Basic residues in the 74-83 and 191-198 segments of protein kinase CK2 catalytic subunit are implicated in negative but not in positive regulation by the beta-subunit

DEFF Research Database (Denmark)

Sarno, S; Vaglio, P; Marin, O

1997-01-01

by the beta-subunit many fold more than that of alpha wild type, while extrastimulation by beta mutant D55L56E57A, observable with alpha wild type, is abolished with these mutants. These data support the conclusion that down regulation by the acidic residues clustered in the N-terminal moiety of beta...... is mediated by basic residues in the 74-83 and in the 191-198 sequences of the alpha-subunit. These are also implicated in substrate recognition consistent with the concept that the N-terminal acidic region of the beta subunit operates as a pseudosubstrate. In contrast, another CK2alpha mutant, V66A, is more...

Differential roles of the glycogen-binding domains of beta subunits in regulation of the Snf1 kinase complex.

Science.gov (United States)

Mangat, Simmanjeet; Chandrashekarappa, Dakshayini; McCartney, Rhonda R; Elbing, Karin; Schmidt, Martin C

2010-01-01

Members of the AMP-activated protein kinase family, including the Snf1 kinase of Saccharomyces cerevisiae, are activated under conditions of nutrient stress. AMP-activated protein kinases are heterotrimeric complexes composed of a catalytic alpha subunit and regulatory beta and gamma subunits. In this study, the role of the beta subunits in the regulation of Snf1 activity was examined. Yeasts express three isoforms of the AMP-activated protein kinase consisting of Snf1 (alpha), Snf4 (gamma), and one of three alternative beta subunits, either Sip1, Sip2, or Gal83. The Gal83 isoform of the Snf1 complex is the most abundant and was analyzed in the greatest detail. All three beta subunits contain a conserved domain referred to as the glycogen-binding domain. The deletion of this domain from Gal83 results in a deregulation of the Snf1 kinase, as judged by a constitutive activity independent of glucose availability. In contrast, the deletion of this homologous domain from the Sip1 and Sip2 subunits had little effect on Snf1 kinase regulation. Therefore, the different Snf1 kinase isoforms are regulated through distinct mechanisms, which may contribute to their specialized roles in different stress response pathways. In addition, the beta subunits are subjected to phosphorylation. The responsible kinases were identified as being Snf1 and casein kinase II. The significance of the phosphorylation is unclear since the deletion of the region containing the phosphorylation sites in Gal83 had little effect on the regulation of Snf1 in response to glucose limitation.

Reconstitution of normal and hyperactivated forms of casein kinase-2 by variably mutated beta-subunits

DEFF Research Database (Denmark)

Boldyreff, B; Meggio, F; Pinna, L A

1993-01-01

Twenty-one mutants of the noncatalytic beta-subunit of human casein kinase-2 have been created, expressed in Escherichia coli, and purified to homogeneity. They are either modified at the autophosphorylation site (mutants beta delta 1-4 and beta A 5,6) or bear variable deletions in their C...

T cell precursor migration towards beta 2-microglobulin is involved in thymus colonization of chicken embryos

DEFF Research Database (Denmark)

Dunon, D; Kaufman, J; Salomonsen, J

1990-01-01

beta 2-microglobulin (beta 2m) attracts hemopoietic precursors from chicken bone marrow cells in vitro. The cell population responding to beta 2m increases during the second period of thymus colonization, which takes place at days 12-14 of incubation. The precursors from 13.5 day old embryos were...... isolated after migration towards beta 2m in vitro and shown to be able to colonize a 13 day old thymus in ovo, where they subsequently acquire thymocyte markers. In contrast these beta 2m responsive precursors did not colonize embryonic bursa, i.e. differentiate into B lymphocytes. During chicken...... embryogenesis, peaks of beta 2m transcripts and of free beta 2m synthesis can only be detected in the thymus. The peak of free beta 2m synthesis in the thymus and the increase of beta 2m responding bone marrow cells both occur concomitantly with the second wave of thymus colonization in chicken embryo, facts...

[Molecular cloning of activin betaA subunit mature peptide from peafowl and its application in taxonomy and phylogeny].

Science.gov (United States)

Zou, Fang-Dong; Tong, Xin-Xin; Yue, Bi-Song

2005-03-01

The sequences of activin gene betaA subunit mature peptide have been amplified from white peafowl, blue peafowl (pavo cristatus) and green peafowl (pavo muticus) genomic DNA by polymerase chain reaction (PCR) with a pair of degenerate primers. The target fragments were cloned into the vector pMD18-T and sequenced. The length of activin gene betaA subunit mature peptide is 345bp, which encoded a peptide of 115 amino acid residues. Sequence analysis of activin gene betaA subunit mature peptide demonstrated that the identity of nucleotide is 98.0% between blue peaflowl and green peafowl, and the identity of that is 98.8% between blue peaflowl and white peafow. Sequences comparison in NCBI revealed that the sequences of activin gene betaA subunit mature peptides of different species are highly conserved during evolution process. In addition, the restriction enzyme map of activins is high similar between white peafowl and blue peafowl. Phylogenetic tree was constructed with Mega 2 and Clustalxldx software. The result showed that white peafowl has a closer relationship to blue peafowl than to green peafowl. Considered the nucleotide differences of peafowls' activin gene betaA subunit mature peptides, a highly conserved region, we supported that white peafowl was derived from blue peafowl, and it is more possible the hybrid but just the product of color mutation, or maybe as a subspecies of Pavo genus.

Differential expression of BK channel isoforms and beta-subunits in rat neuro-vascular tissues

DEFF Research Database (Denmark)

Poulsen, Asser Nyander; Wulf, Helle; Hay-Schmidt, Anders

2009-01-01

We investigated the expression of splice variants and beta-subunits of the BK channel (big conductance Ca(2+)-activated K(+) channel, Slo1, MaxiK, K(Ca)1.1) in rat cerebral blood vessels, meninges, trigeminal ganglion among other tissues. An alpha-subunit splice variant X1(+24) was found expresse...

CD147 is a regulatory subunit of the gamma-secretase complex inAlzheimer's disease amyloid beta-peptide production

Energy Technology Data Exchange (ETDEWEB)

Zhou, Shuxia; Zhou, Hua; Walian, Peter J.; Jap, Bing K.

2005-04-06

{gamma}-secretase is a membrane protein complex that cleaves the {beta}-amyloid precursor protein (APP) within the transmembrane region, following prior processing by {beta}-secretase, producing amyloid {beta}-peptides (A{beta}{sub 40} and A{beta}{sub 42}). Errant production of A{beta}-peptides that substantially increases A{beta}{sub 42} production has been associated with the formation of amyloid plaques in Alzheimer's disease patients. Biophysical and genetic studies indicate that presenilin-1 (Psn-1), which contains the proteolytic active site, and three other membrane proteins, nicastrin (Nct), APH-1, and PEN-2 are required to form the core of the active {gamma}-secretase complex. Here, we report the purification of the native {gamma}-secretase complexes from HeLa cell membranes and the identification of an additional {gamma}-secretase complex subunit, CD147, a transmembrane glycoprotein with two immunoglobulin-like domains. The presence of this subunit as an integral part of the complex itself was confirmed through co-immunoprecipitation studies of the purified protein from HeLa cells and solubilized complexes from other cell lines such as neural cell HCN-1A and HEK293. Depletion of CD147 by RNA interference was found to increase the production of A{beta} peptides without changing the expression level of the other {gamma}-secretase components or APP substrates while CD147 overexpression had no statistically significant effect on amyloid {beta}-peptide production, other {gamma}-secretase components or APP substrates, indicating that the presence of the CD147 subunit within the {gamma}-secretase complex directly down-modulates the production of A{beta}-peptides. {gamma}-secretase was first recognized through its role in the production of the A{beta} peptides that are pathogenic in Alzheimer's disease (AD) (1). {gamma}-secretase is a membrane protein complex with unusual aspartyl protease activity that cleaves a variety of type I membrane proteins

Isolation and characterization of BetaM protein encoded by ATP1B4 - a unique member of the Na,K-ATPase {beta}-subunit gene family

Energy Technology Data Exchange (ETDEWEB)

Pestov, Nikolay B. [Department of Physiology and Pharmacology, University of Toledo College of Medicine, 3000 Arlington Ave., Toledo, OH 43614 (United States); Shemyakin-Ovchinnikov Institute of Bioorganic Chemistry, Moscow 117997 (Russian Federation); Zhao, Hao [Department of Physiology and Pharmacology, University of Toledo College of Medicine, 3000 Arlington Ave., Toledo, OH 43614 (United States); Basrur, Venkatesha [Department of Pathology, University of Michigan Medical School, Ann Arbor, MI 48109 (United States); Modyanov, Nikolai N., E-mail: nikolai.modyanov@utoledo.edu [Department of Physiology and Pharmacology, University of Toledo College of Medicine, 3000 Arlington Ave., Toledo, OH 43614 (United States)

2011-09-09

Highlights: {yields} Structural properties of BetaM and Na,K-ATPase {beta}-subunits are sharply different. {yields} BetaM protein is concentrated in nuclear membrane of skeletal myocytes. {yields} BetaM does not associate with a Na,K-ATPase {alpha}-subunit in skeletal muscle. {yields} Polypeptide chain of the native BetaM is highly sensitive to endogenous proteases. {yields} BetaM in neonatal muscle is a product of alternative splice mRNA variant B. -- Abstract: ATP1B4 genes represent a rare instance of the orthologous gene co-option that radically changed functions of encoded BetaM proteins during vertebrate evolution. In lower vertebrates, this protein is a {beta}-subunit of Na,K-ATPase located in the cell membrane. In placental mammals, BetaM completely lost its ancestral role and through acquisition of two extended Glu-rich clusters into the N-terminal domain gained entirely new properties as a muscle-specific protein of the inner nuclear membrane possessing the ability to regulate gene expression. Strict temporal regulation of BetaM expression, which is the highest in late fetal and early postnatal myocytes, indicates that it plays an essential role in perinatal development. Here we report the first structural characterization of the native eutherian BetaM protein. It should be noted that, in contrast to structurally related Na,K-ATPase {beta}-subunits, the polypeptide chain of BetaM is highly sensitive to endogenous proteases that greatly complicated its isolation. Nevertheless, using a complex of protease inhibitors, a sample of authentic BetaM was isolated from pig neonatal skeletal muscle by a combination of ion-exchange and lectin-affinity chromatography followed by SDS-PAGE. Results of the analysis of the BetaM tryptic digest using MALDI-TOF and ESI-MS/MS mass spectrometry have demonstrated that native BetaM in neonatal skeletal muscle is a product of alternative splice mRNA variant B and comprised of 351 amino acid residues. Isolated BetaM protein was

Release of newly synthesized nucleoplasmic ribosomal subunits or their precursor particles from isolated nuclei of regenerating rat liver

Energy Technology Data Exchange (ETDEWEB)

Usami, K; Ogata, K [Niigata Univ. (Japan). School of Medicine

1930-06-16

The authors present the time course of the labeling of RNA and protein moieties of these particles in vivo as well as the pattern of one-dimensional acrylamide gel electrophoresis of their protein moieties labeled with (/sup 35/S)methionine in vivo, which shows that released 60 S particles are newly synthesized ribosomal large subunits or their precursor particles in the nucleoplasm on their way from the nucleolus to the cytoplasm. It appears likely that released 40 S particles contain newly synthesized ribosomal small subunits or their precursors in the nucleoplasm.

