Duessel, Maya; Nick, Hanspeter; Nelson, Marvin D.; Moats, Rex

2008-01-01

MRI is gaining increasing importance for the noninvasive quantification of organ iron burden. Since transverse relaxation rates depend on iron distribution as well as iron concentration, physiologic and pharmacologic processes that alter iron distribution could change MRI calibration curves. This paper compares the effect of three iron chelators, deferoxamine, deferiprone, and deferasirox on R1 and R2 calibration curves according to two iron loading and chelation strategies. 33 Mongolian gerbils underwent iron loading (iron dextran 500 mg/kg/wk) for 3 weeks followed by 4 weeks of chelation. An additional 56 animals received less aggressive loading (200 mg/kg/week) for 10 weeks, followed by 12 weeks of chelation. R1 and R2 calibration curves were compared to results from 23 iron-loaded animals that had not received chelation. Acute iron loading and chelation biased R1 and R2 from the unchelated reference calibration curves but chelator-specific changes were not observed, suggesting physiologic rather than pharmacologic differences in iron distribution. Long term chelation deferiprone treatment increased liver R1 50% (p<0.01), while long term deferasirox lowered liver R2 30.9% (p<0.0001). The relationship between R1 and R2 and organ iron concentration may depend upon the acuity of iron loading and unloading as well as the iron chelator administered. PMID:18581418

Molecular Cloning and Characterization of Two Pig Vasoactive Intestinal Polypeptide Receptors (VPAC1-R and VPAC2-R)

PubMed Central

He, Xiaping; Meng, Fengyan; Wang, Yajun

2014-01-01

We here report the cloning, tissue expression, and functional analyses of the two pig vasoactive intestinal polypeptide (VIP) receptors (pVPAC1-R and pVPAC2-R). The cloned full-length pVPAC1-R and pVPAC2-R share high structural similarity with their mammalian counterparts. Functional assay revealed that the full-length pVPAC1-R and pVPAC2-R-expressed Chinese hamster ovary (CHO) cells could be activated by pVIP and pPACAP38 potently, indicating that pVPAC1-R and pVPAC2-R are capable of binding VIP and pituitary adenylate cyclase-activating polypeptide (PACAP). In addition to the identification of the transcripts encoding the two full-length receptors, multiple splice transcript variants were isolated. Comparison with the pig genome database revealed that pVPAC1-R and pVPAC2-R share a unique gene structure with 14 exons different from other vertebrates. Reverse transcription and polymerase chain reaction (RT-PCR) assays further showed that the transcript encoding the full-length pVPAC2-R is widely expressed in all adult tissues whereas the splice variants of pVPAC1-R are predominantly expressed in all tissues instead of the transcript encoding the full-length receptor, hinting that pVPAC2-R may play more important roles than pVPAC1-R in mediating VIP and PACAP actions. Our present findings help to elucidate the important role of VIP and PACAP and promote to rethink of their species-specific physiological roles including their actions in regulation of phenotypic traits in pigs. PMID:24520933

miR-29a and miR-29b Contribute to Pancreatic β-Cell-Specific Silencing of Monocarboxylate Transporter 1 (Mct1) ▿ †

PubMed Central

Pullen, Timothy J.; da Silva Xavier, Gabriela; Kelsey, Gavin; Rutter, Guy A.

2011-01-01

In pancreatic β cells, elevated glucose concentrations stimulate mitochondrial oxidative metabolism to raise intracellular ATP/ADP levels, prompting insulin secretion. Unusually low levels of expression of genes encoding the plasma membrane monocarboxylate transporter, MCT1 (SLC16A1), as well as lactate dehydrogenase A (LDHA) ensure that glucose-derived pyruvate is efficiently metabolized by mitochondria, while exogenous lactate or pyruvate is unable to stimulate metabolism and hence insulin secretion inappropriately. We show here that whereas DNA methylation at the Mct1 promoter is unlikely to be involved in cell-type-specific transcriptional repression, three microRNAs (miRNAs), miR-29a, miR-29b, and miR-124, selectively target both human and mouse MCT1 3′ untranslated regions. Mutation of the cognate miR-29 or miR-124 binding sites abolishes the effects of the corresponding miRNAs, demonstrating a direct action of these miRNAs on the MCT1 message. However, despite reports of its expression in the mouse β-cell line MIN6, miR-124 was not detectably expressed in mature mouse islets. In contrast, the three isoforms of miR-29 are highly expressed and enriched in mouse islets. We show that inhibition of miR-29a in primary mouse islets increases Mct1 mRNA levels, demonstrating that miR-29 isoforms contribute to the β-cell-specific silencing of the MCT1 transporter and may thus affect insulin release. PMID:21646425

DIA1R is an X-linked gene related to Deleted In Autism-1.

PubMed

Aziz, Azhari; Harrop, Sean P; Bishop, Naomi E

2011-01-17

Autism spectrum disorders (ASDS) are frequently occurring disorders diagnosed by deficits in three core functional areas: social skills, communication, and behaviours and/or interests. Mental retardation frequently accompanies the most severe forms of ASDs, while overall ASDs are more commonly diagnosed in males. Most ASDs have a genetic origin and one gene recently implicated in the etiology of autism is the Deleted-In-Autism-1 (DIA1) gene. Using a bioinformatics-based approach, we have identified a human gene closely related to DIA1, we term DIA1R (DIA1-Related). While DIA1 is autosomal (chromosome 3, position 3q24), DIA1R localizes to the X chromosome at position Xp11.3 and is known to escape X-inactivation. The gene products are of similar size, with DIA1 encoding 430, and DIA1R 433, residues. At the amino acid level, DIA1 and DIA1R are 62% similar overall (28% identical), and both encode signal peptides for targeting to the secretory pathway. Both genes are ubiquitously expressed, including in fetal and adult brain tissue. Examination of published literature revealed point mutations in DIA1R are associated with X-linked mental retardation (XLMR) and DIA1R deletion is associated with syndromes with ASD-like traits and/or XLMR. Together, these results support a model where the DIA1 and DIA1R gene products regulate molecular traffic through the cellular secretory pathway or affect the function of secreted factors, and functional deficits cause disorders with ASD-like symptoms and/or mental retardation.

Hsp90aa1: a novel target gene of miR-1 in cardiac ischemia/reperfusion injury

PubMed Central

Zhu, Wen Si; Guo, Wei; Zhu, Jie Ning; Tang, Chun Mei; Fu, Yong Heng; Lin, Qiu Xiong; Tan, Ning; Shan, Zhi Xin

2016-01-01

The role of microRNA-1 (miR-1) in ischemia/reperfusion (I/R)-induced injury is not well illustrated. The present study aimed to investigate the expression and potential target of miR-1 in the myocardium of a rat model of I/R. The apoptosis of cardiomyocytes in the ischemic rat myocardium increased on day 1, then attenuated on day 3 and day 7 post-I/R. Heat shot protein 90 (Hsp90) aa1 mRNA expression was decreased post-I/R, and Hsp90aa1 protein level was decreased on day1 post-I/R, but was reversed on day 3 and day 7 post-I/R. MiR-1 was downregulated post-I/R, and repression of miR-1 in cultured neonatal rat ventricular cells (NRVCs) led to an increase of Bcl-2 and decreases of Bax and active caspase-3. Dual luciferase reporter assays revealed that miR-1 interacted with the 310–315 nt site at the 3′UTR of Hsp90aa1, and miR-1 was verified to inhibit Hsp90aa1 expression at the posttranscriptional level. Over-expression of Hsp90aa1 could attenuate oxygen-glucose deprivation (OGD)-induced apoptosis of NRVCs. Additionally, miR-1 mimic, in parallel to Hsp90aa1 siRNA, could enhance OGD-induced apoptosis of NRVCs. Taken together, our results reveal that Hsp90aa1 is a novel target of miR-1, and repression of miR-1 may contribute to the recovery of Hsp90aa1 during myocardial I/R. PMID:27076094

R-134a (1,1,1,2-Tetrafluoroethane) Inhalation Induced Reactive Airways Dysfunction Syndrome.

PubMed

Doshi, Viral; Kham, Nang; Kulkarni, Shreedhar; Kapitan, Kent; Henkle, Joseph; White, Peter

2016-01-01

R-134a (1,1,1,2-tetrafluoroethane) is widely used as a refrigerant and as an aerosol propellant. Inhalation of R-134a can lead to asphyxia, transient confusion, and cardiac arrhythmias. We report a case of reactive airways dysfunction syndrome secondary to R-134a inhalation. A 60-year-old nonsmoking man without a history of lung disease was exposed to an air conditioner refrigerant spill while performing repairs beneath a school bus. Afterward, he experienced worsening shortness of breath with minimal exertion, a productive cough, and wheezing. He was also hypoxic. He was admitted to the hospital for further evaluation. Spirometry showed airflow obstruction with an FEV1 1.97 L (45% predicted). His respiratory status improved with bronchodilators and oral steroids. A repeat spirometry 2 weeks later showed improvement with an FEV1 2.5 L (60% predicted). Six months after the incident, his symptoms had improved, but he was still having shortness of breath on exertion and occasional cough.

R1, a novel repressor of the human monoamine oxidase A.

PubMed

Chen, Kevin; Ou, Xiao-Ming; Chen, Gao; Choi, Si Ho; Shih, Jean C

2005-03-25

Monoamine oxidase catalyzes the oxidative deamination of a number of neurotransmitters. A deficiency in monoamine oxidase A results in aggressive behavior in both humans and mice. Studies on the regulation of monoamine oxidase A gene expression have shown that the Sp1 family is important for monoamine oxidase A expression. To search for novel transcription factors, the sequences of three Sp1 sites in the monoamine oxidase A core promoter were used in the yeast one-hybrid system to screen a human cDNA library. A novel repressor, R1 (RAM2), has been cloned. The R1 cDNA encodes a protein with 454 amino acids and an open reading frame at the 5'-end. The transfection of R1 in a human neuroblastoma cell line, SK-N-BE (2)-C, inhibited the monoamine oxidase A promoter and enzymatic activity. The degree of inhibition of monoamine oxidase A by R1 correlated with the level of R1 protein expression. R1 was also found to repress monoamine oxidase A promoter activity within a natural chromatin environment. A gel-shift assay indicated that the endogenous R1 protein in SK-N-BE (2)-C cells interacted with the R1 binding sequence. R1 also bound directly to the natural monoamine oxidase A promoter in vivo as shown by chromatin immunoprecipitation assay. Immunocytochemical analysis showed that R1 was expressed in both cytosol and nucleus, which suggested a role for R1 in transcriptional regulation. Northern blot analysis revealed the presence of endogenous R1 mRNA in human brain and peripheral tissues. Taken together, this study shows that R1 is a novel repressor that inhibits monoamine oxidase A gene expression.

Recent Advances of Colony-Stimulating Factor-1 Receptor (CSF-1R) Kinase and Its Inhibitors.

PubMed

El-Gamal, Mohammed I; Al-Ameen, Shahad K; Al-Koumi, Dania M; Hamad, Mawadda G; Jalal, Nouran A; Oh, Chang-Hyun

2018-01-17

Colony stimulation factor-1 receptor (CSF-1R), which is also known as FMS kinase, plays an important role in initiating inflammatory, cancer, and bone disorders when it is overstimulated by its ligand, CSF-1. Innate immunity, as well as macrophage differentiation and survival, are regulated by the stimulation of the CSF-1R. Another ligand, interlukin-34 (IL-34), was recently reported to activate the CSF-1R receptor in a different manner. The relationship between CSF-1R and microglia has been reviewed. Both CSF-1 antibodies and small molecule CSF-1R kinase inhibitors have now been tested in animal models and in humans. In this Perspective, we discuss the role of CSF-1 and IL-34 in producing cancer, bone disorders, and inflammation. We also review the newly discovered and improved small molecule kinase inhibitors and monoclonal antibodies that have shown potent activity toward CSF-1R, reported from 2012 until 2017.

miR-7-1 POTENTIATED ESTROGEN RECEPTOR AGONISTS FOR FUNCTIONAL NEUROPROTECTION IN VSC4.1 MOTONEURONS

PubMed Central

CHAKRABARTI, M.; BANIK, N. L.; RAY, S. K.

2013-01-01

Protection of motoneurons is an important goal in the treatment of spinal cord injury (SCI). We tested whether neuroprotective microRNAs (miRs) like miR-206, miR-17, miR-21, miR-7-1, and miR-106a could enhance efficacy of estrogen receptor (ER) agonists such as 1,3,5-tris (4-hydroxyphenyl)-4-propyl-1H-pyrazole (PPT, ERα agonist), Way200070 (WAY, ERβ agonist), and estrogen (EST, ERα and ERβ agonist) in preventing apoptosis in the calcium ionophore (CI) insulted VSC4.1 motoneurons. We determined that 200 nM CI induced 70% cell death. Treatment with 50 nM PPT, 100 nM WAY, and 150 nM EST induced overexpression of ERα, ERβ, and both receptors, respectively, at mRNA and protein levels. Treatment with ER agonists significantly upregulated miR-206, miR-17, and miR-7-1 in the CI insulted VSC4.1 motoneurons. Transfection with miR-206, miR-17, or miR-7-1 mimic potentiated WAY or EST to inhibit apoptosis in the CI insulted VSC4.1 motoneurons. Overexpression of miR-7-1 maximally increased efficacy of WAY and EST for down regulation of pro-apoptotic Bax and upregulation of anti-apoptotic Bcl-2. A search using miRDB indicated that miR-7-1 could inhibit expression of L-type Ca2+ channel protein alpha 1C (CPα1C). miR-7-1 overexpression and WAY or EST treatment down regulated CPα1C but upregulated p-Akt to trigger cell survival signaling. The same therapeutic strategy increased expression of the Ca2+/calmodulin-dependent protein kinase II beta (CaMKIIβ) and the phosphorylated cAMP response element binding protein (p-CREB) so as to promote Bcl-2 transcription. Whole cell membrane potential and mitochondrial membrane potential studies indicated that miR-7-1 highly potentiated EST to preserve functionality in the CI insulted VSC4.1 motoneurons. In conclusion, our data indicated that miR-7-1 most significantly potentiated efficacy of EST for functional neuroprotection and this therapeutic strategy could be used in the future to attenuate apoptosis of motoneurons in SCI. PMID

Relationship between the melanocortin-1 receptor (MC1R) variant R306ter and physiological responses to mechanical or thermal stimuli in Labrador Retriever dogs.

PubMed

Perez, Tania E; Mealey, Katrina L; Burke, Neal S; Grubb, Tamara L; Court, Michael H; Greene, Stephen A

2017-03-01

Variants in the MC1R gene have been associated with red hair color and sensitivity to pain in humans. The study objective was to determine if a relationship exists between MC1R genotype and physiological thermal or mechanical nociceptive thresholds in Labrador Retriever dogs. Prospective experimental study. Thirty-four Labrador Retriever dogs were included in the study following public requests for volunteers. Owner consent was obtained and owners verified that their dog was apparently not experiencing pain and had not been treated for pain during the previous 14 days. The study was approved by the Institutional Animal Care and Use Committee. Nociceptive thresholds were determined from a mean of three thermal and five mechanical replications using commercially available algometers. Each dog was genotyped for the previously described MC1R variant (R306ter). Data were analyzed using one-way anova with post hoc comparisons using Tukey's test (p < 0.05). Thirteen dogs were homozygous wild-type (WT/WT), nine were heterozygous (WT/R306ter), and eight were homozygous variant (R306ter/R306ter) genotype. Four dogs could not be genotyped. A significant difference (p = 0.04) in mechanical nociceptive thresholds was identified between dogs with the WT/WT genotype (12.1±2.1 N) and those with the WT/R306ter genotype (9.2±2.4 N). A difference in mechanical, but not thermal, nociceptive threshold was observed between wild-type and heterozygous MC1R variants. Differences in nociceptive thresholds between homozygous R306ter variants and other genotypes for MC1R were not observed. Compared with the wild-type MC1R genotype, nociceptive sensitivity to mechanical force in dogs with a single variant R306ter allele may be greater. However, in contrast to the reported association between homozygous MC1R variants (associated with red hair color) and nociception in humans, we found no evidence of a similar relationship in dogs with the homozygous variant genotype. Copyright © 2017

Vertical Transmission of the Retrotransposable Elements R1 and R2 during the Evolution of the Drosophila Melanogaster Species Subgroup

PubMed Central

Eickbush, D. G.; Eickbush, T. H.

1995-01-01

R1 and R2 are non-long-terminal repeat retrotransposable elements that insert into specific sequences of insect 28S ribosomal RNA genes. These elements have been extensively described in Drosophila melanogaster. To determine whether these elements have been horizontally or vertically transmitted, we characterized R1 and R2 elements from the seven other members of the melanogaster species subgroup by genomic blotting and nucleotide sequencing. Each species was found to have homogeneous families of R1 and R2 elements with the exception of erecta and orena, which have no R2 elements. The DNA sequences of multiple R1 and R2 copies from each species indicated nucleotide divergence within each species averaged only 0.48% for R1 and 0.35% for R2, well below the level of divergence among the species. Most copies of R1 and R2 (40 of 47) sequenced from the seven species were potentially functional, as indicated by the absence of premature termination codons or translational frameshifts that would destroy the open reading frame of the element. The sequence relationships of both the R1 and R2 elements from the various members of the melanogaster subgroup closely followed that of the species phylogeny, suggesting that R1 and R2 have been stably maintained by vertical transmission since the origin of this species subgroup 17-20 million years ago. The remarkable stability of R1 and R2, compared to what has been suggested for transposable elements that insert at multiple locations in these same species, may be due to their unique specificity for sites in the rRNA gene locus. Under low copy number conditions, when it is essential for any mobile element to transpose, the insertion specificities of R1 and R2 ensure uniform developmentally regulated target sites that can be occupied with little or no detrimental effect on the host. PMID:7713424

Sugar-induced cephalic-phase insulin release is mediated by a T1r2+T1r3-independent taste transduction pathway in mice

PubMed Central

Stano, Sarah; Holter, Marlena; Azenkot, Tali; Goldman, Olivia; Margolskee, Robert F.; Vasselli, Joseph R.; Sclafani, Anthony

2015-01-01

Sensory stimulation from foods elicits cephalic phase responses, which facilitate digestion and nutrient assimilation. One such response, cephalic-phase insulin release (CPIR), enhances glucose tolerance. Little is known about the chemosensory mechanisms that activate CPIR. We studied the contribution of the sweet taste receptor (T1r2+T1r3) to sugar-induced CPIR in C57BL/6 (B6) and T1r3 knockout (KO) mice. First, we measured insulin release and glucose tolerance following oral (i.e., normal ingestion) or intragastric (IG) administration of 2.8 M glucose. Both groups of mice exhibited a CPIR following oral but not IG administration, and this CPIR improved glucose tolerance. Second, we examined the specificity of CPIR. Both mouse groups exhibited a CPIR following oral administration of 1 M glucose and 1 M sucrose but not 1 M fructose or water alone. Third, we studied behavioral attraction to the same three sugar solutions in short-term acceptability tests. B6 mice licked more avidly for the sugar solutions than for water, whereas T1r3 KO mice licked no more for the sugar solutions than for water. Finally, we examined chorda tympani (CT) nerve responses to each of the sugars. Both mouse groups exhibited CT nerve responses to the sugars, although those of B6 mice were stronger. We propose that mice possess two taste transduction pathways for sugars. One mediates behavioral attraction to sugars and requires an intact T1r2+T1r3. The other mediates CPIR but does not require an intact T1r2+T1r3. If the latter taste transduction pathway exists in humans, it should provide opportunities for the development of new treatments for controlling blood sugar. PMID:26157055

Sugar-induced cephalic-phase insulin release is mediated by a T1r2+T1r3-independent taste transduction pathway in mice.