Cortisone Dissociates the Shaker Family K Channels from their Beta Subunit

Energy Technology Data Exchange (ETDEWEB)

Pan, Y.; Weng, J; Kabaleeswaran, V; Li, H; Cao, Y; Bholse, R; Zhou, M

2008-01-01

The Shaker family voltage-dependent potassium channels (Kv1) are expressed in a wide variety of cells and are essential for cellular excitability. In humans, loss-of-function mutations of Kv1 channels lead to hyperexcitability and are directly linked to episodic ataxia and atrial fibrillation. All Kv1 channels assemble with {Beta} subunits (Kv{Beta}s), and certain Kv{Beta}s, for example Kv{Beta}1, have an N-terminal segment that closes the channel by the N-type inactivation mechanism. In principle, dissociation of Kv{Beta}1, although never reported, should eliminate inactivation and thus potentiate Kv1 current. We found that cortisone increases rat Kv1 channel activity by binding to Kv{Beta}1. A crystal structure of the K{Beta}v-cortisone complex was solved to 1.82-{angstrom}resolution and revealed novel cortisone binding sites. Further studies demonstrated that cortisone promotes dissociation of Kv{Beta}. The new mode of channel modulation may be explored by native or synthetic ligands to fine-tune cellular excitability.

Beta-secretase-cleaved amyloid precursor protein in Alzheimer brain: a morphologic study

DEFF Research Database (Denmark)

Sennvik, Kristina; Bogdanovic, N; Volkmann, Inga

2004-01-01

beta-amyloid (Abeta) is the main constituent of senile plaques seen in Alzheimer's disease. Abeta is derived from the amyloid precursor protein (APP) via proteolytic cleavage by proteases beta- and gamma-secretase. In this study, we examined content and localization of beta-secretase-cleaved APP...... the beta-sAPP immunostaining to be stronger and more extensive in gray matter in Alzheimer disease (AD) cases than controls. The axonal beta-sAPP staining was patchy and unevenly distributed for the AD cases, indicating impaired axonal transport. beta-sAPP was also found surrounding senile plaques...

Increased expression of the auxiliary beta(2-subunit of ventricular L-type Ca(2+ channels leads to single-channel activity characteristic of heart failure.

Directory of Open Access Journals (Sweden)

Roger Hullin

2007-03-01

Full Text Available Increased activity of single ventricular L-type Ca(2+-channels (L-VDCC is a hallmark in human heart failure. Recent findings suggest differential modulation by several auxiliary beta-subunits as a possible explanation.By molecular and functional analyses of human and murine ventricles, we find that enhanced L-VDCC activity is accompanied by altered expression pattern of auxiliary L-VDCC beta-subunit gene products. In HEK293-cells we show differential modulation of single L-VDCC activity by coexpression of several human cardiac beta-subunits: Unlike beta(1 or beta(3 isoforms, beta(2a and beta(2b induce a high-activity channel behavior typical of failing myocytes. In accordance, beta(2-subunit mRNA and protein are up-regulated in failing human myocardium. In a model of heart failure we find that mice overexpressing the human cardiac Ca(V1.2 also reveal increased single-channel activity and sarcolemmal beta(2 expression when entering into the maladaptive stage of heart failure. Interestingly, these animals, when still young and non-failing ("Adaptive Phase", reveal the opposite phenotype, viz: reduced single-channel activity accompanied by lowered beta(2 expression. Additional evidence for the cause-effect relationship between beta(2-subunit expression and single L-VDCC activity is provided by newly engineered, double-transgenic mice bearing both constitutive Ca(V1.2 and inducible beta(2 cardiac overexpression. Here in non-failing hearts induction of beta(2-subunit overexpression mimicked the increase of single L-VDCC activity observed in murine and human chronic heart failure.Our study presents evidence of the pathobiochemical relevance of beta(2-subunits for the electrophysiological phenotype of cardiac L-VDCC and thus provides an explanation for the single L-VDCC gating observed in human and murine heart failure.

Purification of the alpha and beta subunits of phosphorylase kinase for structural studies

International Nuclear Information System (INIS)

Sotiroudis, T.G.; Heilmeyer, L.M.G. Jr.; Crabb, J.W.

1987-01-01

Structural analysis of the alpha (Mr, 132,000) and beta (Mr, 113,000) subunits of phosphorylase kinase may provide clues to their yet unknown functions however purification remains problematic. Preparative RP-HPLC procedures yield inconveniently large, dilute solutions and concentration steps are required prior to subunit modification and fragmentation. Concentration of the β subunit usually results in significant losses due to insolubility. Using preparative SDS-polyacrylamide gel electrophoresis, they have purified the α, 7 , and β subunits from rabbit muscle phosphorylase kinase in a soluble and concentrated form suitable for structural studies. Phosphorylase kinase labelled with fluorescein isothiocyanate in the α and α' subunits and fully 14 C-S-carboxymethylated was fractionated on a 5% acrylamide Laemmli slab gel. The subunit bands were visualized by fluorescence and by SDS precipitation then excised and electroeluted in the presence of SDS using an ELUTRAP device. From 4.5 mg of enzyme applied to a 4.5 mm thick gel about 70% of the α subunit and about 90% of the β subunit were typically recovered in less than 1 ml with overnight elution

Isolation and characterization of recombinant human casein kinase II subunits alpha and beta from bacteria

DEFF Research Database (Denmark)

Grankowski, N; Boldyreff, B; Issinger, O G

1991-01-01

cDNA encoding the casein kinase II (CKII) subunits alpha and beta of human origin were expressed in Escherichia coli using expression vector pT7-7. Significant expression was obtained with E. coli BL21(DE3). The CKII subunits accounted for approximately 30% of the bacterial protein; however, most...

Synthesis of beta alumina from aluminum hydroxide and oxyhydroxide precursors

CSIR Research Space (South Africa)

Van Zyl, A

1993-02-01

Full Text Available Two aluminium oxyhydroxides, boehmite and pseudoboehmite, and two aluminium hydroxides, bayerite and gibbsite, have been investigated as precursors for the synthesis of the solid electrolyte, beta alumina. Reaction pathways and products have been...

CK2(beta)tes gene encodes a testis-specific isoform of the regulatory subunit of casein kinase 2 in Drosophila melanogaster

DEFF Research Database (Denmark)

Kalmykova, Alla I; Shevelyov, Yuri Y; Polesskaya, Oksana O

2002-01-01

An earlier described CK2(beta)tes gene of Drosophila melanogaster is shown to encode a male germline specific isoform of regulatory beta subunit of casein kinase 2. Western-analysis using anti-CK2(beta)tes Ig revealed CK2(beta)tes protein in Drosophila testes extract. Expression of a CK2(beta...... and coimmunoprecipitation analysis of protein extract from Drosophila testes, we demonstrated an association between CK2(beta)tes and CK2alpha. Northern-analysis has shown that another regulatory (beta') subunit found recently in D. melanogaster genome is also testis-specific. Thus, we describe the first example of two...

[beta]-hexosaminidase isozymes from cells cotransfected with [alpha] and [beta] cDNA constructs: Analysis of the [alpha]-subunit missense mutation associated with the adult form of Tay-Sachs disease

Energy Technology Data Exchange (ETDEWEB)

Brown, C.A.; Mahuran, D.J. (Univ. of Toronto (Canada))

1993-08-01

In vitro mutagenesis and transient expression in COS cells has been used to associate a missense mutation with a clinical or biochemical phenotype. Mutations affecting the [alpha]-subunit of [beta]-hexosaminidase A ([alpha][beta]) (E.C.3.2.1.52) result in Tay-Sachs disease. Because hexosaminidase A is heterodimeric, analysis of [alpha]-chain mutations is not straightforward. The authors examine three approaches utilizing previously identified mutations affecting [alpha]-chain folding. These involve transfection of (1) the [alpha] cDNA alone; (2) a [beta] cDNA construct encoding a [beta]-subunit substituted at a position homologous to that of the [alpha]-subunit, and (3) both [alpha] and [beta] cDNAs. The latter two procedures amplified residual activity levels over that of patient samples, an effect not previously found with mutations affecting an [open quotes]active[close quotes] [alpha]Arg residue. This effect may help to discriminate between protein-folding and active-site mutations. The authors conclude that, with proper controls, the latter method of cotransfection can be used to evaluate the effects and perhaps to predict the clinical course of some [alpha]-chain mutations. Using this technique, they demonstrate that the adult-onset Tay-Sachs mutation, [alpha]Gly[yields]Ser[sup 269], does not directly affect [alpha][beta] dimerization but exerts an indirect effect on the dimer through destabilizing the folded [alpha]-subunit at physiological temperatures. Two other [alpha] mutations linked to more severe phenotypes appear to inhibit the initial folding of the subunit. 36 refs., 2 figs., 5 tabs.

Spectral and Temporal Properties of the Alpha and Beta Subunits and (alpha Beta) Monomer Isolated from Nostoc SP. Using Picosecond Laser Spectroscopy.

Science.gov (United States)

Dagen, Aaron J.

1985-12-01

The fluorescence decay profiles, relative quantum yield and transmission of the (alpha), (beta) and ((alpha)(beta)) complexes from phycoerythrin isolated from the photosynthetic antenna system of Nostoc sp. and measured by single picosecond laser spectroscopic techniques is studied. The fluorescence decay profiles of all three complexes are found to be intensity independent for the intensity range investigated ((TURN)4 x 10('13) to (TURN)4 x 10('15) photons-cm('-2) per pulse). The apparent decrease in the relative quantum yield of all three complexes as intensity increases is offset by a corresponding increase in the relative transmission. This evidence, along with the intensity independent fluorescence kinetics, suggests that exciton annihilation is absent in these complexes. The decay profiles are fit to models assuming energy transfer amongst fluorescing chromophores. The intraprotein transfer rate is found to be 100 ps in the (alpha) subunit, 666 ps in the (beta) subunit. Constraining these rates to be identical in the monomer results in explaining the monomer kinetics by an increase in the nonradiative rate of the f(,(beta)) chromophore, an apparent result of aggregation effects.

Orientation of the calcium channel beta relative to the alpha(12.2 subunit is critical for its regulation of channel activity.

Directory of Open Access Journals (Sweden)

Iuliia Vitko

Full Text Available BACKGROUND: The Ca(vbeta subunits of high voltage-activated Ca(2+ channels control the trafficking and biophysical properties of the alpha(1 subunit. The Ca(vbeta-alpha(1 interaction site has been mapped by crystallographic studies. Nevertheless, how this interaction leads to channel regulation has not been determined. One hypothesis is that betas regulate channel gating by modulating movements of IS6. A key requirement for this direct-coupling model is that the linker connecting IS6 to the alpha-interaction domain (AID be a rigid structure. METHODOLOGY/PRINCIPAL FINDINGS: The present study tests this hypothesis by altering the flexibility and orientation of this region in alpha(12.2, then testing for Ca(vbeta regulation using whole cell patch clamp electrophysiology. Flexibility was induced by replacement of the middle six amino acids of the IS6-AID linker with glycine (PG6. This mutation abolished beta2a and beta3 subunits ability to shift the voltage dependence of activation and inactivation, and the ability of beta2a to produce non-inactivating currents. Orientation of Ca(vbeta with respect to alpha(12.2 was altered by deletion of 1, 2, or 3 amino acids from the IS6-AID linker (Bdel1, Bdel2, Bdel3, respectively. Again, the ability of Ca(vbeta subunits to regulate these biophysical properties were totally abolished in the Bdel1 and Bdel3 mutants. Functional regulation by Ca(vbeta subunits was rescued in the Bdel2 mutant, indicating that this part of the linker forms beta-sheet. The orientation of beta with respect to alpha was confirmed by the bimolecular fluorescence complementation assay. CONCLUSIONS/SIGNIFICANCE: These results show that the orientation of the Ca(vbeta subunit relative to the alpha(12.2 subunit is critical, and suggests additional points of contact between these subunits are required for Ca(vbeta to regulate channel activity.

Functional labeling of insulin receptor subunits in live cells. Alpha 2 beta 2 species is the major autophosphorylated form

International Nuclear Information System (INIS)

Le Marchand-Brustel, Y.; Ballotti, R.; Gremeaux, T.; Tanti, J.F.; Brandenburg, D.; Van Obberghen, E.

1989-01-01

Both receptor subunits were functionally labeled in order to provide methods allowing, in live cells and in broken cell systems, concomitant evaluation of the insulin receptor dual function, hormone binding, and kinase activity. In cell-free systems, insulin receptors were labeled on their alpha-subunit with 125I-photoreactive insulin, and on their beta-subunit by autophosphorylation. Thereafter, phosphorylated receptors were separated from the complete set of receptors by means of anti-phosphotyrosine antibodies. Using this approach, a subpopulation of receptors was found which had bound insulin, but which were not phosphorylated. Under nonreducing conditions, receptors appeared in three oligomeric species identified as alpha 2 beta 2, alpha 2 beta, and alpha 2. Mainly the alpha 2 beta 2 receptor species was found to be phosphorylated while insulin was bound to alpha 2 beta 2, alpha 2 beta, and alpha 2 forms. In live cells, biosynthetic labeling of insulin receptors was used. Receptors were first labeled with [35S]methionine. Subsequently, the addition of insulin led to receptor autophosphorylation by virtue of the endogenous ATP pool. The total amount of [35S]methionine-labeled receptors was precipitated with antireceptor antibodies, whereas with anti-phosphotyrosine antibodies, only the phosphorylated receptors were isolated. Using this approach we made the two following key findings: (1) Both receptor species, alpha 2 beta 2 and alpha 2 beta, are present in live cells and in comparable amounts. This indicates that the alpha 2 beta form is not a degradation product of the alpha 2 beta 2 form artificially generated during receptor preparation. (2) The alpha 2 beta 2 species is the prevalently autophosphorylated form

Thyroid hormone coordinately regulates Na sup + -K sup + -ATPase. alpha. - and. beta. -subunit mRNA levels in kidney

Energy Technology Data Exchange (ETDEWEB)

McDonough, A.A.; Brown, T.A.; Horowitz, B.; Chiu, R.; Schlotterbeck, J.; Bowen, J.; Schmitt, C.A. (Univ. of Southern California School of Medicine, Los Angeles (USA))

1988-02-01

Synthesis of the sodium pump, Na{sup +}-K{sup +}-ATPase, is regulated by thyroid hormone in responsive tissues. The purpose of this study was to determine if triiodothyronine (T{sub 3}) regulates the concentration of the mRNAs coding for the two enzyme subunits, {alpha} and {beta}, and the time course of the response. A single dose of T{sub 3} was administered to hypothyroid rats that were killed at various times after injection. In the kidney cortexes of the T{sub 3}-injected animals, as well as hypothyroid and euthyroid rats, {alpha}- and {beta}-mRNA concentrations were measured by dot blot using cDNAs corresponding to the two mRNAs; {alpha}-subunit abundance was measured by Western blot using antibodies to the enzyme, and Na{sup +}-K{sup +}-ATPase activity was measured enzymatically. {alpha}- and {beta}-mRNAs increased coordinately to 1.6-fold over hypothyroid levels by 12 h after T{sub 3}. The authors conclude that T{sub 3} regulates Na{sup +}-K{sup +}-ATPase synthesis and activity by coordinately increasing the mRNAs of both the {alpha}- and {beta}-subunits of the enzyme.

The AMP-activated protein kinase beta 1 subunit modulates erythrocyte integrity.

Science.gov (United States)

Cambridge, Emma L; McIntyre, Zoe; Clare, Simon; Arends, Mark J; Goulding, David; Isherwood, Christopher; Caetano, Susana S; Reviriego, Carmen Ballesteros; Swiatkowska, Agnieszka; Kane, Leanne; Harcourt, Katherine; Adams, David J; White, Jacqueline K; Speak, Anneliese O

2017-01-01

Failure to maintain a normal in vivo erythrocyte half-life results in the development of hemolytic anemia. Half-life is affected by numerous factors, including energy balance, electrolyte gradients, reactive oxygen species, and membrane plasticity. The heterotrimeric AMP-activated protein kinase (AMPK) is an evolutionarily conserved serine/threonine kinase that acts as a critical regulator of cellular energy balance. Previous roles for the alpha 1 and gamma 1 subunits in the control of erythrocyte survival have been reported. In the work described here, we studied the role of the beta 1 subunit in erythrocytes and observed microcytic anemia with compensatory extramedullary hematopoiesis together with splenomegaly and increased osmotic resistance. Copyright © 2016 ISEH - International Society for Experimental Hematology. Published by Elsevier Inc. All rights reserved.

Individual variation and hormonal modulation of a sodium channel beta subunit in the electric organ correlate with variation in a social signal.

Science.gov (United States)

Liu, He; Wu, Ming-Ming; Zakon, Harold H

2007-09-01

The sodium channel beta1 subunit affects sodium channel gating and surface density, but little is known about the factors that regulate beta1 expression or its participation in the fine control of cellular excitability. In this study we examined whether graded expression of the beta1 subunit contributes to the gradient in sodium current inactivation, which is tightly controlled and directly related to a social behavior, the electric organ discharge (EOD), in a weakly electric fish Sternopygus macrurus. We found the mRNA and protein levels of beta1 in the electric organ both correlate with EOD frequency. We identified a novel mRNA splice form of this gene and found the splicing preference for this novel splice form also correlates with EOD frequency. Androgen implants lowered EOD frequency and decreased the beta1 mRNA level but did not affect splicing. Coexpression of each splice form in Xenopus oocytes with either the human muscle sodium channel gene, hNav1.4, or a Sternopygus ortholog, smNav1.4b, sped the rate of inactivation of the sodium current and shifted the steady-state inactivation toward less negative membrane potentials. The translational product of the novel mRNA splice form lacks a previously identified important tyrosine residue but still functions normally. The properties of the fish alpha and coexpressed beta1 subunits in the oocyte replicate those of the electric organ's endogenous sodium current. These data highlight the role of ion channel beta subunits in regulating cellular excitability.

Curcumin decreases amyloid-beta peptide levels by attenuating the maturation of amyloid-beta precursor protein.

Science.gov (United States)

Zhang, Can; Browne, Andrew; Child, Daniel; Tanzi, Rudolph E

2010-09-10

Alzheimer disease (AD) is a devastating neurodegenerative disease with no cure. The pathogenesis of AD is believed to be driven primarily by amyloid-beta (Abeta), the principal component of senile plaques. Abeta is an approximately 4-kDa peptide generated via cleavage of the amyloid-beta precursor protein (APP). Curcumin is a compound in the widely used culinary spice, turmeric, which possesses potent and broad biological activities, including anti-inflammatory and antioxidant activities, chemopreventative effects, and effects on protein trafficking. Recent in vivo studies indicate that curcumin is able to reduce Abeta-related pathology in transgenic AD mouse models via unknown molecular mechanisms. Here, we investigated the effects of curcumin on Abeta levels and APP processing in various cell lines and mouse primary cortical neurons. We show for the first time that curcumin potently lowers Abeta levels by attenuating the maturation of APP in the secretory pathway. These data provide a mechanism of action for the ability of curcumin to attenuate amyloid-beta pathology.

Localization of pig Na[sup +], K[sup +]-ATPase [alpha] and [beta] subunit genes to chromosome 4 by radioactive in situ hybridization

Energy Technology Data Exchange (ETDEWEB)

Lahbib-Mansais, Y.; Yerle, M.; Dalens, M.; Chevalet, C.; Gellin, J. (Centre de Recherches de Toulouse (France))

1993-01-01

Two genes coding for Na[sup +],K[sup +] -ATPase [alpha] and [beta] subunits are localized on pig chromosome 4, to the q1.6[yields]q2.3 and 1.3[yields]q2.1 regions, respectively, by radioactive in situ hybridization. According to nucleotide and amino acid sequence comparisons with different human isoforms of Na[sup +] ,K[sup +]-ATPase, these pig [alpha] and [beta] ATPase genes show strong homologies with human [alpha]1 and [beta] subunit ATPase genes, respectively. These results are discussed with respect to comparative mapping data of conserved genes in mammalian species. We showed that the pig cDNA probes encoding ATPase [alpha] and, [beta] genes reveal DNA polymorphism in Meishan an Large White pigs. 35 refs., 4 figs., 2 tabs.

Mapping of the gene encoding the. beta. -amyloid precursor protein and its relationship to the Down syndrome region of chromosome 21

Energy Technology Data Exchange (ETDEWEB)

Patterson, D.; Gardiner, K.; Kao, F.T.; Tanzi, R.; Watkins, P.; Gusella, J.F. (Eleanor Roosevelt Institute for Cancer Research, Denver, CO (USA))

1988-11-01

The gene encoding the {beta}-amyloid precursor protein has been assigned to human chromosome 21, as has a gene responsible for at least some cases of familial Alzheimer disease. Linkage studies strongly suggest that the {beta}-amyloid precursor protein and the product corresponding to familial Alzheimer disease are from two genes, or at least that several million base pairs of DNA separate the markers. The precise location of the {beta}-amyloid precursor protein gene on chromosome 21 has not yet been determined. Here the authors show, by using a somatic-cell/hybrid-cell mapping panel, in situ hybridization, and transverse-alternating-field electrophoresis, that the {beta}-amyloid precursor protein gene is located on chromosome 21 very near the 21q21/21q/22 border and probably within the region of chromosome 21 that, when trisomic, results in Down syndrome.

Casein kinase 2 down-regulation and activation by polybasic peptides are mediated by acidic residues in the 55-64 region of the beta-subunit. A study with calmodulin as phosphorylatable substrate

DEFF Research Database (Denmark)

Meggio, F; Boldyreff, B; Issinger, O G

1994-01-01

to substitute for wild-type beta-subunit as a suppressor of activity toward calmodulin. The only mutations that reduced the ability of the beta-subunit to suppress calmodulin phosphorylation activity, though being compatible with normal reconstitution of CK2 holoenzyme, were those affecting Asp55, Glu57...... are conversely ineffective. The latent "calmodulin kinase" activity of CK2 can also be specifically unmasked by a peptide (alpha[66-86]) reproducing a basic insert of the catalytic subunit. This effect is reversed by equimolar addition of a peptide (beta[55-71]) including the 55-64 acidic stretch of the beta......-subunit. Comparable polylysine stimulation was observed with the holoenzymes reconstituted with either beta wt or the beta mutants capable of assembling with the alpha-subunit, with the notable exception of those bearing Ala substitutions for acidic residues at positions 55, 57, and 59-61. These were nearly...

(Na+ + K+)-ATPase and plasma membrane polarity of intestinal epithelial cells: Presence of a brush border antigen in the distal large intestine that is immunologically related to beta subunit

Energy Technology Data Exchange (ETDEWEB)

Marxer, A.; Stieger, B.; Quaroni, A.; Kashgarian, M.; Hauri, H.P. (Univ. of Basel (Switzerland))

1989-09-01

The previously produced monoclonal antibody IEC 1/48 against cultured rat intestinal crypt cells was extensively characterized and found to be directed against the beta subunit of (Na+ + K+)-ATPase as assessed by immunological and enzymatic criteria. Under nondenaturing conditions the antibody precipitated the alpha-beta enzyme complex (98,000 and 48,000 Mr). This probe, together with the monoclonal antibody C 62.4 against the alpha subunit was used to localize (Na+ + K+)-ATPase in epithelial cells along the rat intestinal tract by immunofluorescence and immunoelectron microscopy. Both antibodies exclusively labeled the basolateral membrane of small intestine and proximal colon epithelial cells. However, in the distal colon, IEC 1/48, but not C 62.4, also labeled the brush border membrane. The cross-reacting beta-subunit-like antigen on the apical cell pole was tightly associated with isolated brush borders but was apparently devoid of (Na+ + K+)-ATPase activity. Subcellular fractionation of colonocytes in conjunction with limited proteolysis and surface radioiodination of intestinal segments suggested that the cross-reacting antigen in the brush border may be very similar to the beta subunit. The results support the notion that in the small intestine and proximal colon the enzyme subunits are exclusively targeted to the basolateral membrane while in the distal colon nonassembled beta subunit or a beta-subunit-like protein is also transported to the apical cell pole.