PubMed

Glendinning, John I; Stano, Sarah; Holter, Marlena; Azenkot, Tali; Goldman, Olivia; Margolskee, Robert F; Vasselli, Joseph R; Sclafani, Anthony

2015-09-01

Sensory stimulation from foods elicits cephalic phase responses, which facilitate digestion and nutrient assimilation. One such response, cephalic-phase insulin release (CPIR), enhances glucose tolerance. Little is known about the chemosensory mechanisms that activate CPIR. We studied the contribution of the sweet taste receptor (T1r2+T1r3) to sugar-induced CPIR in C57BL/6 (B6) and T1r3 knockout (KO) mice. First, we measured insulin release and glucose tolerance following oral (i.e., normal ingestion) or intragastric (IG) administration of 2.8 M glucose. Both groups of mice exhibited a CPIR following oral but not IG administration, and this CPIR improved glucose tolerance. Second, we examined the specificity of CPIR. Both mouse groups exhibited a CPIR following oral administration of 1 M glucose and 1 M sucrose but not 1 M fructose or water alone. Third, we studied behavioral attraction to the same three sugar solutions in short-term acceptability tests. B6 mice licked more avidly for the sugar solutions than for water, whereas T1r3 KO mice licked no more for the sugar solutions than for water. Finally, we examined chorda tympani (CT) nerve responses to each of the sugars. Both mouse groups exhibited CT nerve responses to the sugars, although those of B6 mice were stronger. We propose that mice possess two taste transduction pathways for sugars. One mediates behavioral attraction to sugars and requires an intact T1r2+T1r3. The other mediates CPIR but does not require an intact T1r2+T1r3. If the latter taste transduction pathway exists in humans, it should provide opportunities for the development of new treatments for controlling blood sugar. Copyright © 2015 the American Physiological Society.

MiR-7-1 potentiated estrogen receptor agonists for functional neuroprotection in VSC4.1 motoneurons.

PubMed

Chakrabarti, M; Banik, N L; Ray, S K

2014-01-03

Protection of motoneurons is an important goal in the treatment of spinal cord injury (SCI). We tested whether neuroprotective microRNAs (miRs) like miR-206, miR-17, miR-21, miR-7-1, and miR-106a could enhance efficacy of estrogen receptor (ER) agonists such as 1,3,5-tris (4-hydroxyphenyl)-4-propyl-1H-pyrazole (PPT, ERα agonist), Way200070 (WAY, ERβ agonist), and estrogen (EST, ERα and ERβ agonist) in preventing apoptosis in the calcium ionophore (CI)-insulted ventral spinal cord 4.1 (VSC4.1) motoneurons. We determined that 200 nM CI induced 70% cell death. Treatment with 50 nM PPT, 100 nM WAY, and 150 nM EST induced overexpression of ERα, ERβ, and both receptors, respectively, at mRNA and protein levels. Treatment with ER agonists significantly upregulated miR-206, miR-17, and miR-7-1 in the CI-insulted VSC4.1 motoneurons. Transfection with miR-206, miR-17, or miR-7-1 mimic potentiated WAY or EST to inhibit apoptosis in the CI-insulted VSC4.1 motoneurons. Overexpression of miR-7-1 maximally increased efficacy of WAY and EST for down regulation of pro-apoptotic Bax and upregulation of anti-apoptotic Bcl-2. A search using microRNA database (miRDB) indicated that miR-7-1 could inhibit the expression of L-type Ca(2+) channel protein alpha 1C (CPα1C). miR-7-1 overexpression and WAY or EST treatment down regulated CPα1C but upregulated p-Akt to trigger cell survival signaling. The same therapeutic strategy increased expression of the Ca(2+)/calmodulin-dependent protein kinase II beta (CaMKIIβ) and the phosphorylated cAMP response element binding protein (p-CREB) so as to promote Bcl-2 transcription. Whole cell membrane potential and mitochondrial membrane potential studies indicated that miR-7-1 highly potentiated EST to preserve functionality in the CI-insulted VSC4.1 motoneurons. In conclusion, our data indicated that miR-7-1 most significantly potentiated efficacy of EST for functional neuroprotection and this therapeutic strategy could be used in the future

MiR-608, pre-miR-124-1 and pre-miR26a-1 polymorphisms modify susceptibility and recurrence-free survival in surgically resected CRC individuals.

PubMed

Ying, Hou-Qun; Peng, Hong-Xin; He, Bang-Shun; Pan, Yu-Qin; Wang, Feng; Sun, Hui-Ling; Liu, Xian; Chen, Jie; Lin, Kang; Wang, Shu-Kui

2016-11-15

Genetic variation within microRNA (miRNA) may result in its abnormal folding or aberrant expression, contributing to colorectal turmorigenesis and metastasis. However, the association of six polymorphisms (miR-608 rs4919510, miR-499a rs3746444, miR-146a rs2910164, pre-miR-143 rs41291957, pre-miR-124-1 rs531564 and pre-miR-26a-1 rs7372209) with colorectal cancer (CRC) risk, therapeutic response and survival remains unclear. A retrospective study was carried out to investigate the association in 1358 0-III stage resected CRC patients and 1079 healthy controls using Sequenom's MassARRAY platform. The results showed that rs4919510 was significantly associated with a decreased susceptibility to CRC in co-dominant, allele and recessive genetic models, and the protective role of rs4919510 allele G and genotype GG was more pronounced among stage 0-II cases; significant association between rs531564 and poor RFS was observed in cases undergoing adjuvant chemo-radiotherapy in co-dominant, allele and dominant models; moreover, there was a positive association between rs7372209 and recurrence-free survival in stage II cases in co-dominant and over-dominant models; additionally, a cumulative effect of rs531564 and rs7372209 at-risk genotypes with hazard ratio at 1.30 and 1.95 for one and two at-risk genotypes was examined in stage II cases, respectively. Our findings indicated that rs4919510 allele G and genotype GG were protective factors for 0-II stage CRC, rs7372209 and rs531564 could decrease RFS in II stage individuals and resected CRC patients receiving adjuvant chemo-radiology.

NK1R/5-HT1AR interaction is related to the regulation of melanogenesis.

PubMed

Wu, Huali; Zhao, Yucheng; Huang, Qiaoling; Cai, Minxuan; Pan, Qi; Fu, Mengsi; An, Xiaohong; Xia, Zhenjiang; Liu, Meng; Jin, Yu; He, Ling; Shang, Jing

2018-06-01

Substance P (SP) is a candidate mediator along the brain-skin axis and can mimic the effects of stress to regulate melanogenesis. Previously, we and others have found that the regulation of SP for pigmentary function was mediated by neurokinin 1 receptor (NK1R). Emerging evidence has accumulated that psychologic stress can induce dysfunction in the cutaneous serotonin 5-hydroxytryptamine (5-HT)-5-HT1A/1B receptor system, thereby resulting in skin hypopigmentation. Moreover, NK1R and 5-HTR (except 5-HT3) belong to GPCR. The present study aimed at assessing the possible existence of NK1R-5-HTR interactions and related melanogenic functions. Western blot and PCR detection revealed that SP reduced expression of 5-HT1A receptor via the NK1 receptor. Biochemical analyses showed that NK1R and 5-HT1AR could colocalize and interact in a cell and in the skin. When the N terminus of the NK1R protein was removed NK1R surface targeting was prevented, the interaction between NK1R-5-HT1AR decreased, and the depigmentation caused by SP and WAY100635 could be rescued. Importantly, pharmaceutical coadministration of NK1R agonist (SP) and 5-HT1A antagonist (WAY100635) enhanced the NK1-5-HT1A receptor coimmunoprecipitation along with the depigmentary response. SP and WAY100635 cooperation elicited activation of a signaling cascade (the extracellular, regulated protein kinase p-JNK signaling pathway) and inhibition of p70S6K1 phosphorylation and greatly reduced melanin production in vitro and in vivo in mice and zebrafish. Moreover, the SP-induced depigmentation response did not be occur in 5-htr1aa +/- zebrafish embryos. Taken together, the results of our systemic study increases our knowledge of the roles of NK1R and 5-HT1AR in melanogenesis and provides possible, novel therapeutic strategies for treatment of skin hypo/hyperpigmentation.-Wu, H., Zhao, Y., Huang, Q., Cai, M., Pan, Q., Fu, M., An, X., Xia, Z., Liu, M., Jin, Y., He, L., Shang, J. NK1R/5-HT1AR interaction is related to

PSCs and GLP-1R: occurrence in normal pancreas, acute/chronic pancreatitis and effect of their activation by a GLP-1R agonist

PubMed Central

Nakamura, Taichi; Ito, Tetsuhide; Uchida, Masahiko; Hijioka, Masayuki; Igarashi, Hisato; Oono, Takamasa; Kato, Masaki; Nakamura, Kazuhiko; Suzuki, Koichi; Jensen, Robert T.; Takayanagi, Ryoichi

2013-01-01

Background and Aims There is increasing concern about the development of pancreatitis in patients with diabetes mellitus who received long-term GLP-1 analog treatment. Its pathogenesis is unknown. The effects of GLP-1 agonists on pancreatic endocrine cells is well studied, however there is little information on effects on other pancreatic tissues that might be involved in inflammatory processes. Pancreatic stellate cells (PSCs) can play an important role in pancreatitis, secreting various inflammatory cytokines/chemokines, as well as collagen. In this study, we investigated GLP-1R occurrence in normal pancreas, acute/chronic pancreatitis, and the effects of GLP-1 analog on normal PSCs, their ability to stimulate inflammatory mediator secretion or proliferation. Methods GLP-1R expression/localization in normal pancreas and pancreatitis (acute/chronic) tissues were evaluated with histological/immunohistochemical analysis. PSCs were isolated from male Wistar rats. GLP1R expression and effects of GLP-1 analog on activated PSCs was examined with realtime PCR, MTS assays and Western Blotting. Results In normal pancreas, pancreatic β cells expressed GLP-1R, with only low expression in acinar cells, whereas in acute or chronic pancreatitis, acinar cells, ductal cells and activated PSCs expressed GLP-1R. With activation of normal PSCs, GLP-1R is markedly increased, as is multiple other incretin-related receptors. The GLP-1 analog, liraglutide, did not induce inflammatory genes expression in activated PSCs, but induced proliferation. Liraglutide activated multiple signaling cascades in PSCs, and the ERK pathway mediated the PSCs proliferation. Conclusions GLP-1Rs are expressed in normal pancreas and there is marked enhanced expression in acute/chronic pancreatitis. GLP-1-agonist induced cell proliferation of activated PSCs without increasing release of inflammatory mediators. These results suggest chronic treatment with GLP-1R agonists could lead to proliferation

Differential Regulation of ERK1/2 and mTORC1 Through T1R1/T1R3 in MIN6 Cells.

PubMed

Wauson, Eric M; Guerra, Marcy L; Dyachok, Julia; McGlynn, Kathleen; Giles, Jennifer; Ross, Elliott M; Cobb, Melanie H

2015-08-01

The MAPKs ERK1/2 respond to nutrients and other insulin secretagogues in pancreatic β-cells and mediate nutrient-dependent insulin gene transcription. Nutrients also stimulate the mechanistic target of rapamycin complex 1 (mTORC1) to regulate protein synthesis. We showed previously that activation of both ERK1/2 and mTORC1 in the MIN6 pancreatic β-cell-derived line by extracellular amino acids (AAs) is at least in part mediated by the heterodimeric T1R1/T1R3, a G protein-coupled receptor. We show here that AAs differentially activate these two signaling pathways in MIN6 cells. Pretreatment with pertussis toxin did not prevent the activation of either ERK1/2 or mTORC1 by AAs, indicating that G(I) is not central to either pathway. Although glucagon-like peptide 1, an agonist for a G(s-)coupled receptor, activated ERK1/2 well and mTORC1 to a small extent, AAs had no effect on cytosolic cAMP accumulation. Ca(2+) entry is required for ERK1/2 activation by AAs but is dispensable for AA activation of mTORC1. Pretreatment with UBO-QIC, a selective G(q) inhibitor, reduced the activation of ERK1/2 but had little effect on the activation of mTORC1 by AAs, suggesting a differential requirement for G(q). Inhibition of G(12/13) by the overexpression of the regulator of G protein signaling domain of p115 ρ-guanine nucleotide exchange factor had no effect on mTORC1 activation by AAs, suggesting that these G proteins are also not involved. We conclude that AAs regulate ERK1/2 and mTORC1 through distinct signaling pathways.

Differential Regulation of ERK1/2 and mTORC1 Through T1R1/T1R3 in MIN6 Cells

PubMed Central

Wauson, Eric M.; Guerra, Marcy L.; Dyachok, Julia; McGlynn, Kathleen; Giles, Jennifer; Ross, Elliott M.

2015-01-01

The MAPKs ERK1/2 respond to nutrients and other insulin secretagogues in pancreatic β-cells and mediate nutrient-dependent insulin gene transcription. Nutrients also stimulate the mechanistic target of rapamycin complex 1 (mTORC1) to regulate protein synthesis. We showed previously that activation of both ERK1/2 and mTORC1 in the MIN6 pancreatic β-cell-derived line by extracellular amino acids (AAs) is at least in part mediated by the heterodimeric T1R1/T1R3, a G protein-coupled receptor. We show here that AAs differentially activate these two signaling pathways in MIN6 cells. Pretreatment with pertussis toxin did not prevent the activation of either ERK1/2 or mTORC1 by AAs, indicating that Gi is not central to either pathway. Although glucagon-like peptide 1, an agonist for a Gs-coupled receptor, activated ERK1/2 well and mTORC1 to a small extent, AAs had no effect on cytosolic cAMP accumulation. Ca2+ entry is required for ERK1/2 activation by AAs but is dispensable for AA activation of mTORC1. Pretreatment with UBO-QIC, a selective Gq inhibitor, reduced the activation of ERK1/2 but had little effect on the activation of mTORC1 by AAs, suggesting a differential requirement for Gq. Inhibition of G12/13 by the overexpression of the regulator of G protein signaling domain of p115 ρ-guanine nucleotide exchange factor had no effect on mTORC1 activation by AAs, suggesting that these G proteins are also not involved. We conclude that AAs regulate ERK1/2 and mTORC1 through distinct signaling pathways. PMID:26168033

Taste information derived from T1R-expressing taste cells in mice.

PubMed

Yoshida, Ryusuke; Ninomiya, Yuzo

2016-03-01

The taste system of animals is used to detect valuable nutrients and harmful compounds in foods. In humans and mice, sweet, bitter, salty, sour and umami tastes are considered the five basic taste qualities. Sweet and umami tastes are mediated by G-protein-coupled receptors, belonging to the T1R (taste receptor type 1) family. This family consists of three members (T1R1, T1R2 and T1R3). They function as sweet or umami taste receptors by forming heterodimeric complexes, T1R1+T1R3 (umami) or T1R2+T1R3 (sweet). Receptors for each of the basic tastes are thought to be expressed exclusively in taste bud cells. Sweet (T1R2+T1R3-expressing) taste cells were thought to be segregated from umami (T1R1+T1R3-expressing) taste cells in taste buds. However, recent studies have revealed that a significant portion of taste cells in mice expressed all T1R subunits and responded to both sweet and umami compounds. This suggests that sweet and umami taste cells may not be segregated. Mice are able to discriminate between sweet and umami tastes, and both tastes contribute to behavioural preferences for sweet or umami compounds. There is growing evidence that T1R3 is also involved in behavioural avoidance of calcium tastes in mice, which implies that there may be a further population of T1R-expressing taste cells that mediate aversion to calcium taste. Therefore the simple view of detection and segregation of sweet and umami tastes by T1R-expressing taste cells, in mice, is now open to re-examination. © 2016 Authors; published by Portland Press Limited.

26 CFR 1.414(r)-11 - Definitions and special rules.

Code of Federal Regulations, 2010 CFR

2010-04-01

... 26 Internal Revenue 5 2010-04-01 2010-04-01 false Definitions and special rules. 1.414(r)-11 Section 1.414(r)-11 Internal Revenue INTERNAL REVENUE SERVICE, DEPARTMENT OF THE TREASURY (CONTINUED) INCOME TAX (CONTINUED) INCOME TAXES Pension, Profit-Sharing, Stock Bonus Plans, Etc. § 1.414(r)-11...

Evaluation of R1-6 gateway treatment alternatives for pedestrian crossings.

DOT National Transportation Integrated Search

2016-02-01

A series of studies evaluated the Gateway configuration of R1-6 in-street signs. A : Gateway configuration consists of an R1-6 sign on the centerline, an R1-6 signs on both edges of : the roadway, and R1-6 signs on the lane lines if it is a multilane...

26 CFR 1.414(r)-3 - Separate line of business.

Code of Federal Regulations, 2013 CFR

2013-04-01

... 26 Internal Revenue 5 2013-04-01 2013-04-01 false Separate line of business. 1.414(r)-3 Section 1.414(r)-3 Internal Revenue INTERNAL REVENUE SERVICE, DEPARTMENT OF THE TREASURY (CONTINUED) INCOME TAX (CONTINUED) INCOME TAXES (CONTINUED) Pension, Profit-Sharing, Stock Bonus Plans, Etc. § 1.414(r)-3 Separate...

26 CFR 1.414(r)-3 - Separate line of business.

Code of Federal Regulations, 2011 CFR

2011-04-01

... 26 Internal Revenue 5 2011-04-01 2011-04-01 false Separate line of business. 1.414(r)-3 Section 1.414(r)-3 Internal Revenue INTERNAL REVENUE SERVICE, DEPARTMENT OF THE TREASURY (CONTINUED) INCOME TAX (CONTINUED) INCOME TAXES (CONTINUED) Pension, Profit-Sharing, Stock Bonus Plans, Etc. § 1.414(r)-3 Separate...

26 CFR 1.414(r)-3 - Separate line of business.

Code of Federal Regulations, 2012 CFR

2012-04-01

... 26 Internal Revenue 5 2012-04-01 2011-04-01 true Separate line of business. 1.414(r)-3 Section 1.414(r)-3 Internal Revenue INTERNAL REVENUE SERVICE, DEPARTMENT OF THE TREASURY (CONTINUED) INCOME TAX (CONTINUED) INCOME TAXES (CONTINUED) Pension, Profit-Sharing, Stock Bonus Plans, Etc. § 1.414(r)-3 Separate...

26 CFR 1.414(r)-3 - Separate line of business.

Code of Federal Regulations, 2014 CFR

2014-04-01

... 26 Internal Revenue 5 2014-04-01 2014-04-01 false Separate line of business. 1.414(r)-3 Section 1.414(r)-3 Internal Revenue INTERNAL REVENUE SERVICE, DEPARTMENT OF THE TREASURY (CONTINUED) INCOME TAX (CONTINUED) INCOME TAXES (CONTINUED) Pension, Profit-Sharing, Stock Bonus Plans, Etc. § 1.414(r)-3 Separate...

Optically active antifungal azoles. XII. Synthesis and antifungal activity of the water-soluble prodrugs of 1-[(1R,2R)-2-(2,4-difluorophenyl)-2-hydroxy-1-methyl-3-(1H-1,2,4-triazol-1-yl)propyl]-3-[4-(1H-1-tetrazolyl)phenyl]-2-imidazolidinone.