The alpha3 laminin subunit, alpha6beta4 and alpha3beta1 integrin coordinately regulate wound healing in cultured epithelial cells and in the skin

DEFF Research Database (Denmark)

Goldfinger, L E; Hopkinson, S B; deHart, G W

1999-01-01

Previously, we demonstrated that proteolytic processing within the globular domain of the alpha3 subunit of laminin-5 (LN5) converts LN5 from a cell motility-inducing factor to a protein complex that can trigger the formation of hemidesmosomes, certain cell-matrix attachment sites found in epithe......-inhibiting antibodies, we provide evidence that LN5 and its two integrin receptors (alpha6beta4 and alpha3beta1) appear necessary for wound healing to occur in MCF-10A cell culture wounds. We propose a model for healing of wounded epithelial tissues based on these results....... in epithelial cells. We have prepared a monoclonal antibody (12C4) whose epitope is located toward the carboxy terminus of the globular domain of the alpha3 laminin subunit. This epitope is lost from the alpha3 subunit as a consequence of proteolytic processing. Antibody 12C4 stains throughout the matrix...... the wound site. A similar phenomenon is observed in human skin wounds, since we also detect expression of the unprocessed alpha3 laminin subunit at the leading tip of the sheet of epidermal cells that epithelializes skin wounds in vivo. In addition, using alpha3 laminin subunit and integrin function...

Teaching Glycoproteins with a Classical Paper: Knowledge and Methods in the Course of an Exciting Discovery--The story of Discovering HK-ATPase [Beta]-Subunit

Science.gov (United States)

Zhu, Lixin

2008-01-01

To integrate research into the teaching of glycoproteins, the story of discovering hydrogen-potassium ATPase (HK-ATPase) [beta] subunit is presented in a way covering all the important teaching points. The interaction between the HK-ATPase [alpha] subunit and a glycoprotein of 60-80 kDa was demonstrated to support the existence of the [beta]…

The crystal structure of the complex of Zea mays alpha subunit with a fragment of human beta subunit provides the clue to the architecture of protein kinase CK2 holoenzyme

DEFF Research Database (Denmark)

Battistutta, R; Sarno, S; De Moliner, E

2000-01-01

The crystal structure of a complex between the catalytic alpha subunit of Zea mays CK2 and a 23-mer peptide corresponding the C-terminal sequence 181-203 of the human CK2 regulatory beta subunit has been determined at 3.16-A resolution. The complex, composed of two alpha chains and two peptides, ...

A Trp474Cys mutation in the alpha-subunit of beta-hexosaminidase causes a subacute encephalopathic form of G{sub M2} gangliosidosis, type 1

Energy Technology Data Exchange (ETDEWEB)

Petroulakis, E.; Cao, Z.; Salo, T. [Univ. of Manitoba, Winnipeg (Canada)] [and others

1994-09-01

Mutations in the HEXA gene that encodes the {alpha}-subunit of the heterodimeric lysosomal enzyme {beta}-hexosaminidase A, or Hex A ({alpha}{beta}), cause G{sub M2} gangliosidosis, type 1. The infantile form (Tay-Sachs disease) results when there is no residual Hex A activity, while less severe and more variable clinical phenotypes result when residual Hex A activity is present. A non-Jewish male who presented with an acute psychotic episode at age 16 was diagnosed with a subacute encephalopathic form of G{sub M2} gangliosidosis. At age 19, chronic psychosis with intermittent acute exacerbations remains the most disabling symptom in this patient and his affected brother although both exhibit some ataxia and moderately severe dysarthria. We have found a 4 bp insertion (+TATC 1278) associated with infantile Tay-Sachs disease on one allele; no previously identified mutation was found on the second allele. SSCP analysis detected a shift in exon 13 and sequencing revealed a G1422C mutation in the second allele that results in a Trp474Cys substitution. The presence of the mutation was confirmed by the loss of HaeIII and ScrFI sites in exon 13 PCR products from the subjects and their father. The mutation was introduced into the {alpha}-subunit cDNA and Hex S ({alpha}{alpha}) and Hex A ({alpha}{beta}) were transiently expressed in monkey COS-7 cells. The Trp474Cys mutant protein had approximately 5% and 12% of wild-type Hex S and Hex A activity, respectively. Western blot analysis revealed a small amount of residual mature {alpha}-subunit and a normal level of precursor protein. We conclude that the Trp474Cys mutation is the cause of the Hex A deficiency associated with a subacute (juvenile-onset) phenotype in this patient. Like other mutations in exon 13 of HEXA, it appears to affect intracellular processing. Studies of the defect in intracellular processing are in progress.

Drosophila egghead encodes a beta 1,4-mannosyltransferase predicted to form the immediate precursor glycosphingolipid substrate for brainiac

DEFF Research Database (Denmark)

Wandall, Hans H; Pedersen, Johannes W; Park, Chaeho

2002-01-01

-N-acetylglucosamine:beta Man beta 1,3-N-acetylglucosaminyltransferase (beta 3GlcNAc-transferase) tentatively assigned a key role in biosynthesis of arthroseries glycosphingolipids and forming the trihexosylceramide, GlcNAc beta 1-3Man beta 1-4Glc beta 1-1Cer. In the present study we demonstrate that egghead encodes a Golgi......-located GDP-mannose:beta Glc beta 1,4-mannosyltransferase tentatively assigned a biosynthetic role to form the precursor arthroseries glycosphingolipid substrate for Brainiac, Man beta 1-4Glc beta 1-1Cer. Egghead is unique among eukaryotic glycosyltransferase genes in that homologous genes are limited...

Proteome analysis reveals phosphorylation of ATP synthase beta -subunit in human skeletal muscle and proteins with potential roles in type 2 diabetes

DEFF Research Database (Denmark)

Højlund, Kurt; Wrzesinski, Krzysztof; Larsen, Peter Mose

2003-01-01

quantitate a large number of proteins and their post-translational modifications simultaneously and is a powerful tool to study polygenic diseases like type 2 diabetes. Using this approach on human skeletal muscle biopsies, we have identified eight potential protein markers for type 2 diabetes in the fasting...... synthase beta-subunit phosphoisoform in diabetic muscle correlated inversely with fasting plasma glucose levels. These data suggest a role for phosphorylation of ATP synthase beta-subunit in the regulation of ATP synthesis and that alterations in the regulation of ATP synthesis and cellular stress proteins...

Interaction of the sliding clamp beta-subunit and Hda, a DnaA-related protein.

Science.gov (United States)

Kurz, Mareike; Dalrymple, Brian; Wijffels, Gene; Kongsuwan, Kritaya

2004-06-01

In Escherichia coli, interactions between the replication initiation protein DnaA, the beta subunit of DNA polymerase III (the sliding clamp protein), and Hda, the recently identified DnaA-related protein, are required to convert the active ATP-bound form of DnaA to an inactive ADP-bound form through the accelerated hydrolysis of ATP. This rapid hydrolysis of ATP is proposed to be the main mechanism that blocks multiple initiations during cell cycle and acts as a molecular switch from initiation to replication. However, the biochemical mechanism for this crucial step in DNA synthesis has not been resolved. Using purified Hda and beta proteins in a plate binding assay and Ni-nitrilotriacetic acid pulldown analysis, we show for the first time that Hda directly interacts with beta in vitro. A new beta-binding motif, a hexapeptide with the consensus sequence QL[SP]LPL, related to the previously identified beta-binding pentapeptide motif (QL[SD]LF) was found in the amino terminus of the Hda protein. Mutants of Hda with amino acid changes in the hexapeptide motif are severely defective in their ability to bind beta. A 10-amino-acid peptide containing the E. coli Hda beta-binding motif was shown to compete with Hda for binding to beta in an Hda-beta interaction assay. These results establish that the interaction of Hda with beta is mediated through the hexapeptide sequence. We propose that this interaction may be crucial to the events that lead to the inactivation of DnaA and the prevention of excess initiation of rounds of replication.

Isolation and characterization of BetaM protein encoded by ATP1B4 - a unique member of the Na,K-ATPase β-subunit gene family

International Nuclear Information System (INIS)

Pestov, Nikolay B.; Zhao, Hao; Basrur, Venkatesha; Modyanov, Nikolai N.

2011-01-01

Highlights: → Structural properties of BetaM and Na,K-ATPase β-subunits are sharply different. → BetaM protein is concentrated in nuclear membrane of skeletal myocytes. → BetaM does not associate with a Na,K-ATPase α-subunit in skeletal muscle. → Polypeptide chain of the native BetaM is highly sensitive to endogenous proteases. → BetaM in neonatal muscle is a product of alternative splice mRNA variant B. -- Abstract: ATP1B4 genes represent a rare instance of the orthologous gene co-option that radically changed functions of encoded BetaM proteins during vertebrate evolution. In lower vertebrates, this protein is a β-subunit of Na,K-ATPase located in the cell membrane. In placental mammals, BetaM completely lost its ancestral role and through acquisition of two extended Glu-rich clusters into the N-terminal domain gained entirely new properties as a muscle-specific protein of the inner nuclear membrane possessing the ability to regulate gene expression. Strict temporal regulation of BetaM expression, which is the highest in late fetal and early postnatal myocytes, indicates that it plays an essential role in perinatal development. Here we report the first structural characterization of the native eutherian BetaM protein. It should be noted that, in contrast to structurally related Na,K-ATPase β-subunits, the polypeptide chain of BetaM is highly sensitive to endogenous proteases that greatly complicated its isolation. Nevertheless, using a complex of protease inhibitors, a sample of authentic BetaM was isolated from pig neonatal skeletal muscle by a combination of ion-exchange and lectin-affinity chromatography followed by SDS-PAGE. Results of the analysis of the BetaM tryptic digest using MALDI-TOF and ESI-MS/MS mass spectrometry have demonstrated that native BetaM in neonatal skeletal muscle is a product of alternative splice mRNA variant B and comprised of 351 amino acid residues. Isolated BetaM protein was also characterized by SELDI-TOF mass

Calcium ionophore A23187 specifically decreases the secretion of beta-secretase cleaved amyloid precursor protein during apoptosis in primary rat cortical cultures

DEFF Research Database (Denmark)

Sennvik, K; Benedikz, Eirikur; Fastbom, J

2001-01-01

Alzheimer's disease (AD) is characterized by the degeneration and loss of neurons, intracellular neurofibrillary tangles and the accumulation of extracellular senile plaques consisting mainly of beta-amyloid (A beta). A beta is generated from the amyloid precursor protein (APP) by sequential beta...

Differential regulation of thyrotropin subunit apoprotein and carbohydrate biosynthesis by thyroid hormone

International Nuclear Information System (INIS)

Taylor, T.; Weintraub, B.D.