PubMed

Ichikawa, T; Kitazaki, T; Matsushita, Y; Yamada, M; Hayashi, R; Yamaguchi, M; Kiyota, Y; Okonogi, K; Itoh, K

2001-09-01

1-[(1R,2R)-2-(2,4-Difluorophenyl)-2-hydroxy-1-methyl-3-(1H-1,2,4-triazol-1-yl)propyl]-3-[4-(1H-1-tetrazolyl)phenyl]-2-imidazolidinone (1: TAK-456) was selected as a candidate for clinical trials, but since its water-solubility was insufficient for an injectable formulation, the quaternary triazolium salts 2 were designed as water-soluble prodrugs. Among the prodrugs prepared, 4-acetoxymethyl-1-[(2R,3R)-2-(2,4-difluorophenyl)-2-hydroxy-3-[2-oxo-3-[4-(1H-1-terazolyl)phenyl]-1-imidazolidinyl]butyl]-1H-1,2,4-triazolium chloride (2a: TAK-457) was selected as an injectable candidate for clinical trials based on the results of evaluations on solubility, stability, hemolytic effect and in vivo antifungal activities.

Notoginsenoside R1 attenuates renal ischemia-reperfusion injury in rats.

PubMed

Liu, Wen-Jun; Tang, Hong-Tai; Jia, Yi-Tao; Ma, Bing; Fu, Jin-Feng; Wang, Yu; Lv, Kai-Yang; Xia, Zhao-Fan

2010-09-01

Ischemia-reperfusion (I/R) injury of the kidney is a complex pathophysiological process and a major cause of acute renal failure. It has been shown that I/R injury is related to inflammatory responses and activation of apoptotic pathways. Inhibition of certain elements of inflammatory responses and apoptotic pathway seemed to ameliorate renal I/R injury. As an effective element of Panax notoginseng, NR1 has antioxidant, anti-inflammatory, antiapoptotic, and immune-stimulatory activities. Therefore, we speculate that NR1 can attenuate renal I/R injury. Ischemia-reperfusion injury was induced by renal pedicle ligation followed by reperfusion along with a contralateral nephrectomy. Male Sprague-Dawley rats were randomized to four groups: sham group, I/R control group, NR1-1 group (rats treated with NR1, 20 mg.kg.d) and NR1-2 group (rats treated with NR1, 40 mg.kg.d). All animals were killed 72 h after I/R induction. Blood and renal tissues were collected. Renal dysfunction was observed by the level of serum creatinine and histological evaluation. Apoptosis and inflammatory response in the tissue of kidney were detected mainly with molecular biological methods. NR1 attenuated I/R-induced renal dysfunction as indicated by the level of serum creatinine and histological evaluation. It prevented the I/R-induced increases in the levels of proinflammatory cytokine TNF-alpha, myeloperoxidase activity, phosphorylation of p38, and activation of nuclear factor kappaB with cell apoptosis in the kidney and enhanced expression of antiapoptosis cytokine bcl-2. Treatment with NR1 improves renal function after I/R associated with a significant reduction in cell apoptosis and inflammatory responses, which may be related to p38 and nuclear factor kappaB inhibition.

Induction of an interleukin-1 receptor (IL-1R) on monocytic cells. Evidence that the receptor is not encoded by a T cell-type IL-1R mRNA.

PubMed

Spriggs, M K; Lioubin, P J; Slack, J; Dower, S K; Jonas, U; Cosman, D; Sims, J E; Bauer, J

1990-12-25

Primary human monocytes and the human monocytic cell line THP-1 were induced to express receptors for interleukin-1 alpha (IL-1 alpha) and IL-1 beta. Treatment of primary monocytes with dexamethasone resulted in a 10-fold increase in receptor number over untreated cells, to approximately 2,000 receptors/cell. Treatment of THP-1 cells with phorbol ester followed by prostaglandin E2 and dexamethasone resulted in the expression of approximately 30,000 receptors/cell. Competitive binding assays on THP-1 cells showed that both IL-1 alpha and IL-1 beta bind to the same receptor. The monocyte IL-1R is significantly smaller (63 kDa) than the T cell IL-1R (80 kDa) and is immunologically distinct. However, induction of monocytes and monocytic cell lines leads to the appearance of an abundant mRNA of approximately 5,000 bases which hybridizes to a cDNA probe from the T cell-type IL-1R. Sequence data obtained from a cDNA clone of this mRNA indicate that the message is identical to the T cell IL-1R mRNA throughout the coding region. A smaller mRNA, also homologous to the T cell IL-1R mRNA, accumulated in induced THP-1 cells and has a shorter 3'-untranslated region than the larger. Data are presented which suggest that neither form of this message encodes the 63-kDa IL-1R, but rather that this protein is the product of a separate nonhomologous mRNA.

Turnover of R1 (Type I) and R2 (Type Ii) Retrotransposable Elements in the Ribosomal DNA of Drosophila Melanogaster

PubMed Central

Jakubczak, J. L.; Zenni, M. K.; Woodruff, R. C.; Eickbush, T. H.

1992-01-01

R1 and R2 are distantly related non-long terminal repeat retrotransposable elements each of which inserts into a specific site in the 28S rRNA genes of most insects. We have analyzed aspects of R1 and R2 abundance and sequence variation in 27 geographical isolates of Drosophila melanogaster. The fraction of 28S rRNA genes containing these elements varied greatly between strains, 17-67% for R1 elements and 2-28% for R2 elements. The total percentage of the rDNA repeats inserted ranged from 32 to 77%. The fraction of the rDNA repeats that contained both of these elements suggested that R1 and R2 exhibit neither an inhibition of nor preference for insertion into a 28S gene already containing the other type of element. Based on the conservation of restriction sites in the elements of all strains, and sequence analysis of individual elements from three strains, nucleotide divergence is very low for R1 and R2 elements within or between strains (<0.6%). This sequence uniformity is the expected result of the forces of concerted evolution (unequal crossovers and gene conversion) which act on the rRNA genes themselves. Evidence for the role of retrotransposition in the turnover of R1 and R2 was obtained by using naturally occurring 5' length polymorphisms of the elements as markers for independent transposition events. The pattern of these different length 5' truncations of R1 and R2 was found to be diverse and unique to most strains analyzed. Because recombination can only, with time, amplify or eliminate those length variants already present, the diversity found in each strain suggests that retrotransposition has played a critical role in maintaining these elements in the rDNA repeats of D. melanogaster. PMID:1317313

Longitudinal T1 relaxation rate (R1) captures changes in short-term Mn exposure in welders

PubMed Central

Lewis, Mechelle M.; Flynn, Michael R.; Lee, Eun-Young; Van Buren, Scott; Van Buren, Eric; Du, Guangwei; Fry, Rebecca C.; Herring, Amy H.; Kong, Lan; Mailman, Richard B.; Huang, Xuemei

2016-01-01

Objectives We demonstrated recently that the T1 relaxation rate (R1) captured short-term Mn exposure in welders with chronic, relatively low exposure levels in a cross-sectional study. In the current study, we used a longitudinal design to examine whether R1 values reflect the short-term dynamics of Mn exposure. Methods Twenty-nine welders were evaluated at baseline and 12 months. Occupational questionnaires estimated short-term welding exposure using welding hours in the 90 days prior to each study visit (HrsW90). In addition, blood Mn levels, the pallidal index (PI; globus pallidus T1-weighted intensity (T1WI)/frontal white matter T1WI), and R1 values in brain regions of interest (ROIs) were determined as Mn biomarkers at each visit. Associations between changes in estimated welding exposure and changes in purported Mn biomarkers were assessed by Spearman’s correlations with adjustment for age and baseline R1, HrsW90, and blood Mn values. Results Changes in welding hours (HrsW90: the short-term welding exposure estimate), was associated significantly with changes in R1 values in the putamen (r=0.541, p=0.005), caudate (R=0.453, p=0.023), globus pallidus (R=0.430, p=0.032), amygdala (R=0.461, p=0.020), and hippocampus (R=0.447, p=0.025), but not with changes in blood Mn levels or the PI. Discussion Changes in R1 values correlated with changes in the short-term welding exposure estimate, but not with more traditional measures of Mn exposure (blood Mn levels or PI). These results suggest that R1 may serve as a useful marker to capture the short-term dynamics in Mn brain accumulation related to welding exposure. PMID:27567731

Crosstalk between the IGF-1R/AKT/mTORC1 pathway and the tumor suppressors p53 and p27 determines cisplatin sensitivity and limits the effectiveness of an IGF-1R pathway inhibitor

PubMed Central

Davaadelger, Batzaya; Duan, Lei; Perez, Ricardo E.; Gitelis, Steven; Maki, Carl G.

2016-01-01

The insulin-like growth factor-1 receptor (IGF-1R) signaling pathway is aberrantly activated in multiple cancers and can promote proliferation and chemotherapy resistance. Multiple IGF-1R inhibitors have been developed as potential therapeutics. However, these inhibitors have failed to increase patient survival when given alone or in combination with chemotherapy agents. The reason(s) for the disappointing clinical effect of these inhibitors is not fully understood. Cisplatin (CP) activated the IGF-1R/AKT/mTORC1 pathway and stabilized p53 in osteosarcoma (OS) cells. p53 knockdown reduced IGF-1R/AKT/mTORC1 activation by CP, and IGF-1R inhibition reduced the accumulation of p53. These data demonstrate positive crosstalk between p53 and the IGF-1R/AKT/mTORC1 pathway in response to CP. Further studies showed the effect of IGF-1R inhibition on CP response is dependent on p53 status. In p53 wild-type cells treated with CP, IGF-1R inhibition increased p53s apoptotic function but reduced p53-dependent senescence, and had no effect on long term survival. In contrast, in p53-null/knockdown cells, IGF-1R inhibition reduced apoptosis in response to CP and increased long term survival. These effects were due to p27 since IGF-1R inhibition stabilized p27 in CP-treated cells, and p27 depletion restored apoptosis and reduced long term survival. Together, the results demonstrate 1) p53 expression determines the effect of IGF-1R inhibition on cancer cell CP response, and 2) crosstalk between the IGF-1R/AKT/mTORC1 pathway and p53 and p27 can reduce cancer cell responsiveness to chemotherapy and may ultimately limit the effectiveness of IGF-1R pathway inhibitors in the clinic. PMID:27050276

SUMO-modified insulin-like growth factor 1 receptor (IGF-1R) increases cell cycle progression and cell proliferation.

PubMed

Lin, Yingbo; Liu, Hongyu; Waraky, Ahmed; Haglund, Felix; Agarwal, Prasoon; Jernberg-Wiklund, Helena; Warsito, Dudi; Larsson, Olle

2017-10-01

Increasing number of studies have shown nuclear localization of the insulin-like growth factor 1 receptor (nIGF-1R) in tumor cells and its links to adverse clinical outcome in various cancers. Any obvious cell physiological roles of nIGF-1R have, however, still not been disclosed. Previously, we reported that IGF-1R translocates to cell nucleus and modulates gene expression by binding to enhancers, provided that the receptor is SUMOylated. In this study, we constructed stable transfectants of wild type IGF1R (WT) and triple-SUMO-site-mutated IGF1R (TSM) using igf1r knockout mouse fibroblasts (R-). Cell clones (R-WT and R-TSM) expressing equal amounts of IGF-1R were selected for experiments. Phosphorylation of IGF-1R, Akt, and Erk upon IGF-1 stimulation was equal in R-WT and R-TSM. WT was confirmed to enter nuclei. TSM did also undergo nuclear translocation, although to a lesser extent. This may be explained by that TSM heterodimerizes with insulin receptor, which is known to translocate to cell nuclei. R-WT proliferated substantially faster than R-TSM, which did not differ significantly from the empty vector control. Upon IGF-1 stimulation G1-S-phase progression of R-WT increased from 12 to 38%, compared to 13 to 20% of R-TSM. The G1-S progression of R-WT correlated with increased expression of cyclin D1, A, and CDK2, as well as downregulation of p27. This suggests that SUMO-IGF-1R affects upstream mechanisms that control and coordinate expression of cell cycle regulators. Further studies to identify such SUMO-IGF-1R dependent mechanisms seem important. © 2017 The Authors. Journal of Cellular Physiology Published by Wiley Periodicals Inc.

Csf1r-mApple Transgene Expression and Ligand Binding In Vivo Reveal Dynamics of CSF1R Expression within the Mononuclear Phagocyte System.

PubMed

Hawley, Catherine A; Rojo, Rocio; Raper, Anna; Sauter, Kristin A; Lisowski, Zofia M; Grabert, Kathleen; Bain, Calum C; Davis, Gemma M; Louwe, Pieter A; Ostrowski, Michael C; Hume, David A; Pridans, Clare; Jenkins, Stephen J

2018-03-15

CSF1 is the primary growth factor controlling macrophage numbers, but whether expression of the CSF1 receptor differs between discrete populations of mononuclear phagocytes remains unclear. We have generated a Csf1r -mApple transgenic fluorescent reporter mouse that, in combination with lineage tracing, Alexa Fluor 647-labeled CSF1-Fc and CSF1, and a modified Δ Csf1- enhanced cyan fluorescent protein (ECFP) transgene that lacks a 150 bp segment of the distal promoter, we have used to dissect the differentiation and CSF1 responsiveness of mononuclear phagocyte populations in situ. Consistent with previous Csf1r- driven reporter lines, Csf1r -mApple was expressed in blood monocytes and at higher levels in tissue macrophages, and was readily detectable in whole mounts or with multiphoton microscopy. In the liver and peritoneal cavity, uptake of labeled CSF1 largely reflected transgene expression, with greater receptor activity in mature macrophages than monocytes and tissue-specific expression in conventional dendritic cells. However, CSF1 uptake also differed between subsets of monocytes and discrete populations of tissue macrophages, which in macrophages correlated with their level of dependence on CSF1 receptor signaling for survival rather than degree of transgene expression. A double Δ Csf1r -ECFP- Csf1r -mApple transgenic mouse distinguished subpopulations of microglia in the brain, and permitted imaging of interstitial macrophages distinct from alveolar macrophages, and pulmonary monocytes and conventional dendritic cells. The Csf1r- mApple mice and fluorescently labeled CSF1 will be valuable resources for the study of macrophage and CSF1 biology, which are compatible with existing EGFP-based reporter lines. Copyright © 2018 The Authors.

Unprecedented genomic diversity of AhR1 and AhR2 genes in Atlantic salmon (Salmo salar L.).

PubMed

Hansson, Maria C; Wittzell, Håkan; Persson, Kerstin; von Schantz, Torbjörn

2004-06-24

Aryl hydrocarbon receptor (AhR) genes encode proteins involved in mediating the toxic responses induced by several environmental pollutants. Here, we describe the identification of the first two AhR1 (alpha and beta) genes and two additional AhR2 (alpha and beta) genes in the tetraploid species Atlantic salmon (Salmo salar L.) from a cosmid library screening. Cosmid clones containing genomic salmon AhR sequences were isolated using a cDNA clone containing the coding region of the Atlantic salmon AhR2gamma as a probe. Screening revealed 14 positive clones, from which four were chosen for further analyses. One of the cosmids contained genomic AhR sequences that were highly similar to the rainbow trout (Oncorhynchus mykiss) AhR2alpha and beta genes. SMART RACE amplified two complete, highly similar but not identical AhR type 2 sequences from salmon cDNA, which from phylogenetic analyses were determined as the rainbow trout AhR2alpha and beta orthologs. The salmon AhR2alpha and beta encode proteins of 1071 and 1058 residues, respectively, and encompass characteristic AhR sequence elements like a basic-helix-loop-helix (bHLH) and two PER-ARNT-SIM (PAS) domains. Both genes are transcribed in liver, spleen and muscle tissues of adult salmon. A second cosmid contained partial sequences, which were identical to the previously characterized AhR2gamma gene. The last two cosmids contained partial genomic AhR sequences, which were more similar to other AhR type 1 fish genes than the four characterized salmon AhR2 genes. However, attempts to amplify the corresponding complete cDNA sequences of the inserts proved very difficult, suggesting that these genes are non-functional or very weakly transcribed in the examined tissues. Phylogenetic analyses of the conserved regions did, however, clearly indicate that these two AhRs belong to the AhR type 1 clade and have been assigned as the Atlantic salmon AhR1alpha and AhR1beta genes. Taken together, these findings demonstrate that

Characterization of the compact bicistronic microRNA precursor, miR-1/miR-133, expressed specifically in Ciona muscle tissues.

PubMed

Kusakabe, Rie; Tani, Saori; Nishitsuji, Koki; Shindo, Miyuki; Okamura, Kohji; Miyamoto, Yuki; Nakai, Kenta; Suzuki, Yutaka; Kusakabe, Takehiro G; Inoue, Kunio

2013-01-01

Muscle-specific miR-1/206 and miR-133 families have been suggested to play fundamental roles in skeletal and cardiac myogenesis in vertebrates. To gain insights into the relationships between the divergence of these miRs and muscular tissue types, we investigated the expression patterns of miR-1 and miR-133 in two ascidian Ciona species and compared their genomic structures with those of other chordates. We found that Ciona intestinalis and Ciona savignyi each possess a single copy of the miR-1/miR-133 cluster, which is only 350 nucleotide long. During embryogenesis, Ciona miR-1 and miR-133 are generated as a single continuous primary transcript accumulated in the nuclei of the tail muscle cells, starting at the gastrula stage. In adults, mature miR-133 and miR-1 are differentially expressed in the heart and body wall muscle. Expression of the reporter gene linked to the 850-bp upstream region of the predicted transcription start site confirmed that this region drives the muscle-specific expression of the primary transcript of miR-1/miR-133. In many deuterostome lineages, including that of Ciona, the miR-1/133 cluster is located in the same intron of the mind bomb (mib) gene in reverse orientation. Our results suggest that the origin of genomic organization and muscle-specific regulation of miR-1/133 can be traced back to the ancestor of chordates. Duplication of this miR cluster might have led to the remarkable elaboration in the morphology and function of skeletal muscles in the vertebrate lineage. Copyright © 2012 Elsevier B.V. All rights reserved.

TRPV1 channels and the progesterone receptor Sig-1R interact to regulate pain.

PubMed

Ortíz-Rentería, Miguel; Juárez-Contreras, Rebeca; González-Ramírez, Ricardo; Islas, León D; Sierra-Ramírez, Félix; Llorente, Itzel; Simon, Sidney A; Hiriart, Marcia; Rosenbaum, Tamara; Morales-Lázaro, Sara L

2018-02-13

The Transient Receptor Potential Vanilloid 1 (TRPV1) ion channel is expressed in nociceptors where, when activated by chemical or thermal stimuli, it functions as an important transducer of painful and itch-related stimuli. Although the interaction of TRPV1 with proteins that regulate its function has been previously explored, their modulation by chaperones has not been elucidated, as is the case for other mammalian TRP channels. Here we show that TRPV1 physically interacts with the Sigma 1 Receptor (Sig-1R), a chaperone that binds progesterone, an antagonist of Sig-1R and an important neurosteroid associated to the modulation of pain. Antagonism of Sig-1R by progesterone results in the down-regulation of TRPV1 expression in the plasma membrane of sensory neurons and, consequently, a decrease in capsaicin-induced nociceptive responses. This is observed both in males treated with a synthetic antagonist of Sig-1R and in pregnant females where progesterone levels are elevated. This constitutes a previously undescribed mechanism by which TRPV1-dependent nociception and pain can be regulated.

Transcription factor-dependent chromatin remodeling of Il18r1 during Th1 and Th2 differentiation 1

PubMed Central

Yu, Qing; Chang, Hua-Chen; Ahyi, Ayele-Nati N.; Kaplan, Mark H.

2008-01-01

The IL-18Rα chain is expressed on Th1 but not Th2 cells. We have recently shown that Stat4 is an important component of programming the Il18r1 locus (encoding IL-18Rα) for maximal expression in Th1 cells. Il18r1 is reciprocally repressed during Th2 development. In this report we demonstrate that the establishment of DNase hypersensitivity patterns that are distinct among undifferentiated CD4 T cells, Th1 and Th2 cells. Stat6 is required for the repression of Il18r1 expression and in Stat6-deficient Th2 cultures, mRNA levels, histone acetylation and H3K4 methylation levels are intermediate between levels observed in Th1 and Th2 cells. Despite the repressive effects of IL-4 during Th2 differentiation, we observed only modest binding of Stat6 to the Il18r1 locus. In contrast, we observed robust GATA-3 binding to a central region of the locus where DNase hypersensitivity sites overlapped with conserved non-coding sequences in Il18r1 introns. Ectopic expression of GATA-3 in differentiated Th1 cells repressed Il18r1 mRNA and surface expression of IL-18Rα. These data provide further mechanistic insight into transcription factor dependent establishment of Th subset-specific patterns of gene expression. PMID:18714006

26 CFR 1.414(r)-11 - Definitions and special rules.

Code of Federal Regulations, 2014 CFR

2014-04-01

... 26 Internal Revenue 5 2014-04-01 2014-04-01 false Definitions and special rules. 1.414(r)-11 Section 1.414(r)-11 Internal Revenue INTERNAL REVENUE SERVICE, DEPARTMENT OF THE TREASURY (CONTINUED...(r)-11 Definitions and special rules. (a) In general. This section contains certain definitions and...

26 CFR 1.414(r)-11 - Definitions and special rules.

Code of Federal Regulations, 2012 CFR

2012-04-01

... 26 Internal Revenue 5 2012-04-01 2011-04-01 true Definitions and special rules. 1.414(r)-11 Section 1.414(r)-11 Internal Revenue INTERNAL REVENUE SERVICE, DEPARTMENT OF THE TREASURY (CONTINUED...(r)-11 Definitions and special rules. (a) In general. This section contains certain definitions and...

26 CFR 1.414(r)-11 - Definitions and special rules.

Code of Federal Regulations, 2011 CFR

2011-04-01

... 26 Internal Revenue 5 2011-04-01 2011-04-01 false Definitions and special rules. 1.414(r)-11 Section 1.414(r)-11 Internal Revenue INTERNAL REVENUE SERVICE, DEPARTMENT OF THE TREASURY (CONTINUED...(r)-11 Definitions and special rules. (a) In general. This section contains certain definitions and...

26 CFR 1.414(r)-11 - Definitions and special rules.

Code of Federal Regulations, 2013 CFR

2013-04-01

... 26 Internal Revenue 5 2013-04-01 2013-04-01 false Definitions and special rules. 1.414(r)-11 Section 1.414(r)-11 Internal Revenue INTERNAL REVENUE SERVICE, DEPARTMENT OF THE TREASURY (CONTINUED...(r)-11 Definitions and special rules. (a) In general. This section contains certain definitions and...

The cognition-enhancing activity of E1R, a novel positive allosteric modulator of sigma-1 receptors.

PubMed

Zvejniece, L; Vavers, E; Svalbe, B; Vilskersts, R; Domracheva, I; Vorona, M; Veinberg, G; Misane, I; Stonans, I; Kalvinsh, I; Dambrova, M

2014-02-01

Here, we describe the in vitro and in vivo effects of (4R,5S)-2-(5-methyl-2-oxo-4-phenyl-pyrrolidin-1-yl)-acetamide (E1R), a novel positive allosteric modulator of sigma-1 receptors. E1R was tested for sigma receptor binding activity in a [³H](+)-pentazocine assay, in bradykinin (BK)-induced intracellular Ca²⁺ concentration ([Ca²⁺](i)) assays and in an electrically stimulated rat vas deferens model. E1R's effects on cognitive function were tested using passive avoidance (PA) and Y-maze tests in mice. A selective sigma-1 receptor antagonist (NE-100), was used to study the involvement of the sigma-1 receptor in the effects of E1R. The open-field test was used to detect the effects of E1R on locomotion. Pretreatment with E1R enhanced the selective sigma-1 receptor agonist PRE-084's stimulating effect during a model study employing electrically stimulated rat vasa deferentia and an assay measuring the BK-induced [Ca²⁺](i) increase. Pretreatment with E1R facilitated PA retention in a dose-related manner. Furthermore, E1R alleviated the scopolamine-induced cognitive impairment during the PA and Y-maze tests in mice. The in vivo and in vitro effects of E1R were blocked by treatment with the selective sigma-1 receptor antagonist NE-100. E1R did not affect locomotor activity. E1R is a novel 4,5-disubstituted derivative of piracetam that enhances cognition and demonstrates efficacy against scopolamine-induced cholinergic dysfunction in mice. These effects are attributed to its positive modulatory action on the sigma-1 receptor and this activity may be relevant when developing new drugs for treating cognitive symptoms related to neurodegenerative diseases. © 2013 The British Pharmacological Society.

The cognition-enhancing activity of E1R, a novel positive allosteric modulator of sigma-1 receptors

PubMed Central

Zvejniece, L; Vavers, E; Svalbe, B; Vilskersts, R; Domracheva, I; Vorona, M; Veinberg, G; Misane, I; Stonans, I; Kalvinsh, I; Dambrova, M

2014-01-01

Background and Purpose Here, we describe the in vitro and in vivo effects of (4R,5S)-2-(5-methyl-2-oxo-4-phenyl-pyrrolidin-1-yl)-acetamide (E1R), a novel positive allosteric modulator of sigma-1 receptors. Experimental Approach E1R was tested for sigma receptor binding activity in a [3H](+)-pentazocine assay, in bradykinin (BK)-induced intracellular Ca2+ concentration ([Ca2+]i) assays and in an electrically stimulated rat vas deferens model. E1R's effects on cognitive function were tested using passive avoidance (PA) and Y-maze tests in mice. A selective sigma-1 receptor antagonist (NE-100), was used to study the involvement of the sigma-1 receptor in the effects of E1R. The open-field test was used to detect the effects of E1R on locomotion. Key Results Pretreatment with E1R enhanced the selective sigma-1 receptor agonist PRE-084's stimulating effect during a model study employing electrically stimulated rat vasa deferentia and an assay measuring the BK-induced [Ca2+]i increase. Pretreatment with E1R facilitated PA retention in a dose-related manner. Furthermore, E1R alleviated the scopolamine-induced cognitive impairment during the PA and Y-maze tests in mice. The in vivo and in vitro effects of E1R were blocked by treatment with the selective sigma-1 receptor antagonist NE-100. E1R did not affect locomotor activity. Conclusion and Implications E1R is a novel 4,5-disubstituted derivative of piracetam that enhances cognition and demonstrates efficacy against scopolamine-induced cholinergic dysfunction in mice. These effects are attributed to its positive modulatory action on the sigma-1 receptor and this activity may be relevant when developing new drugs for treating cognitive symptoms related to neurodegenerative diseases. PMID:24490863

Transcription-coupled repair of UV damage in the halophilic archaea.

PubMed

Stantial, Nicole; Dumpe, Jarrod; Pietrosimone, Kathryn; Baltazar, Felicia; Crowley, David J

2016-05-01

Transcription-coupled repair (TCR) is a subpathway of nucleotide excision repair (NER) in which excision repair proteins are targeted to RNA polymerase-arresting lesions located in the transcribed strand of active genes. TCR has been documented in a variety of bacterial and eukaryotic organisms but has yet to be observed in the Archaea. We used Halobacterium sp. NRC-1 and Haloferax volcanii to determine if TCR occurs in the halophilic archaea. Following UV irradiation of exponentially growing cultures, we quantified the rate of repair of cyclobutane pyrimidine dimers in the two strands of the rpoB2B1A1A2 and the trpDFEG operons of Halobacterium sp. NRC-1 and the pts operon of H. volcanii through the use of a Southern blot assay and strand-specific probes. TCR was observed in all three operons and was dependent on the NER gene uvrA in Halobacterium sp. NRC-1, but not in H. volcanii. The halophilic archaea likely employ a novel mechanism for TCR in which an as yet unknown coupling factor recognizes the arrested archaeal RNA polymerase complex and recruits certain NER proteins to complete the process. Copyright © 2016 Elsevier B.V. All rights reserved.

miR-200a controls hepatic stellate cell activation and fibrosis via SIRT1/Notch1 signal pathway.

PubMed

Yang, Jing-Jing; Tao, Hui; Liu, Li-Ping; Hu, Wei; Deng, Zi-Yu; Li, Jun

2017-04-01

miR-200a has been established as a key regulator of HSC activation processes in liver fibrosis. Epigenetic silencing of miR-200a contributing to SIRT1 over-expression has been discussed in breast cancer; however, whether miR-200a controls SIRT1 gene expression in hepatic fibrosis is still unknown. We analyzed miR-200a regulation of SIRT1 expression in CCl 4 -induced liver fibrosis and TGF-β1-mediated activation of HSC. miR-200a, SIRT1, α-SMA, Col1A1, Notch1 and NICD expression were estimated by Western blotting, qRT-PCR and Immunohistochemistry. HSCs were transfected with miR-200a mimic, miR-200a inhibitor and SIRT1-RNAi. Luciferase reporter assays further confirmed the interaction between miR-200a and the SIRT1 mRNA 3'-UTR. Cell proliferation ability was assessed by MTT and cell cycle. We found that treatment activated HSC with miR-200a mimics, restored miR-200a expression and reduced SIRT1 levels. Conversely, treatment activated HSC with miR-200a inhibitors, decreased miR-200a expression and up-regulated SIRT1 levels. Restoration of miR-200a or the knockdown of SIRT1 prevented HSC activation and proliferation. We have established the SIRT1 transcript as subject to regulation by miR-200a, through miR-200a targeting of SIRT1 3'-UTR. Finally, HSC transfected with SIRT1-siRNA increased the levels of Notch1 protein and mRNA expression. Our study demonstrated that miR-200a regulates SIRT1/Notch1 expression during HSC activation and fibrosis.

MiR-506 suppresses liver cancer angiogenesis through targeting sphingosine kinase 1 (SPHK1) mRNA.

PubMed

Lu, Zhanping; Zhang, Weiying; Gao, Shan; Jiang, Qiulei; Xiao, Zelin; Ye, Lihong; Zhang, Xiaodong

MicroRNAs acting as oncogenes or tumor suppressor genes play crucial roles in human cancers. Sphingosine kinase 1 (SPHK1) and its metabolite sphingosine 1-phosphate (S1P) contribute to tumor angiogenesis. We have reported that the down-regulation of miR-506 targeting YAP mRNA results in the hepatocarcinogenesis. In the present study, we report a novel function of miR-506, which suppresses tumor angiogenesis through targeting SPHK1 mRNA in liver cancer. Bioinformatics analysis showed that miR-506 might target 3'-untranslated region (3'UTR) of SPHK1 mRNA. Then, we validated that by luciferase reporter gene assays. MiR-506 was able to reduce the expression of SPHK1 at the levels of mRNA and protein using reverse transcription-polymerase chain reaction and Western blot analysis in hepatoma HepG2 cells. Functionally, human umbilical vein endothelial cell (HUVEC) tube formation assays demonstrated that the forced miR-506 expression remarkably inhibited the production of S1P in the supernatant of hepatoma cells. The supernatant resulted in the inhibition of tumor angiogenesis. Interestingly, the supernatant with overexpression of SPHK1 could rescue the inhibition of angiogenesis of liver cancer mediated by miR-506. Anti-miR-506 increased the production of S1P in the supernatant of hepatoma cells, but the supernatant with silencing of SPHK1 abolished anti-miR-506-induced acceleration of tumor angiogenesis. Clinically, we observed that the levels of miR-506 were negatively related to those of SPHK1 mRNA in liver cancer tissues. Thus, we conclude that miR-506 depresses the angiogenesis of liver cancer through targeting 3'UTR of SPHK1 mRNA. Our finding provides new insights into the mechanism of tumor angiogenesis. Copyright © 2015 Elsevier Inc. All rights reserved.

Ethanol exposure induces neonatal neurodegeneration by enhancing CB1R Exon1 histone H4K8 acetylation and up-regulating CB1R function causing neurobehavioral abnormalities in adult mice.

PubMed

Subbanna, Shivakumar; Nagre, Nagaraja N; Umapathy, Nagavedi S; Pace, Betty S; Basavarajappa, Balapal S

2014-10-31

Ethanol exposure to rodents during postnatal day 7 (P7), which is comparable to the third trimester of human pregnancy, induces long-term potentiation and memory deficits. However, the molecular mechanisms underlying these deficits are still poorly understood. In the present study, we explored the potential role of epigenetic changes at cannabinoid type 1 (CB1R) exon1 and additional CB1R functions, which could promote memory deficits in animal models of fetal alcohol spectrum disorder. We found that ethanol treatment of P7 mice enhances acetylation of H4 on lysine 8 (H4K8ace) at CB1R exon1, CB1R binding as well as the CB1R agonist-stimulated GTPγS binding in the hippocampus and neocortex, two brain regions that are vulnerable to ethanol at P7 and are important for memory formation and storage, respectively. We also found that ethanol inhibits cyclic adenosine monophosphate response element-binding protein (CREB) phosphorylation and activity-regulated cytoskeleton-associated protein (Arc) expression in neonatal and adult mice. The blockade or genetic deletion of CB1Rs prior to ethanol treatment at P7 rescued CREB phosphorylation and Arc expression. CB1R knockout mice exhibited neither ethanol-induced neurodegeneration nor inhibition of CREB phosphorylation or Arc expression. However, both neonatal and adult mice did exhibit enhanced CREB phosphorylation and Arc protein expression. P7 ethanol-treated adult mice exhibited impaired spatial and social recognition memory, which were prevented by the pharmacological blockade or deletion of CB1Rs at P7. Together, these findings suggest that P7 ethanol treatment induces CB1R expression through epigenetic modification of the CB1R gene, and that the enhanced CB1R function induces pCREB, Arc, spatial, and social memory deficits in adult mice. © The Author 2015. Published by Oxford University Press on behalf of CINP.

Dysregulated miR-671-5p / CDR1-AS / CDR1 / VSNL1 axis is involved in glioblastoma multiforme

PubMed Central

Salito, Loredana; Sammito, Mariangela; Banelli, Barbara; Caltabiano, Rosario; Barbagallo, Giuseppe; Zappalà, Agata; Battaglia, Rosalia; Cirnigliaro, Matilde; Lanzafame, Salvatore; Vasquez, Enrico; Parenti, Rosalba; Cicirata, Federico; Di Pietro, Cinzia; Romani, Massimo; Purrello, Michele

2016-01-01

MiR-671-5p is encoded by a gene localized at 7q36.1, a region amplified in human glioblastoma multiforme (GBM), the most malignant brain cancer. To investigate whether expression of miR-671-5p were altered in GBM, we analyzed biopsies from a cohort of forty-five GBM patients and from five GBM cell lines. Our data show significant overexpression of miR-671-5p in both biopsies and cell lines. By exploiting specific miRNA mimics and inhibitors, we demonstrated that miR-671-5p overexpression significantly increases migration and to a less extent proliferation rates of GBM cells. Through a combined in silico and in vitro approach, we identified CDR1-AS, CDR1, VSNL1 as downstream miR-671-5p targets in GBM. Expression of these genes significantly decreased both in GBM biopsies and cell lines and negatively correlated with that of miR-671-5p. Based on our data, we propose that the axis miR-671-5p / CDR1-AS / CDR1 / VSNL1 is functionally altered in GBM cells and is involved in the modification of their biopathological profile. PMID:26683098

Dysregulated miR-671-5p / CDR1-AS / CDR1 / VSNL1 axis is involved in glioblastoma multiforme.

PubMed

Barbagallo, Davide; Condorelli, Angelo; Ragusa, Marco; Salito, Loredana; Sammito, Mariangela; Banelli, Barbara; Caltabiano, Rosario; Barbagallo, Giuseppe; Zappalà, Agata; Battaglia, Rosalia; Cirnigliaro, Matilde; Lanzafame, Salvatore; Vasquez, Enrico; Parenti, Rosalba; Cicirata, Federico; Di Pietro, Cinzia; Romani, Massimo; Purrello, Michele

2016-01-26

MiR-671-5p is encoded by a gene localized at 7q36.1, a region amplified in human glioblastoma multiforme (GBM), the most malignant brain cancer. To investigate whether expression of miR-671-5p were altered in GBM, we analyzed biopsies from a cohort of forty-five GBM patients and from five GBM cell lines. Our data show significant overexpression of miR-671-5p in both biopsies and cell lines. By exploiting specific miRNA mimics and inhibitors, we demonstrated that miR-671-5p overexpression significantly increases migration and to a less extent proliferation rates of GBM cells. Through a combined in silico and in vitro approach, we identified CDR1-AS, CDR1, VSNL1 as downstream miR-671-5p targets in GBM. Expression of these genes significantly decreased both in GBM biopsies and cell lines and negatively correlated with that of miR-671-5p. Based on our data, we propose that the axis miR-671-5p / CDR1-AS / CDR1 / VSNL1 is functionally altered in GBM cells and is involved in the modification of their biopathological profile.

Insulin-like Growth Factor (IGF) Signaling Requires αvβ3-IGF1-IGF Type 1 Receptor (IGF1R) Ternary Complex Formation in Anchorage Independence, and the Complex Formation Does Not Require IGF1R and Src Activation

PubMed Central

Fujita, Masaaki; Takada, Yoko K.; Takada, Yoshikazu

2013-01-01

Integrin αvβ3 plays a role in insulin-like growth factor 1 (IGF1) signaling (integrin-IGF1 receptor (IGF1R) cross-talk) in non-transformed cells in anchorage-dependent conditions. We reported previously that IGF1 directly binds to αvβ3 and induces αvβ3-IGF1-IGF1R ternary complex formation in these conditions. The integrin-binding defective IGF1 mutant (R36E/R37E) is defective in inducing ternary complex formation and IGF signaling, whereas it still binds to IGF1R. We studied if IGF1 can induce signaling in anchorage-independent conditions in transformed Chinese hamster ovary cells that express αvβ3 (β3-CHO) cells. Here we describe that IGF1 signals were more clearly detectable in anchorage-independent conditions (polyHEMA-coated plates) than in anchorage-dependent conditions. This suggests that IGF signaling is masked by signals from cell-matrix interaction in anchorage-dependent conditions. IGF signaling required αvβ3 expression, and R36E/R37E was defective in inducing signals in polyHEMA-coated plates. These results suggest that αvβ3-IGF1 interaction, not αvβ3-extracellular matrix interaction, is essential for IGF signaling. Inhibitors of IGF1R, Src, AKT, and ERK1/2 did not suppress αvβ3-IGF-IGF1R ternary complex formation, suggesting that activation of these kinases are not required for ternary complex formation. Also, mutations of the β3 cytoplasmic tail (Y747F and Y759F) that block β3 tyrosine phosphorylation did not affect IGF1R phosphorylation or AKT activation. We propose a model in which IGF1 binding to IGF1R induces recruitment of integrin αvβ3 to the IGF-IGF1R complex and then β3 and IGF1R are phosphorylated. It is likely that αvβ3 should be together with the IGF1-IGF1R complex for triggering IGF signaling. PMID:23243309

miR-543 promotes gastric cancer cell proliferation by targeting SIRT1

DOE Office of Scientific and Technical Information (OSTI.GOV)

Li, Juan; Dong, Guoying; Wang, Bo

SIRT1, a class III histone deacetylase, exerts inhibitory effects on tumorigenesis and is downregulated in gastric cancer. However, the role of microRNAs in the regulation of SIRT1 in gastric cancer is still largely unknown. Here, we identified miR-543 as a predicted upstream regulator of SIRT1 using 3 different bioinformatics databases. Mimics of miR-543 significantly inhibited the expression of SIRT1, whereas an inhibitor of miR-543 increased SIRT1 expression. MiR-543 directly targeted the 3′-UTR of SIRT1, and both of the two binding sites contributed to the inhibitory effects. In gastric epithelium-derived cell lines, miR-543 promoted cell proliferation and cell cycle progression, andmore » overexpression of SIRT1 rescued the above effects of miR-543. The inhibitory effects of miR-543 on SIRT1 were also validated using clinical gastric cancer samples. Moreover, we found that miR-543 expression was positively associated with tumor size, clinical grade, TNM stage and lymph node metastasis in gastric cancer patients. Our results identify a new regulatory mechanism of miR-543 on SIRT1 expression in gastric cancer, and raise the possibility that the miR-543/SIRT1 pathway may serve as a potential target for the treatment of gastric cancer. - Highlights: • SIRT1 is a novel target of miR-543. • miR-543 promotes gastric cancer cell proliferation and cell cycle progression by targeting SIRT1. • miR-543 is upregulated in GC and positively associated with tumor size, clinical grade, TNM stage and lymph node metastasis. • miR-543 is negatively correlated with SIRT1 expression in gastric cancer tissues.« less

Soluble interleukin-13rα1: a circulating regulator of glucose.