1985-01-01

The regulation of TSH apoprotein and carbohydrate biosynthesis by thyroid hormone was studied by incubating pituitaries from normal and hypothyroid (3 weeks post-thyroidectomy) rats in medium containing [ 14 C]alanine and [ 3 H] glucosamine. After 6 h, samples were sequentially treated with anti-TSH beta to precipitate TSH and free TSH beta, anti-LH beta to clear the sample of LH and free LH beta, then anti-LH alpha to precipitate free alpha-subunit. Total proteins were acid precipitated. All precipitates were subjected to electrophoresis on sodium dodecyl sulfate-polyacrylamide gels, which were then sliced and assayed by scintillation spectrometry. In hypothyroid pituitaries plus medium, [ 14 C]alanine incorporation in combined and free beta-subunits was 26 times normal and considerably greater than the 3.4-fold increase seen in total protein; combined and free alpha-subunits showed no specific increase in apoprotein synthesis. [ 3 H]Glucosamine incorporation in combined alpha- and beta-subunits in hypothyroid samples was 13 and 21 times normal, respectively, and was greater than the 1.9-fold increase in total protein; free alpha-subunit showed no specific increase in carbohydrate synthesis. The glucosamine to alanine ratio, reflecting relative glycosylation of newly synthesized molecules, was increased in hypothyroidism for combined alpha-subunits, but not for combined beta-subunits, free alpha-subunits, or total proteins. In summary, short term hypothyroidism selectively stimulated TSH beta apoprotein synthesis and carbohydrate synthesis of combined alpha- and beta-subunits. Hypothyroidism also increased the relative glycosylation of combined alpha-subunit. Thus, thyroid hormone deficiency appears to alter the rate-limiting step in TSH assembly (i.e. beta-subunit synthesis) as well as the carbohydrate structure of TSH, which may play important roles in its biological function

Ser2 is the autophosphorylation site in the beta subunit from bicistronically expressed human casein kinase-2 and from native rat liver casein kinase-2 beta

DEFF Research Database (Denmark)

Boldyreff, B; James, P; Staudenmann, W

1993-01-01

Human casein kinase-2 (CK-2) subunits alpha and beta were bicistronically expressed in bacteria. The recombinant holoenzyme shared all investigated properties with the native CK-2 from mammalian sources (rat liver, Krebs II mouse ascites tumour cells). Contrary to recombinant human CK-2 produced...

Integrins beta 5, beta 3 and alpha v are apically distributed in endometrial epithelium.

Science.gov (United States)

Aplin, J D; Spanswick, C; Behzad, F; Kimber, S J; Vićovac, L

1996-07-01

Several adhesion molecules have been shown to occur at the surface of endometrial cells. One of these is the integrin alpha v subunit which associates with various beta chains including beta 5. We demonstrate the presence of integrin beta 5 polypeptide in human endometrial epithelial cells throughout the menstrual cycle using immunocytochemistry with monospecific antibodies, and at the mRNA level by thermal amplification from endometrial cDNA. Integrin beta 5 is also found in a population of bone marrow-derived cells. A notable feature of the distribution of the beta 5 subunit in the glandular and luminal epithelium is its apical localization, which may suggest an involvement in implantation. However, no evidence was found for regulated expression of epithelial beta 5. In mouse, the beta 5 subunit is found at both the apical and basal surface of epithelial cells and expression is essentially oestrous cycle-independent. Comparisons are made in both species with the distribution of the alpha v and beta 3 subunits which also localize to the apical epithelium.

First demonstration of cerebrospinal fluid and plasma A beta lowering with oral administration of a beta-site amyloid precursor protein-cleaving enzyme 1 inhibitor in nonhuman primates.

Science.gov (United States)

Sankaranarayanan, Sethu; Holahan, Marie A; Colussi, Dennis; Crouthamel, Ming-Chih; Devanarayan, Viswanath; Ellis, Joan; Espeseth, Amy; Gates, Adam T; Graham, Samuel L; Gregro, Allison R; Hazuda, Daria; Hochman, Jerome H; Holloway, Katharine; Jin, Lixia; Kahana, Jason; Lai, Ming-tain; Lineberger, Janet; McGaughey, Georgia; Moore, Keith P; Nantermet, Philippe; Pietrak, Beth; Price, Eric A; Rajapakse, Hemaka; Stauffer, Shaun; Steinbeiser, Melissa A; Seabrook, Guy; Selnick, Harold G; Shi, Xiao-Ping; Stanton, Matthew G; Swestock, John; Tugusheva, Katherine; Tyler, Keala X; Vacca, Joseph P; Wong, Jacky; Wu, Guoxin; Xu, Min; Cook, Jacquelynn J; Simon, Adam J

2009-01-01

beta-Site amyloid precursor protein (APP)-cleaving enzyme (BACE) 1 cleavage of amyloid precursor protein is an essential step in the generation of the potentially neurotoxic and amyloidogenic A beta 42 peptides in Alzheimer's disease. Although previous mouse studies have shown brain A beta lowering after BACE1 inhibition, extension of such studies to nonhuman primates or man was precluded by poor potency, brain penetration, and pharmacokinetics of available inhibitors. In this study, a novel tertiary carbinamine BACE1 inhibitor, tertiary carbinamine (TC)-1, was assessed in a unique cisterna magna ported rhesus monkey model, where the temporal dynamics of A beta in cerebrospinal fluid (CSF) and plasma could be evaluated. TC-1, a potent inhibitor (IC(50) approximately 0.4 nM), has excellent passive membrane permeability, low susceptibility to P-glycoprotein transport, and lowered brain A beta levels in a mouse model. Intravenous infusion of TC-1 led to a significant but transient lowering of CSF and plasma A beta levels in conscious rhesus monkeys because it underwent CYP3A4-mediated metabolism. Oral codosing of TC-1 with ritonavir, a potent CYP3A4 inhibitor, twice daily over 3.5 days in rhesus monkeys led to sustained plasma TC-1 exposure and a significant and sustained reduction in CSF sAPP beta, A beta 40, A beta 42, and plasma A beta 40 levels. CSF A beta 42 lowering showed an EC(50) of approximately 20 nM with respect to the CSF [TC-1] levels, demonstrating excellent concordance with its potency in a cell-based assay. These results demonstrate the first in vivo proof of concept of CSF A beta lowering after oral administration of a BACE1 inhibitor in a nonhuman primate.

Expression of alpha and beta subunit isoforms of Na,K-ATPase in the mouse inner ear and changes with mutations at the Wv or Sld loci.

Science.gov (United States)

Schulte, B A; Steel, K P

1994-07-01

Mice homozygous for mutations at the viable dominant spotting (Wv) and Steel-dickie (Sld) loci exhibit a similar phenotype which includes deafness. The auditory dysfunction derives from failure of the stria vascularis to develop normally and to generate a high positive endocochlear potential (EP). Because strial function is driven by Na,K-ATPase its expression was investigated in inner ears of Wv/Wv and Sld/Sld mice and their wild-type littermates by immunostaining with antisera against four of the enzyme's subunit isoforms. Wild-type mice from two different genetic backgrounds showed an identical distribution of subunit isoforms among inner ear transport cells. Several epithelial cell types coexpressed the alpha 1 and beta 1 subunits. Vestibular dark cells showed no reactivity for beta 1 but expressed abundant beta 2, whereas, strial marginal cells stained strongly for both beta isoforms. The only qualitative difference between mutant and wild-type mice was the absence of beta 1 subunit in marginal cells of the mutant's stria. However, it is unlikely that this difference accounts for failure of mutants to generate a high EP because the beta 1 subunit is not present in the stria vascularis of either rats or gerbils with normal EP values. Strong immunostaining for Na,K-ATPase in lateral wall fibrocytes of normal mice along with diminished immunoreactivity in the mutants supports the concept that these strategically located transport fibrocytes actively resorb K+ leaked across Reissner's membrane into scala vestibuli or effluxed from hair cells and nerves into scala tympani. It is further speculated that the resorbed K+ normally is siphoned down its concentration gradient into the intrastrial space through gap junctions between fibrocytes and strial basal and intermediate cells where it is recycled back to endolymph via marginal cells. Thus, failure of mutants to generate a positive EP could be explained by the absence of intermediate cells which may form the final

Highly conserved small subunit residues influence rubisco large subunit catalysis.

Science.gov (United States)

Genkov, Todor; Spreitzer, Robert J

2009-10-30

The chloroplast enzyme ribulose 1,5-bisphosphate carboxylase/oxygenase (Rubisco) catalyzes the rate-limiting step of photosynthetic CO(2) fixation. With a deeper understanding of its structure-function relationships and competitive inhibition by O(2), it may be possible to engineer an increase in agricultural productivity and renewable energy. The chloroplast-encoded large subunits form the active site, but the nuclear-encoded small subunits can also influence catalytic efficiency and CO(2)/O(2) specificity. To further define the role of the small subunit in Rubisco function, the 10 most conserved residues in all small subunits were substituted with alanine by transformation of a Chlamydomonas reinhardtii mutant that lacks the small subunit gene family. All the mutant strains were able to grow photosynthetically, indicating that none of the residues is essential for function. Three of the substitutions have little or no effect (S16A, P19A, and E92A), one primarily affects holoenzyme stability (L18A), and the remainder affect catalysis with or without some level of associated structural instability (Y32A, E43A, W73A, L78A, P79A, and F81A). Y32A and E43A cause decreases in CO(2)/O(2) specificity. Based on the x-ray crystal structure of Chlamydomonas Rubisco, all but one (Glu-92) of the conserved residues are in contact with large subunits and cluster near the amino- or carboxyl-terminal ends of large subunit alpha-helix 8, which is a structural element of the alpha/beta-barrel active site. Small subunit residues Glu-43 and Trp-73 identify a possible structural connection between active site alpha-helix 8 and the highly variable small subunit loop between beta-strands A and B, which can also influence Rubisco CO(2)/O(2) specificity.

The regulatory beta-subunit of protein kinase CK2 regulates cell-cycle progression at the onset of mitosis

DEFF Research Database (Denmark)

Yde, C W; Olsen, B B; Meek, D

2008-01-01

25 dual-specificity phosphatase family members. In somatic cells, Wee1 is downregulated by phosphorylation and ubiquitin-mediated degradation to ensure rapid activation of CDK1 at the beginning of M phase. Here, we show that downregulation of the regulatory beta-subunit of protein kinase CK2 by RNA...

Alternative-splicing in the exon-10 region of GABA(A receptor beta(2 subunit gene: relationships between novel isoforms and psychotic disorders.

Directory of Open Access Journals (Sweden)

Cunyou Zhao

Full Text Available BACKGROUND: Non-coding single nucleotide polymorphisms (SNPs in GABRB2, the gene for beta(2-subunit of gamma-aminobutyric acid type A (GABA(A receptor, have been associated with schizophrenia (SCZ and quantitatively correlated to mRNA expression and alternative splicing. METHODS AND FINDINGS: Expression of the Exon 10 region of GABRB2 from minigene constructs revealed this region to be an "alternative splicing hotspot" that readily gave rise to differently spliced isoforms depending on intron sequences. This led to a search in human brain cDNA libraries, and the discovery of two novel isoforms, beta(2S1 and beta(2S2, bearing variations in the neighborhood of Exon-10. Quantitative real-time PCR analysis of postmortem brain samples showed increased beta(2S1 expression and decreased beta(2S2 expression in both SCZ and bipolar disorder (BPD compared to controls. Disease-control differences were significantly correlated with SNP rs187269 in BPD males for both beta(2S1 and beta(2S2 expressions, and significantly correlated with SNPs rs2546620 and rs187269 in SCZ males for beta(2S2 expression. Moreover, site-directed mutagenesis indicated that Thr(365, a potential phosphorylation site in Exon-10, played a key role in determining the time profile of the ATP-dependent electrophysiological current run-down. CONCLUSION: This study therefore provided experimental evidence for the importance of non-coding sequences in the Exon-10 region in GABRB2 with respect to beta(2-subunit splicing diversity and the etiologies of SCZ and BPD.

The beta subunit modulates bypass and termination at UV lesions during in vitro replication with DNA polymerase III holoenzyme of Escherichia coli

International Nuclear Information System (INIS)

Shavitt, O.; Livneh, Z.