PubMed

Rachmin, Inbal; O'Meara, Caitlin C; Ricci-Blair, Elisabeth M; Feng, Yilin; Christensen, Emily M; Duffy, Jeanne F; Zitting, Kirsi M; Czeisler, Charles A; Pancoast, James R; Cannon, Christopher P; O'Donoghue, Michelle L; Morrow, David A; Lee, Richard T

2017-12-01

Soluble IL-13 receptor-α1, or sIL13rα1, is a soluble protein that binds to interleukin-13 (IL-13) that has been previously described in mice. The function of sIL13rα1 remains unclear, but it has been hypothesized to act as a decoy receptor for IL-13. Recent studies have identified a role for IL-13 in glucose metabolism, suggesting that a decoy receptor for IL-13 might increase circulating glucose levels. Here, we report that delivery of sIL13rα1 to mice by either gene transfer or recombinant protein decreases blood glucose levels. Surprisingly, the glucose-lowering effect of sIL13rα1 was preserved in mice lacking IL-13, demonstrating that IL-13 was not required for the effect. In contrast, deletion of IL-4 in mice eliminated the hypoglycemic effect of sIL13rα1. In humans, endogenous blood levels of IL13rα1 varied substantially, although there were no differences between diabetic and nondiabetic patients. There was no circadian variation of sIL13rα1 in normal human volunteers. Delivery of sIL13rα1 fused to a fragment crystallizable (Fc) domain provided sustained glucose lowering in mice on a high-fat diet, suggesting a potential therapeutic strategy. These data reveal sIL13rα1 as a circulating human protein with an unexpected role in glucose metabolism. Copyright © 2017 the American Physiological Society.

Significant reduction of acute cardiac allograft rejection by selective janus kinase-1/3 inhibition using R507 and R545.

PubMed

Deuse, Tobias; Hua, Xiaoqin; Taylor, Vanessa; Stubbendorff, Mandy; Baluom, Muhammad; Chen, Yan; Park, Gary; Velden, Joachim; Streichert, Thomas; Reichenspurner, Hermann; Robbins, Robert C; Schrepfer, Sonja

2012-10-15

Selective inhibition of lymphocyte activation through abrogation of signal 3-cytokine transduction emerges as a new strategy for immunosuppression. This is the first report on the novel Janus kinase (JAK)1/3 inhibitors R507 and R545 for prevention of acute allograft rejection. Pharmacokinetic and in vitro enzyme inhibition assays were performed to characterize the drugs. Heterotopic Brown Norway-Lewis heart transplantations were performed to study acute cardiac allograft rejection, graft survival, suppression of cellular host responsiveness, and antibody production. Therapeutic and subtherapeutic doses of R507 (60 and 15 mg/kg 2 times per day) and R545 (20 and 5 mg/kg 2 times per day) were compared with those of tacrolimus (Tac; 4 and 1 mg/kg once per day). Plasma levels of R507 and R545 were sustained high for several hours. Cell-based enzyme assays showed selective inhibition of JAK1/3-dependent pathways with 20-fold or greater selectivity over JAK2 and Tyrosine kinase 2 kinases. After heart transplantation, both JAK1/3 inhibitors reduced early mononuclear graft infiltration, even significantly more potent than Tac. Intragraft interferon-γ release was significantly reduced by R507 and R545, and for interleukin-10 suppression, they were even significantly more potent than Tac. Both JAK1/3 inhibitors and Tac were similarly effective in reducing the host Th1 and Th2, but not Th17, responsiveness and similarly prevented donor-specific immunoglobulin M antibody production. Subtherapeutic and therapeutic R507 and R545 doses prolonged the mean graft survival and were similarly effective as 1 and 4 mg/kg Tac, respectively. In combination regimens, however, only R507 showed highly beneficial synergistic drug interactions with Tac. Both R507 and R545 are potent novel immunosuppressants with favorable pharmacokinetics and high JAK1/3 selectivity, but only R507 synergistically interacts with Tac.

Crystal structures of the diastereomeric salt pair of the prostaglandin intermediate 1 R, 2 S(+)- cis-2-hydroxycyclopent-4-enylacetic acid with S- and R- 1-phenylethylamine

NASA Astrophysics Data System (ADS)

Czugler, Mátyás; Csöregh, Ingeborg; Kálmán, Alajos; Faigl, Ferenc; Ács, Mária

1989-05-01

Crystal structures of an enantiomeric salt pair formed between 1 R,2 S- cis-2-hydroxycyclopent-4-enylacetic acid ( S-HCA) and R(+)-1-phenylethylamine ( R-PEA) and the corresponding S-PEA salt have been determined by X-ray crystallography. The S-HCA: R-PEA 1:1 salt ( R-HCA-PEA hereafter) is orthorhombic, P2 12 12 1, with the unit-cell parameters a = 5.806(1), b = 9.261(1), c = 27.624(2) Å and R = 0.056 for 1162 reflections at ambient temperature. The S-HCA: S-PEA 1:1 salt ( S-HCAPEA) is also orthorhombic, P2 12 12 1, with the unit-cell data a = 6.034(2), b = 11.840(7), c = 20.198(11) Å at 170 K, R = 0.082 for 1196 data measured at low temperatures (170 K). The R-HCAPEA salt has its two components assembled into an elongated rod-like shape via two-dimensional hydrogen bonding between cations and anions thus forming a well-ordered crystal. In contrast, the cation and anion in the S-HCAPEA salt forms a more globular aggregate and displays orientation disorder in the five-membered ring part of the anion and maintains an essentially one-dimensional hydrogen-bond network, while the total number of hydrogen bonds between cationic and anionic species remains three in both crystals.

Three novel serum biomarkers, miR-1, miR-133a, and miR-206 for Limb-girdle muscular dystrophy, Facioscapulohumeral muscular dystrophy, and Becker muscular dystrophy.

PubMed

Matsuzaka, Yasunari; Kishi, Soichiro; Aoki, Yoshitsugu; Komaki, Hirofumi; Oya, Yasushi; Takeda, Shin-Ichi; Hashido, Kazuo

2014-11-01

Muscular dystrophies are a clinically and genetically heterogeneous group of inherited myogenic disorders. In clinical tests for these diseases, creatine kinase (CK) is generally used as diagnostic blood-based biomarker. However, because CK levels can be altered by various other factors, such as vigorous exercise, etc., false positive is observed. Therefore, three microRNAs (miRNAs), miR-1, miR-133a, and miR-206, were previously reported as alternative biomarkers for duchenne muscular dystrophy (DMD). However, no alternative biomarkers have been established for the other muscular dystrophies. We, therefore, evaluated whether these miR-1, miR-133a, and miR-206 can be used as powerful biomarkers using the serum from muscular dystrophy patients including DMD, myotonic dystrophy 1 (DM1), limb-girdle muscular dystrophy (LGMD), facioscapulohumeral muscular dystrophy (FSHD), becker muscular dystrophy (BMD), and distal myopathy with rimmed vacuoles (DMRV) by qualitative polymerase chain reaction (PCR) amplification assay. Statistical analysis indicated that all these miRNA levels in serum represented no significant differences between all muscle disorders examined in this study and controls by Bonferroni correction. However, some of these indicated significant differences without correction for testing multiple diseases (P < 0.05). The median values of miR-1 levels in the serum of patients with LGMD, FSHD, and BMD were approximately 5.5, 3.3 and 1.7 compared to that in controls, 0.68, respectively. Similarly, those of miR-133a and miR-206 levels in the serum of BMD patients were about 2.5 and 2.1 compared to those in controls, 1.03 and 1.32, respectively. Taken together, our data demonstrate that levels of miR-1, miR-133a, and miR-206 in serum of BMD and miR-1 in sera of LGMD and FSHD patients showed no significant differences compared with those of controls by Bonferroni correction. However, the results might need increase in sample sizes to evaluate these three miRNAs as

Decoy Oligonucleotide Rescues IGF1R Expression from MicroRNA-223 Suppression

PubMed Central

Wang, Rong; He, Bao Mei; Qi, Bing; Xu, Chang Jun; Wu, Xing Zhong

2013-01-01

A mature miRNA generally suppresses hundreds of mRNA targets. To evaluate the selective effect of synthetic oligonucleotide decoys on hsa-miR-223 activity, reporters containing 3’ untranslated regions (UTR) of IGF1R, FOXO1, POLR3G, FOXO3, CDC27, FBXW7 and PAXIP1 mRNAs were constructed for the luciferase assay. The oligonucleotide decoys were designed and synthesized according to mature miR-223 sequence and its target mRNA sequence. Quantitative RT-PCR & western analysis were used to measure miR-223-targeted mRNA expression, Interestingly, apart from the antisense oligonucleotide, decoy nucleotides which were complementary to the 5’, central or 3’ region of mature miR-223 suppressed miR-223 targeting the 3’UTR of IGF1R, FOXO1, FOXO3, CDC27, POLR3G, and FBXW7 mRNAs and rescued the expression of these genes to varying degrees from miR-223 suppression at both mRNA and protein levels. All decoys had no effect on PAXIP1 which was not targeted by miR-223. The decoy 1 that was based on the sequence of IGF1R 3’UTR rescued the expression of IGF1R more significantly than other decoy nucleotides except the antisense decoy 4. Decoy 1 also rescued the expression of FOXO3 and POLR3G of which their 3’UTRs have similar binding sites for miR-223 with IGF1R 3’UTR. However decoy 1 failed to recover Sp1, CDC27 and FBXW7 expression. These data support that the sequence-specific decoy oligonucleotides might represent exogenous competing RNA which selectively inhibits microRNA targeting. PMID:24324762

Decoy oligonucleotide rescues IGF1R expression from MicroRNA-223 suppression.

PubMed

Wu, Li Hui; Cai, Qian Qian; Dong, Yi Wei; Wang, Rong; He, Bao Mei; Qi, Bing; Xu, Chang Jun; Wu, Xing Zhong

2013-01-01

A mature miRNA generally suppresses hundreds of mRNA targets. To evaluate the selective effect of synthetic oligonucleotide decoys on hsa-miR-223 activity, reporters containing 3' untranslated regions (UTR) of IGF1R, FOXO1, POLR3G, FOXO3, CDC27, FBXW7 and PAXIP1 mRNAs were constructed for the luciferase assay. The oligonucleotide decoys were designed and synthesized according to mature miR-223 sequence and its target mRNA sequence. Quantitative RT-PCR & western analysis were used to measure miR-223-targeted mRNA expression, Interestingly, apart from the antisense oligonucleotide, decoy nucleotides which were complementary to the 5', central or 3' region of mature miR-223 suppressed miR-223 targeting the 3'UTR of IGF1R, FOXO1, FOXO3, CDC27, POLR3G, and FBXW7 mRNAs and rescued the expression of these genes to varying degrees from miR-223 suppression at both mRNA and protein levels. All decoys had no effect on PAXIP1 which was not targeted by miR-223. The decoy 1 that was based on the sequence of IGF1R 3'UTR rescued the expression of IGF1R more significantly than other decoy nucleotides except the antisense decoy 4. Decoy 1 also rescued the expression of FOXO3 and POLR3G of which their 3'UTRs have similar binding sites for miR-223 with IGF1R 3'UTR. However decoy 1 failed to recover Sp1, CDC27 and FBXW7 expression. These data support that the sequence-specific decoy oligonucleotides might represent exogenous competing RNA which selectively inhibits microRNA targeting.

Interaction of Extreme Halophilic Archaea With the Evaporites of the Solar Salterns Guerrero Negro Baja California, Mexico

NASA Astrophysics Data System (ADS)

Tamez, P.; Lopez-Cortés, A.

2008-12-01

Hypersaline environments have been significant reservoirs for the long-term evolution of specifically adapted microorganisms. Characterized to have higher salt concentrations (up to 35 g/L), they are worldwide distributed and have a commercial significance. Exportadora de Sal, Guerrero Negro, Mexico has a multipond salterns system designed to harvest common salt (NaCl) from sea water. To achieve this purpose, sea water is pumped through a set of shallow ponds where water evaporates and salts concentrate. Sequential precipitation of CaCO3, CaSO4 2H2O and NaCl occurs in a mineral formations call it evaporites. In the interior of those gypsum-encrusted and halite-encrusted minerals, communities of extremely salt-loving archaea prosper. Previous studies have showed the influence of Haloarchaeal cells in the formation of larger fluid inclusions than crystals formed in sterile salt solutions. S-layer envelopes and cells of Haloarcula strain SP8807 contributed to the nucleation of new crystals of NaCl. Given the significance of the scope in phylogenetic archaeal diversity research, this study had a polyphasic approach. SEM micrographs from a 21- 31% (w/v) gradient salt multipond system evaporites, gave an insight profile of the extreme halophilic archaeal communities thriving in the surface of the gypsum and halite evaporites. Halite crystals were form after 21 days of incubation in solid medium with archaeal cells. Both culture and non-culture dependent methods, Nested-PCR-DGGE analysis and sequencing of 16S rDNA amplified fragment genes from environmental samples and isolated strains were used for this purpose. We isolate three strains from Pond 9 (21.07% total salt concentration) and one strain from Cristallizer 20 (25.15% total salt concentration). 16S rDNA signaling gave 99% of similarity with Halogeometricum borinquense, sequence AF002984, two other strains were 99% of similarity with Halobacterium salinarum, sequence AJ496185 these strains shown different colony

miR-22 and miR-29a Are Members of the Androgen Receptor Cistrome Modulating LAMC1 and Mcl-1 in Prostate Cancer.

PubMed

Pasqualini, Lorenza; Bu, Huajie; Puhr, Martin; Narisu, Narisu; Rainer, Johannes; Schlick, Bettina; Schäfer, Georg; Angelova, Mihaela; Trajanoski, Zlatko; Börno, Stefan T; Schweiger, Michal R; Fuchsberger, Christian; Klocker, Helmut

2015-07-01

The normal prostate as well as early stages and advanced prostate cancer (PCa) require a functional androgen receptor (AR) for growth and survival. The recent discovery of microRNAs (miRNAs) as novel effector molecules of AR disclosed the existence of an intricate network between AR, miRNAs and downstream target genes. In this study DUCaP cells, characterized by high content of wild-type AR and robust AR transcriptional activity, were chosen as the main experimental model. By integrative analysis of chromatin immunoprecipitation-sequencing (ChIP-seq) and microarray expression profiling data, miRNAs putatively bound and significantly regulated by AR were identified. A direct AR regulation of miR-22, miR-29a, and miR-17-92 cluster along with their host genes was confirmed. Interestingly, endogenous levels of miR-22 and miR-29a were found to be reduced in PCa cells expressing AR. In primary tumor samples, miR-22 and miR-29a were less abundant in the cancerous tissue compared with the benign counterpart. This specific expression pattern was associated with a differential DNA methylation of the genomic AR binding sites. The identification of laminin gamma 1 (LAMC1) and myeloid cell leukemia 1 (MCL1) as direct targets of miR-22 and miR-29a, respectively, suggested a tumor-suppressive role of these miRNAs. Indeed, transfection of miRNA mimics in PCa cells induced apoptosis and diminished cell migration and viability. Collectively, these data provide additional information regarding the complex regulatory machinery that guides miRNAs activity in PCa, highlighting an important contribution of miRNAs in the AR signaling.

CYP2R1 mutations causing vitamin D-deficiency rickets.

PubMed

Thacher, Tom D; Levine, Michael A

2017-10-01

CYP2R1 is the principal hepatic 25-hydroxylase responsible for the hydroxylation of parent vitamin D to 25-hydroxyvitamin D [25(OH)D]. Serum concentrations of 25(OH)D reflect vitamin D status, because 25(OH)D is the major circulating metabolite of vitamin D. The 1α-hydroxylation of 25(OH)D in the kidney by CYP27B1 generates the fully active vitamin D metabolite, 1,25-dihydroxyvitamin D (1,25(OH) 2 D). The human CYP2R1 gene, located at 11p15.2, has five exons, coding for an enzyme with 501 amino acids. In Cyp2r1-/- knockout mice, serum 25(OH)D levels were reduced by more than 50% compared wild-type mice. Genetic polymorphisms of CYP2R1 account for some of the individual variability of circulating 25(OH)D values in the population. We review the evidence that inactivating mutations in CYP2R1 can lead to a novel form of vitamin D-deficiency rickets resulting from impaired 25-hydroxylation of vitamin D. We sequenced the promoter, exons and intron-exon flanking regions of the CYP2R1 gene in members of 12 Nigerian families with rickets in more than one family member. We found missense mutations (L99P and K242N) in affected members of 2 of 12 families. The L99P mutation had previously been reported as a homozygous defect in an unrelated child of Nigerian origin with rickets. In silico analyses predicted impaired CYP2R1 folding or reduced interaction with substrate vitamin D by L99P and K242N mutations, respectively. In vitro studies of the mutant CYP2R1 proteins in HEK293 cells confirmed normal expression levels but completely absent or markedly reduced 25-hydroxylase activity by the L99P and K242N mutations, respectively. Heterozygous subjects had more moderate biochemical and clinical features of vitamin D deficiency than homozygous subjects. After an oral bolus dose of 50,000 IU of vitamin D 2 or vitamin D 3 , heterozygous subjects had lower increases in serum 25(OH)D than control subjects, and homozygous subjects had minimal increases, supporting a semidominant

Sus scrofa miR-204 and miR-4331 Negatively Regulate Swine H1N1/2009 Influenza A Virus Replication by Targeting Viral HA and NS, Respectively.

PubMed

Zhang, Shishuo; Wang, Ruifang; Su, Huijuan; Wang, Biaoxiong; Sizhu, Suolang; Lei, Zhixin; Jin, Meilin; Chen, Huanchun; Cao, Jiyue; Zhou, Hongbo

2017-04-03

The prevalence of swine pandemic H1N1/2009 influenza A virus (SIV-H1N1/2009) in pigs has the potential to generate novel reassortant viruses, posing a great threat to human health. Cellular microRNAs (miRNAs) have been proven as promising small molecules for regulating influenza A virus replication by directly targeting viral genomic RNA. In this study, we predicted potential Sus scrofa (ssc-, swine) miRNAs targeting the genomic RNA of SIV-H1N1/2009 by RegRNA 2.0, and identified ssc-miR-204 and ssc-miR-4331 to target viral HA and NS respectively through dual-luciferase reporter assays. The messenger RNA (mRNA) levels of viral HA and NS were significantly suppressed when newborn pig trachea (NPTr) cells respectively overexpressed ssc-miR-204 and ssc-miR-4331 and were infected with SIV-H1N1/2009, whereas the suppression effect could be restored when respectively decreasing endogenous ssc-miR-204 and ssc-miR-4331 with inhibitors. Because of the importance of viral HA and NS in the life cycle of influenza A virus, ssc-miR-204 and ssc-miR-4331 exhibited an inhibition effect on SIV-H1N1/2009 replication. The antiviral effect was sequence-specific of SIV-H1N1/2009, for the target sites in HA and NS of H5N1 or H9N2 influenza A virus were not conserved. Furthermore, SIV-H1N1/2009 infection reversely downregulated the expression of ssc-miR-204 and ssc-miR-4331, which might facilitate the virus replication in the host. In summary, this work will provide us some important clues for controlling the prevalence of SIV-H1N1/2009 in pig populations.

Expression of miR-155, miR-146a, and miR-326 in T1D patients from Chile: relationship with autoimmunity and inflammatory markers.

PubMed

García-Díaz, Diego F; Pizarro, Carolina; Camacho-Guillén, Patricia; Codner, Ethel; Soto, Néstor; Pérez-Bravo, Francisco

2018-02-01

Objective The aim of this research was to analyze the expression profile of miR-155, miR-146a, and miR-326 in peripheral blood mononuclear cells (PBMC) of 47 patients with type 1 diabetes mellitus (T1D) and 39 control subjects, as well as the possible association with autoimmune or inflammatory markers. Subjects and methods Expression profile of miRs by means of qPCR using TaqMan probes. Autoantibodies and inflammatory markers by ELISA. Statistical analysis using bivariate correlation. Results The analysis of the results shows an increase in the expression of miR-155 in T1D patients in basal conditions compared to the controls (p < 0.001) and a decreased expression level of miR-326 (p < 0.01) and miR-146a (p < 0.05) compared T1D patients to the controls. miR-155 was the only miRs associated with autoinmmunity (ZnT8) and inflammatory status (vCAM). Conclusion Our data show a possible role of miR-155 related to autoimmunity and inflammation in Chilean patients with T1D.

NgR1: A Tunable Sensor Regulating Memory Formation, Synaptic, and Dendritic Plasticity.

PubMed

Karlsson, Tobias E; Smedfors, Gabriella; Brodin, Alvin T S; Åberg, Elin; Mattsson, Anna; Högbeck, Isabelle; Wellfelt, Katrin; Josephson, Anna; Brené, Stefan; Olson, Lars

2016-04-01

Nogo receptor 1 (NgR1) is expressed in forebrain neurons and mediates nerve growth inhibition in response to Nogo and other ligands. Neuronal activity downregulates NgR1 and the inability to downregulate NgR1 impairs long-term memory. We investigated behavior in a serial behavioral paradigm in mice that overexpress or lack NgR1, finding impaired locomotor behavior and recognition memory in mice lacking NgR1 and impaired sequential spatial learning in NgR1 overexpressing mice. We also investigated a role for NgR1 in drug-mediated sensitization and found that repeated cocaine exposure caused stronger locomotor responses but limited development of stereotypies in NgR1 overexpressing mice. This suggests that NgR1-regulated synaptic plasticity is needed to develop stereotypies. Ex vivo magnetic resonance imaging and diffusion tensor imaging analyses of NgR1 overexpressing brains did not reveal any major alterations. NgR1 overexpression resulted in significantly reduced density of mature spines and dendritic complexity. NgR1 overexpression also altered cocaine-induced effects on spine plasticity. Our results show that NgR1 is a negative regulator of both structural synaptic plasticity and dendritic complexity in a brain region-specific manner, and highlight anterior cingulate cortex as a key area for memory-related plasticity. © The Author 2016. Published by Oxford University Press.

NgR1: A Tunable Sensor Regulating Memory Formation, Synaptic, and Dendritic Plasticity

PubMed Central

Karlsson, Tobias E.; Smedfors, Gabriella; Brodin, Alvin T. S.; Åberg, Elin; Mattsson, Anna; Högbeck, Isabelle; Wellfelt, Katrin; Josephson, Anna; Brené, Stefan; Olson, Lars

2016-01-01

Nogo receptor 1 (NgR1) is expressed in forebrain neurons and mediates nerve growth inhibition in response to Nogo and other ligands. Neuronal activity downregulates NgR1 and the inability to downregulate NgR1 impairs long-term memory. We investigated behavior in a serial behavioral paradigm in mice that overexpress or lack NgR1, finding impaired locomotor behavior and recognition memory in mice lacking NgR1 and impaired sequential spatial learning in NgR1 overexpressing mice. We also investigated a role for NgR1 in drug-mediated sensitization and found that repeated cocaine exposure caused stronger locomotor responses but limited development of stereotypies in NgR1 overexpressing mice. This suggests that NgR1-regulated synaptic plasticity is needed to develop stereotypies. Ex vivo magnetic resonance imaging and diffusion tensor imaging analyses of NgR1 overexpressing brains did not reveal any major alterations. NgR1 overexpression resulted in significantly reduced density of mature spines and dendritic complexity. NgR1 overexpression also altered cocaine-induced effects on spine plasticity. Our results show that NgR1 is a negative regulator of both structural synaptic plasticity and dendritic complexity in a brain region-specific manner, and highlight anterior cingulate cortex as a key area for memory-related plasticity. PMID:26838771

miR-125b-1-3p inhibits trophoblast cell invasion by targeting sphingosine-1-phosphate receptor 1 in preeclampsia.

PubMed

Li, Qinghua; Pan, Zhifang; Wang, Xuejian; Gao, Zhiqin; Ren, Chune; Yang, Weiwei

2014-10-10

Preeclampsia (PE) is the leading cause of maternal and perinatal mortality and morbidity. Understanding the molecular mechanisms underlying placentation facilitates the development of better intervention of this disease. MicroRNAs are strongly implicated in the pathogenesis of this syndrome. In current study, we found that miR-125b-1-3p was elevated in placentas derived from preeclampsia patients. Transfection of miR-125b-1-3p mimics significantly inhibited the invasiveness of human trophoblast cells, whereas miR-125b-1-3p inhibitor enhanced trophoblast cell invasion. Luciferase assays identified that S1PR1 was a novel direct target of miR-125b-1-3p in the placenta. Overexpression of S1PR1 could reverse the inhibitory effect of miR-125b-1-3p on the invasion of trophoblast cells. These findings suggested that abnormal expression of miR-125b-1-3p might contribute to the pathogenesis of preeclampsia. Copyright © 2014 Elsevier Inc. All rights reserved.

Generalized Second-Order Partial Derivatives of 1/r

ERIC Educational Resources Information Center

Hnizdo, V.

2011-01-01

The generalized second-order partial derivatives of 1/r, where r is the radial distance in three dimensions (3D), are obtained using a result of the potential theory of classical analysis. Some non-spherical-regularization alternatives to the standard spherical-regularization expression for the derivatives are derived. The utility of a…

A Schiff base connectivity switch in sensory rhodopsin signaling

PubMed Central

Sineshchekov, Oleg A.; Sasaki, Jun; Phillips, Brian J.; Spudich, John L.

2008-01-01

Sensory rhodopsin I (SRI) in Halobacterium salinarum acts as a receptor for single-quantum attractant and two-quantum repellent phototaxis, transmitting light stimuli via its bound transducer HtrI. Signal-inverting mutations in the SRI–HtrI complex reverse the single-quantum response from attractant to repellent. Fast intramolecular charge movements reported here reveal that the unphotolyzed SRI–HtrI complex exists in two conformational states, which differ by their connection of the retinylidene Schiff base in the SRI photoactive site to inner or outer half-channels. In single-quantum photochemical reactions, the conformer with the Schiff base connected to the cytoplasmic (CP) half-channel generates an attractant signal, whereas the conformer with the Schiff base connected to the extracellular (EC) half-channel generates a repellent signal. In the wild-type complex the conformer equilibrium is poised strongly in favor of that with CP-accessible Schiff base. Signal-inverting mutations shift the equilibrium in favor of the EC-accessible Schiff base form, and suppressor mutations shift the equilibrium back toward the CP-accessible Schiff base form, restoring the wild-type phenotype. Our data show that the sign of the behavioral response directly correlates with the state of the connectivity switch, not with the direction of proton movements or changes in acceptor pKa. These findings identify a shared fundamental process in the mechanisms of transport and signaling by the rhodopsin family. Furthermore, the effects of mutations in the HtrI subunit of the complex on SRI Schiff base connectivity indicate that the two proteins are tightly coupled to form a single unit that undergoes a concerted conformational transition. PMID:18852467

A Schiff base connectivity switch in sensory rhodopsin signaling.

PubMed

Sineshchekov, Oleg A; Sasaki, Jun; Phillips, Brian J; Spudich, John L

2008-10-21

Sensory rhodopsin I (SRI) in Halobacterium salinarum acts as a receptor for single-quantum attractant and two-quantum repellent phototaxis, transmitting light stimuli via its bound transducer HtrI. Signal-inverting mutations in the SRI-HtrI complex reverse the single-quantum response from attractant to repellent. Fast intramolecular charge movements reported here reveal that the unphotolyzed SRI-HtrI complex exists in two conformational states, which differ by their connection of the retinylidene Schiff base in the SRI photoactive site to inner or outer half-channels. In single-quantum photochemical reactions, the conformer with the Schiff base connected to the cytoplasmic (CP) half-channel generates an attractant signal, whereas the conformer with the Schiff base connected to the extracellular (EC) half-channel generates a repellent signal. In the wild-type complex the conformer equilibrium is poised strongly in favor of that with CP-accessible Schiff base. Signal-inverting mutations shift the equilibrium in favor of the EC-accessible Schiff base form, and suppressor mutations shift the equilibrium back toward the CP-accessible Schiff base form, restoring the wild-type phenotype. Our data show that the sign of the behavioral response directly correlates with the state of the connectivity switch, not with the direction of proton movements or changes in acceptor pK(a). These findings identify a shared fundamental process in the mechanisms of transport and signaling by the rhodopsin family. Furthermore, the effects of mutations in the HtrI subunit of the complex on SRI Schiff base connectivity indicate that the two proteins are tightly coupled to form a single unit that undergoes a concerted conformational transition.

Electron impact excitation of the 1{sup 1}{ital S}{r_arrow}3{sup 1}{ital P} transition in helium

DOE Office of Scientific and Technical Information (OSTI.GOV)

Khakoo, M.A.; Roundy, D.; Rugamas, F.

1995-07-03

In the first direct application of the electron-photon coincidence technique for differential cross-section measurements, experimentally determined ratios of the differential cross sections for the electron impact excitation of the 1{sup 1}{ital S}{r_arrow}2{sup 1}{ital P} to the 1{sup 1}{ital S}{r_arrow}3{sup 1}{ital P} transitions are presented at 30 and 40 eV incident electron energies. Differential cross sections for the 1{sup 1}{ital S}{r_arrow}3{sup 1}{ital P} transitions are derived by normalizing these ratios to available experimental differential cross sections for the 1{sup 1}{ital S}{r_arrow}2{sup 1}{ital P} transition.

Purification, crystallization and preliminary X-ray diffraction analysis of the IL-20-IL-20R1-IL-20R2 complex

DOE Office of Scientific and Technical Information (OSTI.GOV)

Logsdon, Naomi J.; Allen, Christopher E.; Rajashankar, Kanagalaghatta R.

2012-02-08

Interleukin-20 (IL-20) is an IL-10-family cytokine that regulates innate and adaptive immunity in skin and other tissues. In addition to protecting the host from various external pathogens, dysregulated IL-20 signaling has been shown to contribute to the pathogenesis of human psoriasis. IL-20 signals through two cell-surface receptor heterodimers, IL-20R1-IL-20R2 and IL-22R1-IL-20R2. In this report, crystals of the IL-20-IL-20R1-IL-20R2 ternary complex have been grown from polyethylene glycol solutions. The crystals belonged to space group P4{sub 1}2{sub 1}2 or P4{sub 3}2{sub 1}2, with unit-cell parameters a = 111, c = 135 {angstrom}, and diffracted X-rays to 3 {angstrom} resolution. The crystallographic asymmetricmore » unit contains one IL-20-IL-20R1-IL-20R2 complex, corresponding to a solvent content of approximately 54%.« less

circ-SHKBP1 Regulates the Angiogenesis of U87 Glioma-Exposed Endothelial Cells through miR-544a/FOXP1 and miR-379/FOXP2 Pathways.

PubMed

He, Qianru; Zhao, Lini; Liu, Yunhui; Liu, Xiaobai; Zheng, Jian; Yu, Hai; Cai, Heng; Ma, Jun; Liu, Libo; Wang, Ping; Li, Zhen; Xue, Yixue

2018-03-02

Circular RNAs (circRNAs) are a type of endogenous non-coding RNAs, which have been considered to mediate diverse tumorigenesis including angiogenesis. The present study aims to elucidate the potential role and molecular mechanism of circ-SHKBP1 in regulating the angiogenesis of U87 glioma-exposed endothelial cells (GECs). The expression of circ-SHKBP1, but not linear SHKBP1, was significantly upregulated in GECs compared with astrocyte-exposed endothelial cells (AECs). circ-SHKBP1 knockdown inhibited the viability, migration, and tube formation of GECs dramatically. The expressions of miR-379/miR-544a were downregulated in GECs, and circ-SHKBP1 functionally targeted miR-544a/miR-379 in an RNA-induced silencing complex (RISC) manner. Dual-luciferase reporter assay demonstrated that forkhead box P1/P2 (FOXP1/FOXP2) were targets of miR-544a/miR-379. The expressions of FOXP1/FOXP2 were upregulated in GECs, and silencing of FOXP1/FOXP2 inhibited the viability, migration, and tube formation of GECs. Meanwhile, FOXP1/FOXP2 promoted angiogenic factor with G patch and FHA domains 1 (AGGF1) expression at the transcriptional level. Furthermore, knockdown of AGGF1 suppressed the viability, migration, and tube formation of GECs via phosphatidylinositol 3-kinase (PI3K)/AKT and extracellular signal-regulated kinase (ERK)1/2 pathways. Taken together, the present study demonstrated that circ-SHKBP1 regulated the angiogenesis of GECs through miR-544a/FOXP1 and miR-379/FOXP2 pathways, and these findings might provide a potential target and effective strategy for combined therapy of gliomas. Copyright © 2017 The Authors. Published by Elsevier Inc. All rights reserved.

MiR-23b controls ALDH1A1 expression in cervical cancer stem cells.

PubMed

Wang, Weiwen; Li, Yang; Liu, Na; Gao, Yu; Li, Long

2017-04-27

Cancer stem cells has been widely investigated due to its essential role in cancer progression and drug resistance. Here, we try to find a new therapeutic target for cervical cancer stem cells. We detected ALDH1A1-associated miRNAs expression in our isolated tumorspheres and their corresponding parental cells. Sphere formation assay was also used to determine stemness after cells were manipulated with miR-23b plasmid or miR-23b inhibitor. We found that miR-23b was under-expressed in cervical cancer stem cells to maintain high levels of ALDH1A1. Introduction of miR-23b into cervical cancer cells could alter stemness and cisplatin sensitivity. miR-23b plays key role in maintaining stemness of cervical cancer stem cells and can be developed as therapeutic target to better fight against cervical cancer.

The importance of the presence of a 5'-ribonucleotide and the contribution of the T1R1 + T1R3 heterodimer and an additional low-affinity receptor in the taste detection of L-glutamate as assessed psychophysically.

PubMed

Smith, Kimberly R; Spector, Alan C

2014-09-24

The molecular receptors underlying the purported "umami" taste quality commonly associated with l-glutamate have been controversial. Evidence supports the involvement of the T1R1 + T1R3 heterodimer, a GPCR broadly tuned to l-amino acids, but variants of two mGluRs expressed in taste buds have also been implicated. Using a rigorous psychophysical taste-testing paradigm, we demonstrated impaired, if not eliminated, detection of MSG in WT and T1R1, T1R2, T1R3, and T1R2 + T1R3 KO mice when the contribution of sodium was minimized by the epithelial sodium channel blocker amiloride. When inosine 5'-monophosphate (IMP), a ribonucleotide that potentiates the l-glutamate signal through the T1R1 + T1R3 heterodimer, was added, the WT and T1R2 KO mice were able to detect the compound stimulus across all MSG (+amiloride) concentrations due, in part, to the taste of IMP. In contrast, mice lacking T1R1 or T1R3 could not detect IMP alone, yet some were able to detect MSG + amiloride + IMP, but only at the higher MSG concentrations. Interestingly, the sensitivity of T1R1 KO mice to another l-amino acid, lysine, was unimpaired, suggesting that some l-amino acids can be detected through T1R1 + T1R3-independent receptors without sensitivity loss. Given that IMP is not thought to affect mGluRs, behavioral detection of l-glutamate appears to require the contribution of the T1R1 + T1R3 receptor. However, the partial competence observed in some T1R1 and T1R3 KO mice when MSG + amiloride + IMP was tested suggests that a T1R1 or T1R3 homodimer or an unidentified protein, perhaps in conjunction with T1R1 or T1R3, can serve as a low-affinity taste receptor for l-glutamate in the presence of IMP. Copyright © 2014 the authors 0270-6474/14/3413234-12$15.00/0.

A monoclonal antibody IMab-1 specifically recognizes IDH1R132H, the most common glioma-derived mutation.

PubMed

Kato, Yukinari; Jin, Genglin; Kuan, Chien-Tsun; McLendon, Roger E; Yan, Hai; Bigner, Darell D

2009-12-18

IDH1 (isocitrate dehydrogenase 1) mutations have been identified as early and frequent genetic alterations in astrocytomas, oligodendrogliomas, and oligoastrocytomas as well as secondary glioblastomas. In contrast, primary glioblastomas very rarely contain IDH1 mutations, although primary and secondary glioblastomas are histologically indistinguishable. The IDH1 mutations are remarkably specific to a single codon in the conserved and functionally important Arg132 in IDH1. In gliomas, the most frequent IDH1 mutations (>90%) were G395A (R132H). In this study, we immunized mice with R132H-containing IDH1 (IDH1(R132H)) peptide. After cell fusion using Sendai virus envelope, the monoclonal antibodies (mAbs), which specifically reacted with IDH1(R132H), were screened in ELISA. One of the mAbs, IMab-1 reacted with the IDH1(R132H) peptide, but not with wild type IDH1 (IDH1(wt)) peptide in ELISA. In Western-blot analysis, IMab-1 reacted with only the IDH1(R132H) protein, not IDH1(wt) protein or the other IDH1 mutants, indicating that IMab-1 is IDH1(R132H)-specific. Furthermore, IMab-1 specifically stained the IDH1(R132H)-expressing cells in astrocytomas in immunohistochemistry, whereas it did not react with IDH1(R132H)-negative primary glioblastoma sections. In conclusion, we established an anti-IDH1(R132H)-specific monoclonal antibody IMab-1, which should be significantly useful for diagnosis and biological evaluation of mutation-bearing gliomas.