1989-01-01

The cycling time of DNA polymerase III holoenzyme during replication of UV-irradiated single-stranded (ss) DNA was longer than with unirradiated DNA (8 versus 3 min, respectively), most likely due to slow dissociation from lesion-terminated nascent DNA strands. Initiation of elongation on primed ssDNA was not significantly inhibited by the presence of UV lesions as indicated by the identical distribution of replication products synthesized at early and late reaction times and by the identical duration of the initial synthesis bursts on both unirradiated and UV-irradiated DNA templates. When replication was performed with DNA polymerase III* supplemented with increasing quantities of purified beta 2 subunit, the cycling time on UV-irradiated DNA decreased from 14.8 min at 1.7 nM beta 2 down to 6 min at 170 nM beta 2, a concentration in which beta 2 was in large excess over the polymerase. In parallel to the reduction in cycling time, also the bypass frequency of cyclobutane-photodimers decreased with increasing beta 2 concentration, and at 170 nM beta 2, bypass of photodimers was essentially eliminated. It has been shown that polymerase complexes with more than one beta 2 per polymerase molecule were formed at high beta 2 concentrations. It is plausible that polymerase complexes obtained under high beta 2 concentration dissociate from lesion-terminated primers faster than polymerase complexes formed at a low beta 2 concentration. This is expected to favor termination over bypass at pyrimidine photodimers and thus decrease their bypass frequency. These results suggest that the beta 2 subunit might act as a sensor for obstacles to replication caused by DNA damage, and that it terminates elongation at these sites by promoting dissociation. The intracellular concentration of beta 2 was estimated to be 250 nM

Concentration of carcinoembryonic antigen alpha-fetoprotein and beta-subunit of human chorionic gonadotropin in the serum of coke oven workers

Energy Technology Data Exchange (ETDEWEB)

Snit, M. [Silesian Medical Academy, Zabrze (Poland)

1993-01-01

Increased levels of carcinoembryonic antigen and {alpha}-fetoprotein were found in blood serum of coke oven workers, and also to some extent in smokers and in residents of industrial cities. The {beta} subunit of chorionic gonadotropin was barely detectable.

RFLPs for ATP1BL1 (. beta. subunit Na sup + /K sup + ATPase pseudogene) on chromosome 4

Energy Technology Data Exchange (ETDEWEB)

Georgiou, C.; Shull, M. (Univ. of Iowa Hospitals, Iowa City (USA)); Lingrel, J.B.; Murray, J.C.; Lane, L.K. (Univ. of Cincinnati College of Medicine, OH (USA))

1989-11-11

{beta}51-1(1.4) contains a 1.4kb EcoRI fragment, free of repetitive elements, from the {beta} subunit Na{sup +}/K{sup +} ATPase pseudogene (ATP1BL1). The vector is pUG18. EcoRI identifies 2 allelic bands of 7.0 and 14.0 kb. KpnI identifies 2 allelic bands of 19.0 and 23.0 kb. The probe was localized to chromosome 4 by linkage to chromosome 4 markers (D4S35, KIT) and somatic cell hybrid analysis. Co-dominant segregation was shown in 32 and 16 CEPH families for EcoRI and KpnI respectively.

Two ways of legumin-precursor processing in conifers. Characterization and evolutionary relationships of Metasequoia cDNAs representing two divergent legumin gene subfamilies.

Science.gov (United States)

Häger, K P; Wind, C

1997-06-15

Subunit monomers and oligomers of crystalloid-type legumins are major components of SDS-soluble fractions from Metasequoia glyptostroboides (Dawn redwood, Taxodiaceae) seed proteins. The subunits are made up of disulfide linked alpha-polypeptides and beta-polypeptides with molecular masses of 33 kDa and 23-25 kDa, respectively. Unusually for legumins, those from Metasequoia are glycosylated and the carbohydrate moieties are residing in the C-terminal region of the respective beta-polypeptides. A Metasequoia endosperm cDNA library has been constructed and legumin-encoding transcripts representing two divergent gene subfamilies have been characterized. Intersubfamily comparisons reveal 75% identity at the amino acid level and the values range from 53-35% when the legumin precursors deduced were compared with those from angiosperms. The predicted sequences together with data from amino acid sequencing prove that post-translational processing of Metasequoia prolegumins is directed to two different processing sites, each of them specific for one of the legumin subfamilies. The sites involved differ in their relative position and in the junction to be cleaved: Metasequoia legumin precursors MgLeg18 and MgLeg26 contain the conventional post-translational Asn-Gly processing site, which is generally regarded as highly conserved. In contrast, the MgLeg4 precursor is lacking this site and post-translational cleavage is directed to an unusual Asn-Thr processing site located in its hypervariable region, causing N-terminal extension of the beta-polypeptide relative to those hitherto known. Evidence is given that the unusual variant of processing also occurs in other conifers. Phylogenetic analysis reveals the precursors concerned as representatives of a distinct legumin subfamily, originating from duplication of an ancestral gene prior to or at the beginning of Taxodiaceae diversification.

Efficient autophosphorylation and phosphorylation of the beta-subunit by casein kinase-2 require the integrity of an acidic cluster 50 residues downstream from the phosphoacceptor site

DEFF Research Database (Denmark)

Boldyreff, B; Meggio, F; Pinna, L A

1994-01-01

Various beta-mutants were investigated either as subunits or as substrates for casein kinase 2 (CK-2), in the absence of presence of polylysine. A total of 21 beta-mutants were characterized for their susceptibility to autophosphorylation, by combining them in equimolar amounts with the recombina...

Subunits of the Snf1 kinase heterotrimer show interdependence for association and activity.

Science.gov (United States)

Elbing, Karin; Rubenstein, Eric M; McCartney, Rhonda R; Schmidt, Martin C

2006-09-08

The Snf1 kinase and its mammalian orthologue, the AMP-activated protein kinase (AMPK), function as heterotrimers composed of a catalytic alpha-subunit and two non-catalytic subunits, beta and gamma. The beta-subunit is thought to hold the complex together and control subcellular localization whereas the gamma-subunit plays a regulatory role by binding to and blocking the function of an auto-inhibitory domain (AID) present in the alpha-subunit. In addition, catalytic activity requires phosphorylation by a distinct upstream kinase. In yeast, any one of three Snf1-activating kinases, Sak1, Tos3, or Elm1, can fulfill this role. We have previously shown that Sak1 is the only Snf1-activating kinase that forms a stable complex with Snf1. Here we show that the formation of the Sak1.Snf1 complex requires the beta- and gamma-subunits in vivo. However, formation of the Sak1.Snf1 complex is not necessary for glucose-regulated phosphorylation of the Snf1 activation loop. Snf1 kinase purified from cells lacking the beta-subunits do not contain any gamma-subunit, indicating that the Snf1 kinase does not form a stable alphagamma dimer in vivo. In vitro kinase assays using purified full-length and truncated Snf1 proteins demonstrate that the kinase domain, which lacks the AID, is significantly more active than the full-length Snf1 protein. Addition of purified beta- and gamma-subunits could stimulate the kinase activity of the full-length alpha-subunit but only when all three subunits were present, suggesting an interdependence of all three subunits for assembly of a functional complex.

In cellulo examination of a beta-alpha hybrid construct of beta-hexosaminidase A subunits, reported to interact with the GM2 activator protein and hydrolyze GM2 ganglioside.

Directory of Open Access Journals (Sweden)

Incilay Sinici

Full Text Available The hydrolysis in lysosomes of GM2 ganglioside to GM3 ganglioside requires the correct synthesis, intracellular assembly and transport of three separate gene products; i.e., the alpha and beta subunits of heterodimeric beta-hexosaminidase A, E.C. # 3.2.1.52 (encoded by the HEXA and HEXB genes, respectively, and the GM2-activator protein (GM2AP, encoded by the GM2A gene. Mutations in any one of these genes can result in one of three neurodegenerative diseases collectively known as GM2 gangliosidosis (HEXA, Tay-Sachs disease, MIM # 272800; HEXB, Sandhoff disease, MIM # 268800; and GM2A, AB-variant form, MIM # 272750. Elements of both of the hexosaminidase A subunits are needed to productively interact with the GM2 ganglioside-GM2AP complex in the lysosome. Some of these elements have been predicted from the crystal structures of hexosaminidase and the activator. Recently a hybrid of the two subunits has been constructed and reported to be capable of forming homodimers that can perform this reaction in vivo, which could greatly simplify vector-mediated gene transfer approaches for Tay-Sachs or Sandhoff diseases. A cDNA encoding a hybrid hexosaminidase subunit capable of dimerizing and hydrolyzing GM2 ganglioside could be incorporated into a single vector, whereas packaging both subunits of hexosaminidase A into vectors, such as adeno-associated virus, would be impractical due to size constraints. In this report we examine the previously published hybrid construct (H1 and a new more extensive hybrid (H2, with our documented in cellulo (live cell- based assay utilizing a fluorescent GM2 ganglioside derivative. Unfortunately when Tay-Sachs cells were transfected with either the H1 or H2 hybrid construct and then were fed the GM2 derivative, no significant increase in its turnover was detected. In vitro assays with the isolated H1 or H2 homodimers confirmed that neither was capable of human GM2AP-dependent hydrolysis of GM2 ganglioside.

New insights into structure-function relationship of the DHPR beta1a subunit in skeletal muscle excitation-contraction coupling using zebrafish 'relaxed' as an expression system

International Nuclear Information System (INIS)

Dayal, A.

2010-01-01

The paralyzed zebrafish strain relaxed carries a null mutation for the skeletal muscle dihydropyridine receptor (DHPR) [beta]1a subunit. The lack of [beta]1a not only impedes functional [alpha]1S membrane expression but also precludes the skeletal muscle-specific ultrastructural arrangement of DHPRs into tetrads opposite ryanodine receptor (RyR1), coherent with the absence of skeletal muscle excitation-contraction (EC) coupling. With the plethora of experimental approaches feasible with zebrafish model organism and importantly with the [beta]1-null mutation having a monogenetic inheritance and because of the survival of the relaxed larvae for some days, we were able to establish the zebrafish relaxed as an expression system. Linking in vitro to in vivo observations, a clear differentiation between the major functional roles of [beta] subunits in EC coupling was feasible. The skeletal muscle [beta]1a subunit was able to restore all parameters of EC coupling upon expression in relaxed myotubes and larvae. Expression of the phylogenetically closest isoform to [beta]1a, the cardiac/neuronal [beta]2a subunit or the most distant neuronal [beta]M from the housefly in relaxed myotubes and larvae was likewise able to fully restore [alpha]1S triad targeting and facilitate charge movement. However, efficient tetrad formation and thus intact DHPR-RyR1 coupling was exclusively promoted by the [beta]1a isoform. Consequently, we postulated a model according to which [beta]1a acts as a unique allosteric modifier of [alpha]1S conformation crucial for skeletal muscle EC coupling. Therefore, unique structural elements in [beta]1a must be present which endow it with this exclusive property. Earlier, a unique hydrophobic heptad repeat motif (LVV) in the [beta]1a C-terminus was postulated by others to be essential for skeletal muscle EC coupling. We wanted to address the question if the proposed [beta]1a heptad repeat motif could be an active element of the DHPR-RyR1 signal transduction

Structural and optical characterization of Eu3+ doped beta-Ga2O3 nanoparticles using a liquid-phase precursor method.

Science.gov (United States)

Kim, Moung-O; Kang, Bongkyun; Yoon, Daeho

2013-08-01

Eu3+ doped beta-Ga2O3 and non-doped beta-Ga2O3 nanoparticles were synthesized at 800 degrees C using a liquid-phase precursor (LPP) method, with different annealing times and Eu3+ ion concentrations. Eu3+ doped beta-Ga2O3 nanoparticles showed broad XRD peaks, revealing a second phase compared with the non-doped beta-Ga2O3 nanoparticles. The cathode luminescence (CL) spectra of beta-Ga2O3 and Eu3+ doped beta-Ga2O3 nanoparticles showed a broad band emission (300-500 nm) of imperfection and two component emissions. The luminescence quenching properties of Eu3+ dopant ion concentration appeared gradually beyond 5 mol% in our investigation.