Cytoadhesion to gC1qR through Plasmodium falciparum Erythrocyte Membrane Protein 1 in Severe Malaria

PubMed Central

Magallón-Tejada, Ariel; Machevo, Sónia; Cisteró, Pau; Lavstsen, Thomas; Aide, Pedro; Jiménez, Alfons; Turner, Louise; Gupta, Himanshu; De Las Salas, Briegel; Mandomando, Inacio; Wang, Christian W.; Petersen, Jens E. V.; Muñoz, Jose; Gascón, Joaquim; Macete, Eusebio; Alonso, Pedro L.; Chitnis, Chetan E.

2016-01-01

Cytoadhesion of Plasmodium falciparum infected erythrocytes to gC1qR has been associated with severe malaria, but the parasite ligand involved is currently unknown. To assess if binding to gC1qR is mediated through the P. falciparum erythrocyte membrane protein 1 (PfEMP1) family, we analyzed by static binding assays and qPCR the cytoadhesion and var gene transcriptional profile of 86 P. falciparum isolates from Mozambican children with severe and uncomplicated malaria, as well as of a P. falciparum 3D7 line selected for binding to gC1qR (Pf3D7gC1qR). Transcript levels of DC8 correlated positively with cytoadhesion to gC1qR (rho = 0.287, P = 0.007), were higher in isolates from children with severe anemia than with uncomplicated malaria, as well as in isolates from Europeans presenting a first episode of malaria (n = 21) than Mozambican adults (n = 25), and were associated with an increased IgG recognition of infected erythrocytes by flow cytometry. Pf3D7gC1qR overexpressed the DC8 type PFD0020c (5.3-fold transcript levels relative to Seryl-tRNA-synthetase gene) compared to the unselected line (0.001-fold). DBLβ12 from PFD0020c bound to gC1qR in ELISA-based binding assays and polyclonal antibodies against this domain were able to inhibit binding to gC1qR of Pf3D7gC1qR and four Mozambican P. falciparum isolates by 50%. Our results show that DC8-type PfEMP1s mediate binding to gC1qR through conserved surface epitopes in DBLβ12 domain which can be inhibited by strain-transcending functional antibodies. This study supports a key role for gC1qR in malaria-associated endovascular pathogenesis and suggests the feasibility of designing interventions against severe malaria targeting this specific interaction. PMID:27835682

Using R in Taverna: RShell v1.2

PubMed Central

Wassink, Ingo; Rauwerda, Han; Neerincx, Pieter BT; Vet, Paul E van der; Breit, Timo M; Leunissen, Jack AM; Nijholt, Anton

2009-01-01

Background R is the statistical language commonly used by many life scientists in (omics) data analysis. At the same time, these complex analyses benefit from a workflow approach, such as used by the open source workflow management system Taverna. However, Taverna had limited support for R, because it supported just a few data types and only a single output. Also, there was no support for graphical output and persistent sessions. Altogether this made using R in Taverna impractical. Findings We have developed an R plugin for Taverna: RShell, which provides R functionality within workflows designed in Taverna. In order to fully support the R language, our RShell plugin directly uses the R interpreter. The RShell plugin consists of a Taverna processor for R scripts and an RShell Session Manager that communicates with the R server. We made the RShell processor highly configurable allowing the user to define multiple inputs and outputs. Also, various data types are supported, such as strings, numeric data and images. To limit data transport between multiple RShell processors, the RShell plugin also supports persistent sessions. Here, we will describe the architecture of RShell and the new features that are introduced in version 1.2, i.e.: i) Support for R up to and including R version 2.9; ii) Support for persistent sessions to limit data transfer; iii) Support for vector graphics output through PDF; iv)Syntax highlighting of the R code; v) Improved usability through fewer port types. Our new RShell processor is backwards compatible with workflows that use older versions of the RShell processor. We demonstrate the value of the RShell processor by a use-case workflow that maps oligonucleotide probes designed with DNA sequence information from Vega onto the Ensembl genome assembly. Conclusion Our RShell plugin enables Taverna users to employ R scripts within their workflows in a highly configurable way. PMID:19607662

47 CFR 95.201 - (R/C Rule 1) What is the Radio Control (R/C) Radio Service?

Code of Federal Regulations, 2010 CFR

2010-10-01

... 47 Telecommunication 5 2010-10-01 2010-10-01 false (R/C Rule 1) What is the Radio Control (R/C) Radio Service? 95.201 Section 95.201 Telecommunication FEDERAL COMMUNICATIONS COMMISSION (CONTINUED) SAFETY AND SPECIAL RADIO SERVICES PERSONAL RADIO SERVICES Radio Control (R/C) Radio Service General...

Physicochemical properties of hydroxylated polychlorinated biphenyls aid in predicting their interactions with rat sulfotransferase 1A1 (rSULT1A1)

PubMed Central

Liu, Yungang; Lehmler, Hans-Joachim; Robertson, Larry W.; Duffel, Michael W.

2010-01-01

Hydroxylated metabolites of polychlorinated biphenyls (OHPCBs) interact with rat sulfotransferase 1A1 (rSULT1A1) as substrates and inhibitors. Previous studies have shown that there are complex and incompletely understood structure-activity relationships governing the interaction of rSULT1A1 with these molecules. Furthermore, modification of the enzyme with glutathione disulfide (GSSG) results in the conversion of some OHPCBs from inhibitors to substrates. We have now examined estimated values for the acid-dissociation constant (Ka) and the octanol-water distribution coefficient (D), as well as experimentally determined dissociation constants for enzyme complexes, to assist in the prediction of interactions of OHPCBs with rSULT1A1. Under reducing conditions, initial velocities for rSULT1A1-catalyzed sulfation exhibited a positive correlation with pKa and a negative correlation with log D of the OHPCBs. IC50 values of inhibitory OHPCBs decreased with decreasing pKa values for both the glutathione (GSH)-pretreated and GSSG-pretreated forms of rSULT1A1. Comparison of GSH- and GSSG-pretreated forms of rSULT1A1 with respect to binding of OHPCB in the presence and absence of adenosine 3’,5’-diphosphate (PAP) revealed that the dissociation constants with the two redox states of the enzyme were similar for each OHPCB. Thus, pKa and log D values are useful in predicting the binding of OHPCBs to the two redox forms of rSULT1A1 as well as the rates of sulfation of those OHPCBs that are substrates. However, the differences in substrate specificity for OHPCBs that are seen with changes in redox status of the enzyme are not directly related to specific structural effects of individual OHPCBs within inhibitory enzyme-PAP-OHPCB complexes. PMID:21130751

Halophilic life on Mars ?

NASA Astrophysics Data System (ADS)

Stan-Lotter, Helga; Fendrihan, Sergiu; Dornmayr-Pfaffenhuemer, Marion; Holzinger, Anita; Polacsek, Tatjana K.; Legat, Andrea; Grösbacher, Michael; Weigl, Andreas

2010-05-01

Background: The search for extraterrestrial life has been declared as a goal for the 21th century by several space agencies. Potential candidates are microorganisms on or in the surface of moons and planets, such as Mars. Extremely halophilic archaea (haloarchaea) are of astrobiological interest since viable strains have been isolated from million years old salt deposits (1) and halite has been found in Martian meteorites and in surface pools. Therefore, haloarchaeal responses to simulated and real space conditions were explored. Immuno assays for a potential Life Marker Chip experiment were developed with antisera against the universal enzyme ATP synthase. Methods: The focus of these studies was on the application of fluorescent probes since they provide strong signals, and detection devices are suitable for miniaturization. Viability of haloarchaeal strains (Halococcus dombrowskii and Halobacterium salinarum NRC-1) was probed with the LIVE/DEAD BacLight™ kit and the BacLight™ Bacterial Membrane Potential kit. Cyclobutane pyrimidine dimers (CPD) in the DNA, following exposure to simulated and real space conditions (UV irradiation from 200 - 400 nm; 18 months exposure on the International Space Station [ISS] within the ADAPT experiment by Dr. P. Rettberg), were detected with fluorescent Alexa-Fluor-488-coupled antibodies. Immuno assays with antisera against the A-ATPase subunits from Halorubrum saccharovorum were carried out with the highly sensitive Immun-Star ™ WesternC ™ chemiluminescent kit (Bio-Rad). Results: Using the LIVE/DEAD BacLight™ kit, the D37 (dose of 37% survival) for Hcc. dombrowskii and Hbt. salinarum NRC-1, following exposure to UV (200-400 nm) was about 400 kJ/m2, when cells were embedded in halite and about 1 kJ/m2, when cells were in liquid cultures. Fluorescent staining indicated a slightly higher cellular activity than that which was derived from the determination of colony forming units. Assessment of viability with the Bac

Do polymorphisms in the TAS1R1 gene contribute to broader differences in human taste intensity?

PubMed

Rawal, Shristi; Hayes, John E; Wallace, Margaret R; Bartoshuk, Linda M; Duffy, Valerie B

2013-10-01

The TAS1R genes encode heterodimeric receptors that mediate umami (hTAS1R1 + hTAS1R3) and sweet (hTAS1R2 + hTAS1R3) sensations. The question of interest for this study is if TAS1R1 variation associates with differences in overall taste intensity. We leveraged an existing database of adults (n = 92, primarily European American) to test associations between 2 TAS1R1 single nucleotide polymorphisms (SNPs) (intronic rs17492553, C/T and exonic rs34160967, G/A) and intensity of 4 prototypical tastants (NaCl, sucrose, citric acid, and quinine), applied regionally to fungiform and circumvallate loci, and sampled with the whole mouth. Both SNPs were associated with modest shifts in perceived intensities across all taste qualities. Three genotype groups were represented for the intronic SNP-minor allele homozygotes (TT) averaged 40% lower intensities than did CC homozygotes for all regionally applied tastants, as well as whole-mouth NaCl and citric acid. Similar, but less pronounced, intensity differences were seen for the exonic SNP (GG homozygotes reported greater intensities than did the AA/AG group). Our predominantly European American cohort had a low frequency of AA homozygotes, which may have attenuated the SNP-related differences in perceived intensity. These preliminary findings, if replicated, could add TAS1R1 polymorphisms to the repertoire of genotypic and phenotypic markers of heightened taste sensation.

Do Polymorphisms in the TAS1R1 Gene Contribute to Broader Differences in Human Taste Intensity?

PubMed Central

2013-01-01

The TAS1R genes encode heterodimeric receptors that mediate umami (hTAS1R1 + hTAS1R3) and sweet (hTAS1R2 + hTAS1R3) sensations. The question of interest for this study is if TAS1R1 variation associates with differences in overall taste intensity. We leveraged an existing database of adults (n = 92, primarily European American) to test associations between 2 TAS1R1 single nucleotide polymorphisms (SNPs) (intronic rs17492553, C/T and exonic rs34160967, G/A) and intensity of 4 prototypical tastants (NaCl, sucrose, citric acid, and quinine), applied regionally to fungiform and circumvallate loci, and sampled with the whole mouth. Both SNPs were associated with modest shifts in perceived intensities across all taste qualities. Three genotype groups were represented for the intronic SNP—minor allele homozygotes (TT) averaged 40% lower intensities than did CC homozygotes for all regionally applied tastants, as well as whole-mouth NaCl and citric acid. Similar, but less pronounced, intensity differences were seen for the exonic SNP (GG homozygotes reported greater intensities than did the AA/AG group). Our predominantly European American cohort had a low frequency of AA homozygotes, which may have attenuated the SNP-related differences in perceived intensity. These preliminary findings, if replicated, could add TAS1R1 polymorphisms to the repertoire of genotypic and phenotypic markers of heightened taste sensation. PMID:24000232

MiR-146a negatively regulates dectin-1-induced inflammatory responses

PubMed Central

Duan, Zhimin; Liu, Caixia; Zeng, Rong

2017-01-01

Dectin-1 is the critical sensor for β-glucan from Candida which is the most common human fungal pathogen and cause superficial and system infection. MicroRNAs (miRNAs) play crucial roles in regulating innate immunity. However, the functional role of miRNAs in inflammatory response dependent on the activation of dectin-1 pathway has not been defined. In the present study, we found insoluble β-glucan from the cell wall of Candida albicans (CaIG) was able to increase the production of of IL-6 and TNFα through Dectin-1-Syk-NF-κB and p38MAPK pathway. MiRNAs profiles combined with real-time PCR validation revealed that miR-146a, miR-30-5p, miR-210-3p expression level were increased in THP-1 cells treated with CaIG. The interaction between Dectin-1 and CaIG resulted in an long lasting increase of miR-146a expression dependent on Dectin-1-Syk-NF-κB, p38MAPK, contrasting with a rapid and transient increase of IL-6 and TNFα. Overexpression of miR-146a significantly suppressed the production of IL-6 and TNFα. MiR-146a mimics inhibited CaIG-induced activity of p-IκBα and translocation of NF-κB p65. Luciferase reporter assays showed miR-146a inhibited NF-κB promoter-binding activity. Together, our data suggest miR-146a may play the potent negative feedback regulator in inflammatory response following Dectin-1 stimulation. PMID:28454101

Actin binding by Hip1 (huntingtin-interacting protein 1) and Hip1R (Hip1-related protein) is regulated by clathrin light chain.

PubMed

Wilbur, Jeremy D; Chen, Chih-Ying; Manalo, Venus; Hwang, Peter K; Fletterick, Robert J; Brodsky, Frances M

2008-11-21

The huntingtin-interacting protein family members (Hip1 and Hip1R in mammals and Sla2p in yeast) link clathrin-mediated membrane traffic to actin cytoskeleton dynamics. Genetic data in yeast have implicated the light chain subunit of clathrin in regulating this link. To test this hypothesis, the biophysical properties of mammalian Hip1 and Hip1R and their interaction with clathrin light chain and actin were analyzed. The coiled-coil domains (clathrin light chain-binding) of Hip1 and Hip1R were found to be stable homodimers with no propensity to heterodimerize in vitro. Homodimers were also predominant in vivo, accounting for cellular segregation of Hip1 and Hip1R functions. Coiled-coil domains of Hip1 and Hip1R differed in their stability and flexibility, correlating with slightly different affinities for clathrin light chain and more markedly with effects of clathrin light chain binding on Hip protein-actin interactions. Clathrin light chain binding induced a compact conformation of both Hip1 and Hip1R and significantly reduced actin binding by their THATCH domains. Thus, clathrin is a negative regulator of Hip-actin interactions. These observations necessarily change models proposed for Hip protein function.

The Importance of the Presence of a 5′-Ribonucleotide and the Contribution of the T1R1 + T1R3 Heterodimer and an Additional Low-Affinity Receptor in the Taste Detection of l-Glutamate as Assessed Psychophysically

PubMed Central

Smith, Kimberly R.

2014-01-01

The molecular receptors underlying the purported “umami” taste quality commonly associated with l-glutamate have been controversial. Evidence supports the involvement of the T1R1 + T1R3 heterodimer, a GPCR broadly tuned to l-amino acids, but variants of two mGluRs expressed in taste buds have also been implicated. Using a rigorous psychophysical taste-testing paradigm, we demonstrated impaired, if not eliminated, detection of MSG in WT and T1R1, T1R2, T1R3, and T1R2 + T1R3 KO mice when the contribution of sodium was minimized by the epithelial sodium channel blocker amiloride. When inosine 5′-monophosphate (IMP), a ribonucleotide that potentiates the l-glutamate signal through the T1R1 + T1R3 heterodimer, was added, the WT and T1R2 KO mice were able to detect the compound stimulus across all MSG (+amiloride) concentrations due, in part, to the taste of IMP. In contrast, mice lacking T1R1 or T1R3 could not detect IMP alone, yet some were able to detect MSG + amiloride + IMP, but only at the higher MSG concentrations. Interestingly, the sensitivity of T1R1 KO mice to another l-amino acid, lysine, was unimpaired, suggesting that some l-amino acids can be detected through T1R1 + T1R3-independent receptors without sensitivity loss. Given that IMP is not thought to affect mGluRs, behavioral detection of l-glutamate appears to require the contribution of the T1R1 + T1R3 receptor. However, the partial competence observed in some T1R1 and T1R3 KO mice when MSG + amiloride + IMP was tested suggests that a T1R1 or T1R3 homodimer or an unidentified protein, perhaps in conjunction with T1R1 or T1R3, can serve as a low-affinity taste receptor for l-glutamate in the presence of IMP. PMID:25253867

Reactivity-worth estimates of the OSMOSE samples in the MINERVE reactor R1-MOX, R2-UO2 and MORGANE/R configurations.

DOE Office of Scientific and Technical Information (OSTI.GOV)

Zhong, Z.; Klann, R. T.; Nuclear Engineering Division

2007-08-03

An initial series of calculations of the reactivity-worth of the OSMOSE samples in the MINERVE reactor with the R2-UO2 and MORGANE/R core configuration were completed. The calculation model was generated using the lattice physics code DRAGON. In addition, an initial comparison of calculated values to experimental measurements was performed based on preliminary results for the R1-MOX configuration.

Cross-reactivity of anti-PLA2R1 autoantibodies to rabbit and mouse PLA2R1 antigens and development of two novel ELISAs with different diagnostic performances in idiopathic membranous nephropathy.

PubMed

Seitz-Polski, Barbara; Dolla, Guillaume; Payré, Christine; Tomas, Nicola M; Lochouarn, Marine; Jeammet, Louise; Mariat, Christophe; Krummel, Thierry; Burtey, Stéphane; Courivaud, Cécile; Schlumberger, Wolfgang; Zorzi, Kévin; Benzaken, Sylvia; Bernard, Ghislaine; Esnault, Vincent L M; Lambeau, Gérard

2015-11-01

About 70% of patients with idiopathic membranous nephropathy (iMN) have autoantibodies to the phospholipase A2 receptor PLA2R1. We screened sera from iMN patients for their cross-reactivity to human (h), rabbit (rb) and mouse (m) PLA2R1 by western blot (WB) and antigen-specific ELISAs. All iMN patients recognized hPLA2R1 and rbPLA2R1 by WB, and a rbPLA2R1 ELISA was as sensitive as the standardized hPLA2R1 ELISA to monitor anti-PLA2R1 in patients with active disease or in drug-induced remission. In contrast, only 51% of patients were reactive to mPLA2R1 by WB, and a maximum of 78% were weakly to highly positive in the mPLA2R1 ELISA, suggesting that iMN patients exhibit different subsets of anti-PLA2R1 autoantibodies against epitopes that are shared or not among PLA2R1 orthologs. In a cohort of 41 patients with a mean follow-up of 42 months from anti-PLA2R1 assay, the detection of anti-mPLA2R1 autoantibodies was an independent predictor of clinical outcome in multivariate analysis (p = 0.009), and a ROC curve analysis identified a threshold of 605 RU/mL above which 100% of patients (12 patients) had a poor renal outcome (p < 0.001). A similar threshold could not be defined in hPLA2R1 and rbPLA2R1 ELISAs. We conclude that rbPLA2R1 is an alternative antigen to hPLA2R1 to measure anti-PLA2R1 in active disease while mPLA2R1 is a unique antigen that can detect a subset of anti-PLA2R1 autoantibodies present at high levels (>605 RU/mL) only in iMN patients at risk of poor prognosis, and is thus useful to predict iMN outcome. Copyright © 2015 Elsevier B.V. and Société Française de Biochimie et Biologie Moléculaire (SFBBM). All rights reserved.