Expression of the benign HEXA mutations, Arg247Trp and Arg249Trp, associated with beta-hexosaminidase A pseudodeficiency

Energy Technology Data Exchange (ETDEWEB)

Cao, Z.; Petroulakis, E.; Salo, T. [Univ. of Manitoba (Canada)] [and others

1994-09-01

{beta}-Hexosaminidase (Hex A) is a heterodimer of {alpha} and {beta} subunits encoded by the HEXA and HEXB genes, respectively. Mutations in the HEXA gene typically cause Tay-Sachs disease or less severe forms of G{sub M2} gangliosidosis. However, two benign mutations (Arg247Trp and Arg249Trp) in the {alpha}-subunit of Hex A account for Hex A deficiency in {approximately}36% of non-Jewish enzyme-defined Tay-Sachs disease carriers. These mutations do not result in any apparent clinical phenotype in individuals who are genetic compounds with a second disease-causing mutation. We expressed the {alpha}-subunit harboring each of the benign mutations separately to study activity toward the synthetic substrate, 4-MUGS, for comparison to activity from enzymes containing mutations associated with other forms of G{sub M2} gangliosidosis. The C739T (Arg247Trp;benign), C745T (Arg 249Trp; benign), G805A (Gly269Ser; adult-onset), G749A (Gly250Asp; juvenile), and C508T (Arg170Trp; infantile) mutations were introduced into the {alpha}-subunit cDNA. These were transfected alone, or with the {beta}-subunit cDNA, to generate Hex S ({alpha}{alpha}) or Hex A ({alpha}{beta}), respectively. The activities were monitored using 4-MUGS, and the levels of {alpha}-subunit protein were assessed by Western blotting. Repeated experiments show that the benign mutations produce approximately 35% of normal Hex S and 40% of normal Hex A activity. This level is much higher than that of Hex A harbouring the Gly169Ser adult-onset mutation (12%). A sequential decrease in expressed Hex A activity is observed as mutations associated with more severe phenotypes are expressed. The benign mutations also result in lower levels of mature {alpha}-subunit protein compared to normal, and slightly reduced levels of {alpha}-subunit precursor protein. The Hex A deficiency resulting from benign mutations is not as great as that associated with disease-causing mutations.

scsB, a cDNA encoding the hydrogenosomal beta subunit of succinyl-CoA synthetase from the anaerobic fungus Neocallimastix frontalis

NARCIS (Netherlands)

Brondijk, THC; Durand, R; vanderGiezen, M; Gottschal, JC; Prins, RA; Fevre, M

1996-01-01

A clone containing a Neocallimastix frontalis cDNA assumed to encode the beta subunit of succinyl-CoA synthetase (SCSB) was identified by sequence homology with prokaryotic and eukaryotic counterparts. An open reading frame of 1311 bp was found. The deduced 437 amino acid sequence showed a high

Levels of alpha- and beta-secretase cleaved amyloid precursor protein in the cerebrospinal fluid of Alzheimer's disease patients

DEFF Research Database (Denmark)

Sennvik, K; Fastbom, J; Blomberg, M

2000-01-01

Alternative cleavage of the amyloid precursor protein (APP) results in generation and secretion of both soluble APP (sAPP) and beta-amyloid (Abeta). Abeta is the main component of the amyloid depositions in the brains of Alzheimer's disease (AD) patients. Using Western blotting, we compared...... the levels of alpha-secretase cleaved sAPP, beta-secretase cleaved sAPP and total sAPP, in cerebrospinal fluid (CSF) from 13 sporadic AD patients and 13 healthy controls. Our findings show significant amounts of beta-secretase cleaved sAPP in CSF. There was no statistically significant difference...... in the levels of beta-secretase cleaved sAPP between AD patients and controls. The levels of alpha-secretase cleaved sAPP and total sAPP were, however, found to be significantly lower in the AD patients than in the controls....

Functional isotypes are not encoded by the constant region genes of the beta subunit of the T cell receptor for antigen/major histocompatibility complex

OpenAIRE

1984-01-01

Human T cell clones and a cDNA probe specific for constant regions of the beta subunit of the antigen/major histocompatibility complex (MHC) receptor, TiC beta 1 and TiC beta 2, were employed to determine whether these genes were differentially used by functional classes of T lymphocytes. DNA from 10 interleukin-2-dependent T cell clones including class I and class II MHC-specific cytotoxic T lymphocytes (n = 6), T4+ inducer T lymphocytes (n = 2), and T8+ suppressor T lymphocytes (n = 2) show...

Synthesis and characterization of human recombinant thyrotropin (rec-hTSH) with a chimeric {beta}-subunit (rec-hTSH{beta}-CTPE hCG{beta}); Sintese e caracterizacao do hormonio tireotrofico humano recombinante (rec-hTSH) contendo uma subunidade {beta} quimerica (rec-hTSH{beta}-CTPE hCG{beta})

Energy Technology Data Exchange (ETDEWEB)

Murata, Yoko

1995-12-31

Recombinant hTSH is now successfully being used in clinical studies of thyroid cancer. Because of its therapeutic potential, we have constructed a longer acting analog of hTSH by fusing the carboxy terminal extension peptide (CTEP) of hCG{beta} onto hTSH{beta}. When coexpressed either with {alpha}-subunit complementary DNA or {alpha}-minigene in African green monkey (Cos-7) and human embryonic kidney (293) cells, the chimera was fully bioactive in vitro and exhibited enhanced in vivo potency associated with a prolonged plasma half-life. The addition of 29 amino acids with 4 O-linked oligosaccharide chains did not affect the assembly and secretion of chimeric TSH. Wild type (WT) and chimeric hTSH secreted by Cos-7 and 293 cells displayed wide differences in their plasma half-lives, presumably due to the difference in the terminal sialic acid and sulfate of their oligosaccharide chains. Chimeric and WT hTSH secreted by both cell lines demonstrated similar bioactivity in cAMP production, with some differences in [{sup 3} H]-thymidine incorporation. Chimeric hTSH secreted by Cos-7 appears to be more active than that secreted by 293 cells, as judged by growth assay. Cos-7 produced chimeric hTSH showed the maximum increase in half-life, indicating the importance of sialic acid in prolonging half-life and in vivo potency. Sulfation of both subunits, predominantly {beta} and to a lesser extent {alpha}, appears to be responsible, at least in part, for the increased metabolic clearance of WT and chimeric TSH secreted by 293 cells. Apart from its therapeutic potential, chimeric TSH produced in various cell lines can be used as a tool to delineate the roles of sulfate and sialic acid in the in vivo clearance and, thereby in the in vivo bioactivity. (author). 104 refs., 23 figs., 3 tabs.

Loss of function of ATXN1 increases amyloid beta-protein levels by potentiating beta-secretase processing of beta-amyloid precursor protein.

Science.gov (United States)

Zhang, Can; Browne, Andrew; Child, Daniel; Divito, Jason R; Stevenson, Jesse A; Tanzi, Rudolph E

2010-03-19

Alzheimer disease (AD) is a devastating neurodegenerative disease with complex and strong genetic inheritance. Four genes have been established to either cause familial early onset AD (APP, PSEN1, and PSEN2) or to increase susceptibility for late onset AD (APOE). To date approximately 80% of the late onset AD genetic variance remains elusive. Recently our genome-wide association screen identified four novel late onset AD candidate genes. Ataxin 1 (ATXN1) is one of these four AD candidate genes and has been indicated to be the disease gene for spinocerebellar ataxia type 1, which is also a neurodegenerative disease. Mounting evidence suggests that the excessive accumulation of Abeta, the proteolytic product of beta-amyloid precursor protein (APP), is the primary AD pathological event. In this study, we ask whether ATXN1 may lead to AD pathogenesis by affecting Abeta and APP processing utilizing RNA interference in a human neuronal cell model and mouse primary cortical neurons. We show that knock-down of ATXN1 significantly increases the levels of both Abeta40 and Abeta42. This effect could be rescued with concurrent overexpression of ATXN1. Moreover, overexpression of ATXN1 decreased Abeta levels. Regarding the underlying molecular mechanism, we show that the effect of ATXN1 expression on Abeta levels is modulated via beta-secretase cleavage of APP. Taken together, ATXN1 functions as a genetic risk modifier that contributes to AD pathogenesis through a loss-of-function mechanism by regulating beta-secretase cleavage of APP and Abeta levels.

The Val{sup 192}Leu mutation in the {alpha}-subunit of {beta}-hexosaminidase A is not associated with the B1-variant form of Tay-Sachs disease

Energy Technology Data Exchange (ETDEWEB)

Hou, Y.; Vavougios, G.; Hinek, A. [Univ. of Toronto (Canada)] [and others

1996-07-01

Substitution mutations adversely affecting the {alpha}-subunit of {beta}-hexosaminidase A ({alpha}{beta}) (EC 3.2.1.52) result in Tay-Sachs disease. The majority affect the initial folding of the pro-{alpha} chain in the endoplasmic reticulum, resulting in its retention and degradation. A much less common occurrence is a mutation that specifically affects an {open_quotes}active-site{close_quotes} residue necessary for substrate binding and/or catalysis. In this case, hexosaminidase A is present in the lysosome, but it lacks all {alpha}-specific activity. This biochemical phenotype is referred to as the {open_quotes}B1-variant form{close_quotes} of Tay-Sachs disease. Kinetic analysis of suspected B1-variant mutations is complex because hexosaminidase A is heterodimeric and both subunits possess similar active sites. In this report, we examine a previously identified B1-variant mutation, {alpha}-Val{sup 192}Leu. Chinese hamster ovary cells were permanently cotransfected with an {alpha}-cDNA-construct encoding the substitution and a mutant {beta}-cDNA ({beta}-Arg{sup 211}Lys), encoding a {beta}-subunit that is inactive but normal in all other respects. We were surprised to find that the Val{sup 192}Leu substitution produced a pro-{alpha} chain that did not form {alpha}-{beta} dimers and was not transported to the lysosome. Finally, we reexamined the hexosaminidase activity and protein levels in the fibroblasts from the original patient. These data were also not consistent with the biochemical phenotype of the B1 variant of Tay-Sachs disease previously reported to be present. Thus, we conclude that the Val{sup 192}Leu substitution does not specifically affect the {alpha}-active site. 23 refs., 4 figs., 2 tabs.

Molecular characterization of cDNAs encoding G protein alpha and beta subunits and study of their temporal and spatial expression patterns in Nicotiana plumbaginifolia Viv.

Science.gov (United States)

Kaydamov, C; Tewes, A; Adler, K; Manteuffel, R

2000-04-25

We have isolated cDNA sequences encoding alpha and beta subunits of potential G proteins from a cDNA library prepared from somatic embryos of Nicotiana plumbaginifolia Viv. at early developmental stages. The predicted NPGPA1 and NPGPB1 gene products are 75-98% identical to the known respective plant alpha and beta subunits. Southern hybridizations indicate that NPGPA1 is probably a single-copy gene, whereas at least two copies of NPGPB1 exist in the N. plumbaginifolia genome. Northern analyses reveal that both NPGPA1 and NPGPB1 mRNA are expressed in all embryogenic stages and plant tissues examined and their expression is obviously regulated by the plant hormone auxin. Immunohistological localization of NPGPalpha1 and NPGPbeta1 preferentially on plasma and endoplasmic reticulum membranes and their immunochemical detection exclusively in microsomal cell fractions implicate membrane association of both proteins. The temporal and spatial expression patterns of NPGPA1 and NPGPB1 show conformity as well as differences. This could account for not only cooperative, but also individual activities of both subunits during embryogenesis and plant development.