[Expression of miR-22 and miR-150 in type 1 diabetes mellitus: Possible relationship with autoimmunity and clinical characteristics].

PubMed

Estrella, Santiago; Garcia-Diaz, Diego F; Codner, Ethel; Camacho-Guillén, Patricia; Pérez-Bravo, Francisco

2016-09-16

Type 1 diabetes (T1D) is an autoimmune disease of complex aetiology. Several microRNAs (miR) have been linked to the pathogenesis of autoimmune diseases. To analyze the possible association of miR-22 and miR-150 with autoimmunity and clinical severity of T1D. The study was performed in peripheral blood mononuclear cells of 20 patients with T1D and 20 control subjects. The expression of miR-22 and miR-150 was performed in peripheral blood mononuclear cells using TaqMan probes to different glucose concentrations (baseline, 11mm, 25mm). Our results suggest that the expression of miR-22 is increased in T1D patients compared to the controls. This effect was observed in baseline glucose conditions and decreased in 11 and 25mM of glucose. The expression of miR-150 was lower in T1D patients versus the controls. There was no correlation between the autoimmune profile and the two studied miRNAs. miR-22 (baseline condition) and miR-150 (11mM condition) or the ketoacidosis component. miR-22 and 150 were not associated with the autoimmune component present in T1D patients. Copyright © 2016 Elsevier España, S.L.U. All rights reserved.

HLA-DQA1 and PLA2R1 Polymorphisms and Risk of Idiopathic Membranous Nephropathy

PubMed Central

Bullich, Gemma; Ballarín, José; Oliver, Artur; Ayasreh, Nadia; Silva, Irene; Santín, Sheila; Díaz-Encarnación, Montserrat M.; Torra, Roser

2014-01-01

Summary Background and objectives Single nucleotide polymorphisms (SNPs) within HLA complex class II HLA-DQ α-chain 1 (HLA-DQA1) and M-type phospholipase A2 receptor (PLA2R1) genes were identified as strong risk factors for idiopathic membranous nephropathy (IMN) development in a recent genome-wide association study. Copy number variants (CNVs) within the Fc gamma receptor III (FCGR3) locus have been associated with several autoimmune diseases, but their role in IMN has not been studied. This study aimed to validate the association of HLA-DQA1 and PLA2R1 risk alleles with IMN in a Spanish cohort, test the putative association of FCGR3A and FCGR3B CNVs with IMN, and assess the use of these genetic factors to predict the clinical outcome of the disease. Design, settings, participants, & measurements A Spanish cohort of 89 IMN patients and 286 matched controls without nephropathy was recruited between October of 2009 and July of 2012. Case-control studies for SNPs within HLA-DQA1 (rs2187668) and PLA2R1 (rs4664308) genes and CNVs for FCGR3A and FCGR3B genes were performed. The contribution of these polymorphisms to predict clinical outcome and renal function decline was analyzed. Results This study validated the association of these HLA-DQA1 and PLA2R1 SNPs with IMN in a Spanish cohort and its increased risk when combining both risk genotypes. No significant association was found between FCGR3 CNVs and IMN. These results revealed that HLA-DQA1 and PLA2R1 genotype combination adjusted for baseline proteinuria strongly predicted response to immunosuppressive therapy. HLA-DQA1 genotype adjusted for proteinuria was also linked with renal function decline. Conclusion This study confirms that HLA-DQA1 and PLA2R1 genotypes are risk factors for IMN, whereas no association was identified for FCGR3 CNVs. This study provides, for the first time, evidence of the contribution of these HLA-DQA1 and PLA2R1 polymorphisms in predicting IMN response to immunosuppressors and disease

HLA-DQA1 and PLA2R1 polymorphisms and risk of idiopathic membranous nephropathy.

PubMed

Bullich, Gemma; Ballarín, José; Oliver, Artur; Ayasreh, Nadia; Silva, Irene; Santín, Sheila; Díaz-Encarnación, Montserrat M; Torra, Roser; Ars, Elisabet

2014-02-01

Single nucleotide polymorphisms (SNPs) within HLA complex class II HLA-DQ α-chain 1 (HLA-DQA1) and M-type phospholipase A2 receptor (PLA2R1) genes were identified as strong risk factors for idiopathic membranous nephropathy (IMN) development in a recent genome-wide association study. Copy number variants (CNVs) within the Fc gamma receptor III (FCGR3) locus have been associated with several autoimmune diseases, but their role in IMN has not been studied. This study aimed to validate the association of HLA-DQA1 and PLA2R1 risk alleles with IMN in a Spanish cohort, test the putative association of FCGR3A and FCGR3B CNVs with IMN, and assess the use of these genetic factors to predict the clinical outcome of the disease. A Spanish cohort of 89 IMN patients and 286 matched controls without nephropathy was recruited between October of 2009 and July of 2012. Case-control studies for SNPs within HLA-DQA1 (rs2187668) and PLA2R1 (rs4664308) genes and CNVs for FCGR3A and FCGR3B genes were performed. The contribution of these polymorphisms to predict clinical outcome and renal function decline was analyzed. This study validated the association of these HLA-DQA1 and PLA2R1 SNPs with IMN in a Spanish cohort and its increased risk when combining both risk genotypes. No significant association was found between FCGR3 CNVs and IMN. These results revealed that HLA-DQA1 and PLA2R1 genotype combination adjusted for baseline proteinuria strongly predicted response to immunosuppressive therapy. HLA-DQA1 genotype adjusted for proteinuria was also linked with renal function decline. This study confirms that HLA-DQA1 and PLA2R1 genotypes are risk factors for IMN, whereas no association was identified for FCGR3 CNVs. This study provides, for the first time, evidence of the contribution of these HLA-DQA1 and PLA2R1 polymorphisms in predicting IMN response to immunosuppressors and disease progression. Future studies are needed to validate and identify prognostic markers.

Monoamine oxidase A and repressor R1 are involved in apoptotic signaling pathway.

PubMed

Ou, Xiao-Ming; Chen, Kevin; Shih, Jean C

2006-07-18

Monoamine oxidase A (MAO A) degrades serotonin, norepinephrine, and dopamine and produces reactive oxygen that may cause neuronal cell death. We have previously reported that a novel transcription factor R1 (RAM2/CDCA7L/JPO2) inhibits the MAO A promoter and enzymatic activities. This study reports the roles of MAO A and R1 in apoptosis and proliferation. We have found that in serum starvation-induced apoptosis, p38 kinase, MAO A, and caspase-3 were increased, whereas Bcl-2 and R1 were reduced. Using a p38 kinase inhibitor, R1 overexpression, and MAO A inhibitor, we have shown that MAO A and R1 are downstream of p38 kinase and Bcl-2, but upstream of caspase-3. Inhibition of MAO A prevents cell apoptosis. This notion was further supported by the finding that serum starvation-induced apoptosis is reduced in cortical brain cells from MAO A-deficient mice compared with WT. In addition, we found that MAO A and R1 are involved in the c-Myc-induced proliferative signaling pathway in the presence of serum. Immunoprecipitation and immunohistochemistry experiments indicate that the oncogene c-Myc colocalizes with R1 and induces R1 gene expression. Using R1 overexpression, R1 small interfering RNA, and a MAO A inhibitor, we found that R1 and MAO A act upstream of cyclin D1 and E2F1. In summary, this study demonstrates the functions of MAO A and its repressor R1 in apoptotic signaling pathways.

Bmi-1 confers adaptive radioresistance to KYSE-150R esophageal carcinoma cells

DOE Office of Scientific and Technical Information (OSTI.GOV)

Wang, Guanyu; Liu, Luying; Sharma, Sherven

2012-08-24

Highlights: Black-Right-Pointing-Pointer Adaptive radioresistant KYSE-150R cells expressed high level of Bmi-1. Black-Right-Pointing-Pointer Bmi-1 depletion sensitized KYSE-150R cells to RT. Black-Right-Pointing-Pointer Bmi-1 depletion increased the generation of ROS in KYSE-150R cells exposed to radiation. Black-Right-Pointing-Pointer Bmi-1 depletion impaired DNA repair capacities in KYSE-150R cells exposed to radiation. -- Abstract: Radiotherapy (RT) is a major modality of cancer treatment. However, tumors often acquire radioresistance, which causes RT to fail. The exact mechanisms by which tumor cells subjected to fractionated irradiation (FIR) develop an adaptive radioresistance are largely unknown. Using the radioresistant KYSE-150R esophageal squamous cell carcinoma (ESCC) model, which was derived frommore » KYSE-150 parental cells using FIR, the role of Bmi-1 in mediating the radioadaptive response of ESCC cells to RT was investigated. The results showed that the level of Bmi-1 expression was significantly higher in KYSE-150R cells than in the KYSE-150 parental cells. Bmi-1 depletion sensitized the KYSE-150R cells to RT mainly through the induction of apoptosis, partly through the induction of senescence. A clonogenic cell survival assay showed that Bmi-1 depletion significantly decreased the radiation survival fraction in KYSE-150R cells. Furthermore, Bmi-1 depletion increased the generation of reactive oxygen species (ROS) and the expression of oxidase genes (Lpo, Noxo1 and Alox15) in KYSE-150R cells exposed to irradiation. DNA repair capacities assessed by {gamma}-H2AX foci formation were also impaired in the Bmi-1 down-regulated KYSE-150R cells. These results suggest that Bmi-1 plays an important role in tumor radioadaptive resistance under FIR and may be a potent molecular target for enhancing the efficacy of fractionated RT.« less

Degenerative phenotypes caused by the combined deficiency of murine HIP1 and HIP1r are rescued by human HIP1.

PubMed

Bradley, Sarah V; Hyun, Teresa S; Oravecz-Wilson, Katherine I; Li, Lina; Waldorff, Erik I; Ermilov, Alexander N; Goldstein, Steven A; Zhang, Claire X; Drubin, David G; Varela, Kate; Parlow, Al; Dlugosz, Andrzej A; Ross, Theodora S

2007-06-01

The members of the huntingtin-interacting protein-1 (HIP1) family, HIP1 and HIP1-related (HIP1r), are multi-domain proteins that interact with inositol lipids, clathrin and actin. HIP1 is over-expressed in a variety of cancers and both HIP1 and HIP1r prolong the half-life of multiple growth factor receptors. To better understand the physiological importance of the HIP1 family in vivo, we have analyzed a large cohort of double Hip1/Hip1r knockout (DKO) mice. All DKO mice were dwarfed, afflicted with severe vertebral defects and died in early adulthood. These phenotypes were not observed during early adulthood in the single Hip1 or Hip1r knockouts, indicating that HIP1 and HIP1r compensate for one another. Despite the ability of HIP1 and HIP1r to modulate growth factor receptor levels when over-expressed, studies herein using DKO fibroblasts indicate that the HIP1 family is not necessary for endocytosis but is necessary for the maintenance of diverse adult tissues in vivo. To test if human HIP1 can function similar to mouse HIP1, transgenic mice with 'ubiquitous' expression of the human HIP1 cDNA were generated and crossed with DKO mice. Strikingly, the compound human HIP1 transgenic DKO mice were completely free from dwarfism and spinal defects. This successful rescue demonstrates that the human HIP1 protein shares some interchangeable functions with both HIP1 and HIP1r in vivo. In addition, we conclude that the degenerative phenotypes seen in the DKO mice are due mainly to HIP1 and HIP1r protein deficiency rather than altered expression of neighboring genes or disrupted intronic elements.

Hypersensitivity to Tomato (Lycopersicon esculentum) in Peach-Allergic Patients: rPrup 3 and rPrup 1 Are Predictive of Symptom Severity.

PubMed

Mascheri, A; Farioli, L; Pravettoni, V; Piantanida, M; Stafylaraki, C; Scibilia, J; Mirone, C; Preziosi, D; Nichelatti, M; Pastorello, E A

2015-01-01

Background: The role of allergens in the severity of tomato allergy symptoms has not yet been studied. To evaluate the relationship between severe allergic reactions to peach and tomato and between tomato allergy symptoms and the pattern of IgE positivity for rPru p 1, rPru p 3, rPru p 4, rBetv 1, rBetv 2, rBetv4, rPhl p 1, and rPhl p 12 in order to identify the role of recombinant allergens in the severity of reactions to tomato. We studied peach-allergic patients with clinical reactions to tomato by performing an open food challenge, skin prick test, and determination of serum specific IgE to tomato and to recombinant peach, birch, and grass allergens. Statistical analysis was carried out to evaluate the relationship between the severity of tomato symptoms and IgE positivity to the different allergens and to peach-induced symptoms. We found a significant association between severe reactions to tomato and severe reactions to peach (P = .01 7) and levels of IgE to rPru p3 (P = .029) and between mild tomato allergy symptoms and levels of IgE to rPru p1 (P = .047), anti-rBetv 1 (P = .0414), anti-rBetv 2 (P = .0457), and Phleum pratense (P = .0022). We observed a significant relationship between peach and symptoms of tomato allergy. IgE positivity for rPru p3 seems to be a surrogate biochemical marker for severe tomato allergy, whereas the presence of anti-rPru p 1 IgE may be an indicator of mild tomato allergy.

Pancreatic Fibroblasts Stimulate the Motility of Pancreatic Cancer Cells through IGF1/IGF1R Signaling under Hypoxia.

PubMed

Hirakawa, Toshiki; Yashiro, Masakazu; Doi, Yosuke; Kinoshita, Haruhito; Morisaki, Tamami; Fukuoka, Tatsunari; Hasegawa, Tsuyoshi; Kimura, Kenjiro; Amano, Ryosuke; Hirakawa, Kosei

2016-01-01

Pancreatic ductal adenocarcinoma (PDAC) is characterized by its hypovascularity, with an extremely poor prognosis because of its highly invasive nature. PDAC proliferates with abundant stromal cells, suggesting that its invasive activity might be controlled by intercellular interactions between cancer cells and fibroblasts. Using four PDAC cell lines and two pancreas cancer-associated fibroblasts (CAFs), the expression of insulin-like growth factor-1 (IGF1) and IGF1 receptor (IGF1R) was evaluated by RT-PCR, FACScan, western blot, or ELISA. Correlation between IGF1R and the hypoxia marker carbonic anhydrase 9 (CA9) was examined by immunohistochemical staining of 120 pancreatic specimens. The effects of CAFs, IGF1, and IGF1R inhibitors on the motility of cancer cells were examined by wound-healing assay or invasion assay under normoxia (20% O2) and hypoxia (1% O2). IGF1R expression was significantly higher in RWP-1, MiaPaCa-2, and OCUP-AT cells than in Panc-1 cells. Hypoxia increased the expression level of IGF1R in RWP-1, MiaPaCa-2, and OCUP-AT cells. CA9 expression was correlated with IGF1R expression in pancreatic specimens. CAFs produced IGF1 under hypoxia, but PDAC cells did not. A conditioned medium from CAFs, which expressed αSMA, stimulated the migration and invasion ability of MiaPaCa-2, RWP-1, and OCUP-AT cells. The motility of all PDAC cells was greater under hypoxia than under normoxia. The motility-stimulating ability of CAFs was decreased by IGF1R inhibitors. These findings might suggest that pancreas CAFs stimulate the invasion activity of PDAC cells through paracrine IGF1/IGF1R signaling, especially under hypoxia. Therefore the targeting of IGF1R signaling might represent a promising therapeutic approach in IGF1R-dependent PDAC.

Synthesis and Pharmacological Characterization of C4-(Thiotriazolyl)-substituted-2-aminobicyclo[3.1.0]hexane-2,6-dicarboxylates. Identification of (1R,2S,4R,5R,6R)-2-Amino-4-(1H-1,2,4-triazol-3-ylsulfanyl)bicyclo[3.1.0]hexane-2,6-dicarboxylic Acid (LY2812223), a Highly Potent, Functionally Selective mGlu2 Receptor Agonist.

PubMed

Monn, James A; Prieto, Lourdes; Taboada, Lorena; Hao, Junliang; Reinhard, Matthew R; Henry, Steven S; Beadle, Christopher D; Walton, Lesley; Man, Teresa; Rudyk, Helene; Clark, Barry; Tupper, David; Baker, S Richard; Lamas, Carlos; Montero, Carlos; Marcos, Alicia; Blanco, Jaime; Bures, Mark; Clawson, David K; Atwell, Shane; Lu, Frances; Wang, Jing; Russell, Marijane; Heinz, Beverly A; Wang, Xushan; Carter, Joan H; Getman, Brian G; Catlow, John T; Swanson, Steven; Johnson, Bryan G; Shaw, David B; McKinzie, David L

2015-09-24

Identification of orthosteric mGlu(2/3) receptor agonists capable of discriminating between individual mGlu2 and mGlu3 subtypes has been highly challenging owing to the glutamate-site sequence homology between these proteins. Herein we detail the preparation and characterization of a series of molecules related to (1S,2S,5R,6S)-2-aminobicyclo[3.1.0]hexane-2,6-dicarboxylate 1 (LY354740) bearing C4-thiotriazole substituents. On the basis of second messenger responses in cells expressing other recombinant human mGlu2/3 subtypes, a number of high potency and efficacy mGlu2 receptor agonists exhibiting low potency mGlu3 partial agonist/antagonist activity were identified. From this, (1R,2S,4R,5R,6R)-2-amino-4-(1H-1,2,4-triazol-3-ylsulfanyl)bicyclo[3.1.0]hexane-2,6-dicarboxylic acid 14a (LY2812223) was further characterized. Cocrystallization of 14a with the amino terminal domains of hmGlu2 and hmGlu3 combined with site-directed mutation studies has clarified the underlying molecular basis of this unique pharmacology. Evaluation of 14a in a rat model responsive to mGlu2 receptor activation coupled with a measure of central drug disposition provides evidence that this molecule engages and activates central mGlu2 receptors in vivo.

miR-340 alleviates chemoresistance of osteosarcoma cells by targeting ZEB1.

PubMed

Yan, Haibin; Zhang, Bingyun; Fang, Chongbin; Chen, Liqiu

2018-06-01

Chemoresistance during treatment of osteosarcoma (OS) is attracting more and more attention as the main clinical obstacle. The purpose of this study was to elucidate the role of miR-340 in chemoresistance of OS. Plasmid construction and transfection, miRNA arrays, PCR analyses, and western blot analysis, as well as MTT, apoptosis, and luciferase assays were carried out in MG-63 cells and MG-63/cisplatin (DDP)-resistant cells. The results showed that miR-340 was downregulated in OS tissues and drug-resistant OS cells. Moreover, a negative correlation was observed between miR-340 and ZEB1 expression in OS tissues. Forced expression of miR-340 in drug-resistant OS cells significantly reduced multidrug resistance-1 and P-gp expression. Overexpression of miR-340 enhanced sensitivity to DDP by inhibiting viability and promoting apoptosis. The luciferase assay and western blot analysis identified ZEB1 as a direct target of miR-340, and miR-340 negatively regulated ZEB1 expression. Ectopic expression of ZEB1 reversed the effects of miR-340 on P-gp expression, cell viability, and apoptosis. miR-340 alleviated chemoresistance of OS cells by targeting ZEB1. Our results indicate that targeting miR-340 may be a potential therapeutic approach to treat drug-resistant OS.

miR-1 is increased in pulmonary hypertension and downregulates Kv1.5 channels in rat pulmonary arteries.

PubMed

Mondejar-Parreño, Gema; Callejo, María; Barreira, Bianca; Morales-Cano, Daniel; Esquivel-Ruiz, Sergio; Moreno, Laura; Cogolludo, Angel; Perez-Vizcaino, Francisco

2018-05-02