Small-angle neutron scattering from the reconstituted TF sub 1 of H sup + -ATPase from thermophilic bacterium PS3 with deuterated subunits

Energy Technology Data Exchange (ETDEWEB)

Ito, Yuji [Univ. of Tokyo (Japan) Brookhaven National Lab., Upton, NY (United States); Harada, Mitsuo [Univ. of Tokyo (Japan); Ohta, Shigeo; Kagawa, Yasuo; Aono, Osamu [Jichi Medical School, Tochigi (Japan); Schefer, J; Schoenborn, B P [Brookhaven National Lab., Upton (United States)

1990-01-01

Subunits {alpha}, {beta} and {gamma} of adenosine triphosphatase (H{sup +}-ATPase) from the thermophilic bacterium PS3 (TF{sub 1}) have been over-expressed in Escherichia coli. {alpha} and {beta} subunits deuterated to the level of 90% were obtained by culturing E. coli in {sup 2}H{sub 2}O medium. Both the subunits and the reconstituted {alpha}{beta}{gamma} complex, TF{sub 1}, which contain the deuterated components in various combinations, were studied in solution by small-angle neutron scattering. The individual shapes of the subunits and their organization in the {alpha}{beta}{gamma}-TF{sub 1} complex were examined using the techniques of selective deuteration and contrast variation. The {alpha} and {beta} subunits are well approximated as ellipsoids of revolution having minor semi-axes of 20{center dot}4({plus minus}0{center dot}4) and 20{center dot}0({plus minus}0{center dot}2) {angstrom}, and major semi-axes of 53{center dot}0({plus minus}1{center dot}4) and 55{center dot}8({plus minus}0{center dot}9) {angstrom}, respectively. In the TF{sub 1} complex, three {beta} subunits are aligned to form an equilateral triangle, with their major axes tilted by 35{degree} with respect to the 3-fold axis of the complex. The {beta}-{beta} distance is about 53 {angstrom}. Three {alpha} subunits are similarly arranged, positioned between the {beta} subunits, and with their direction of tilt opposite to that of the {beta} subunits. The centers of the {alpha} and {beta} subunits lie in the same plane, forming a hexagon. Adjacent subunits overlap in this model, suggesting that they are not simple ellipsoids of revolution.

Mechanisms and regulation of TSH glycosylation

International Nuclear Information System (INIS)

Gesundheit, N.; Weintraub, B.D.

1986-01-01

Some differences in carbohydrate composition of secreted TSH-alpha, TSH-beta, and free alpha-subunits are examined. In order to explore whether each of the secreted TSH subunits has a distinctive carbohydrate composition, the authors incubated hypothyroid mouse tritiated sugar precursors: tritium-glucosamine; tritium-mannose; tritium-l-fucose; and tritium-N-acetylmannosamine, a specific precursor of the sialic acid tritium-N-acetylneuraminic acid. It was shown that although the two TSH subunits, alpha and beta, combine soon after synthesis and are secreted as a dimeric protein, there are notable differences in the carbohydrate processing of the two subunits

Transcriptional regulators of Na, K-ATPase subunits

OpenAIRE

Zhiqin eLi; Sigrid A Langhans

2015-01-01

The Na,K-ATPase classically serves as an ion pump creating an electrochemical gradient across the plasma membrane that is essential for transepithelial transport, nutrient uptake and membrane potential. In addition, Na,K-ATPase also functions as a receptor, a signal transducer and a cell adhesion molecule. With such diverse roles, it is understandable that the Na,K-ATPase subunits, the catalytic alpha-subunit, the beta-subunit and the FXYD proteins, are controlled extensively during developme...

The search for mutations in the gene for the beta subunit of the cGMP phosphodiesterase (PDEB) in patients with autosomal recessive retinitis pigmentosa

DEFF Research Database (Denmark)

Riess, O; Noerremoelle, A; Weber, B

1992-01-01

The finding of a mutation in the beta subunit of the cyclic GMP (cGMP) phosphodiesterase gene causing retinal degeneration in mice (the Pdeb gene) prompted a search for disease-causing mutations in the human phosphodiesterase gene (PDEB gene) in patients with retinitis pigmentosa. All 22 exons...

Understanding the mechanisms of ATPase beta family genes for cellular thermotolerance in crossbred bulls.

Science.gov (United States)

Deb, Rajib; Sajjanar, Basavaraj; Singh, Umesh; Alex, Rani; Raja, T V; Alyethodi, Rafeeque R; Kumar, Sushil; Sengar, Gyanendra; Sharma, Sheetal; Singh, Rani; Prakash, B

2015-12-01

Na+/K+-ATPase is an integral membrane protein composed of a large catalytic subunit (alpha), a smaller glycoprotein subunit (beta), and gamma subunit. The beta subunit is essential for ion recognition as well as maintenance of the membrane integrity. Present study was aimed to analyze the expression pattern of ATPase beta subunit genes (ATPase B1, ATPase B2, and ATPase B3) among the crossbred bulls under different ambient temperatures (20-44 °C). The present study was also aimed to look into the relationship of HSP70 with the ATPase beta family genes. Our results demonstrated that among beta family genes, transcript abundance of ATPase B1 and ATPase B2 is significantly (P ATPase Β1, ATPase B2, and ATPase B3 is highly correlated (P ATPase beta family genes for cellular thermotolerance in cattle.

A role for 12/15 lipoxygenase in the amyloid beta precursor protein metabolism.

Science.gov (United States)

Succol, Francesca; Praticò, Domenico

2007-10-01

12/15 Lipoxygenase (12/15LO) protein levels and activity are increased in pathologically affected regions of Alzheimer's disease (AD) brains, compared with controls. Its metabolic products are elevated in cerebrospinal fluid of patients with AD and individuals with mild cognitive impairment, suggesting that this enzyme may be involved early in AD pathogenesis. Herein, we investigate the effect of pharmacologic inhibition of 12/15LO on the amyloid beta precursor protein (APP) metabolism. To this end, we used CHO and N2A cells stably expressing human APP with the Swedish mutant, and two structurally distinct and selective 12/15LO inhibitors, PD146176 and CDC. Our results demonstrated that both drugs dose-dependently reduced Abeta formation without affecting total APP levels. Interestingly, in the same cells we observed a significant reduction in secreted (s)APPbeta and beta-secretase (BACE), but not sAPPalpha and ADAM10 protein levels. Together, these data show for the first time that this enzymatic pathway influences Abeta formation whereby modulating the BACE proteolytic cascade. We conclude that specific pharmacologic inhibition of 12/15LO could represent a novel therapeutic target for treating or preventing AD pathology in humans.

Influence of the. cap alpha. -,. beta. -, and. gamma. -subunits of the energy-transducing adenosine triphosphatase from Micrococcus lysodeikticus in the immunochemical properties of the protein and in their reconstitution studied by a radioimmunoassay method

Energy Technology Data Exchange (ETDEWEB)

Larraga, V; Mollinedo, F; Rubio, N; Munoz, E [Unidad de Biomembranas, Instituto de Inmunologia y Biologia Microbiana, Madrid (Spain)

1981-03-01

A sensitive radioimmunoassay was developed for the energy-transducing adenosine triphosphatase (F/sub 1/-ATPase, EC 3.6.1.3) of Micrococcus lysodeikticus and the assay was extended to the ..cap alpha..-, ..beta..-, and ..gamma..-subunits of the enzyme. These subunits were isolated and cross-reactions studied.

The autophosphorylation and p34cdc2 phosphorylation sites of casein kinase-2 beta-subunit are not essential for reconstituting the fully-active heterotetrameric holoenzyme

DEFF Research Database (Denmark)

Meggio, F; Boldyreff, B; Issinger, O G

1993-01-01

Two mutants of human casein kinase-2 beta-subunit with short deletions at either their amino (delta 1-4) or carboxy (delta 209-215) terminal side have been created that have lost the capability to undergo autophosphorylation and p34cdc2 mediated phosphorylation, respectively. Both mutants give rise...

Serotonin Transporter (5-HTT) and gamma-Aminobutyric Acid Receptor Subunit beta3 (GABRB3) Gene Polymorphisms are not Associated with Autism in the IMGSA Families

DEFF Research Database (Denmark)

Maestrini, E.; Lai, C.; Marlow, A.

1999-01-01

Previous studies have suggested that the serotonin transporter (5-HTT) gene and the gamma-aminobutyric acid receptor subunit beta3 (GABRB3) gene, or other genes in the 15q11-q13 region, are possibly involved in susceptibility to autism. To test this hypothesis we performed an association study on...

Early continuous white noise exposure alters l-alpha-amino-3-hydroxy-5-methyl-4-isoxazole propionic acid receptor subunit glutamate receptor 2 and gamma-aminobutyric acid type a receptor subunit beta3 protein expression in rat auditory cortex.

Science.gov (United States)

Xu, Jinghong; Yu, Liping; Zhang, Jiping; Cai, Rui; Sun, Xinde

2010-02-15

Auditory experience during the postnatal critical period is essential for the normal maturation of auditory function. Previous studies have shown that rearing infant rat pups under conditions of continuous moderate-level noise delayed the emergence of adult-like topographic representational order and the refinement of response selectivity in the primary auditory cortex (A1) beyond normal developmental benchmarks and indefinitely blocked the closure of a brief, critical-period window. To gain insight into the molecular mechanisms of these physiological changes after noise rearing, we studied expression of the AMPA receptor subunit GluR2 and GABA(A) receptor subunit beta3 in the auditory cortex after noise rearing. Our results show that continuous moderate-level noise rearing during the early stages of development decreases the expression levels of GluR2 and GABA(A)beta3. Furthermore, noise rearing also induced a significant decrease in the level of GABA(A) receptors relative to AMPA receptors. However, in adult rats, noise rearing did not have significant effects on GluR2 and GABA(A)beta3 expression or the ratio between the two units. These changes could have a role in the cellular mechanisms involved in the delayed maturation of auditory receptive field structure and topographic organization of A1 after noise rearing. Copyright 2009 Wiley-Liss, Inc.

A common polymorphic allele of the LH beta-subunit gene is associated with higher exogenous FSH consumption during controlled ovarian stimulation for assisted reproductive technology

DEFF Research Database (Denmark)

Alviggi, Carlo; Pettersson, Kim; Longobardi, Salvatore

2013-01-01

BACKGROUND: V-betaLH is a common genetic variant of LH caused by two polymorphic base changes in the beta subunit gene, altering the amino acid sequence (Trp8Arg and Ile15Thr). In a previous-preliminary trial performed in women undergoing IVF, it was demonstrated that carriers of v-betaLH show sub......-optimal ovarian response to a standard long GnRH-agonist down -regulation protocol when stimulated with pure recombinant FSH (r-hFSH). The aim of this study was to confirm the hypothesis that women with v-betaLH display hypo-sensitivity to exogenous FSH in a larger IVF population and to explore the frequency...... of this variant in a Danish female population. METHODS: In the present study, the effect of v-betaLH was retrospectively investigated in a larger series of women undergoing controlled ovarian stimulation (COS) and, for the first time, in a Danish IVF population. A total of 220 normogonadotrophic women following...

Characterization and charge distribution of the asparagine-linked oligosaccharides on secreted mouse thyrotropin and free alpha-subunits

International Nuclear Information System (INIS)

Gesundheit, N.; Gyves, P.W.; DeCherney, G.S.; Stannard, B.S.; Winston, R.L.; Weintraub, B.D.

1989-01-01

Mouse hemipituitaries in vitro secrete TSH, composed of an alpha-beta heterodimer, as well as excess (free) alpha-subunits. By dual metabolic labeling with [35S]sulfate and [3H]mannose, we have characterized oligosaccharides from secreted TSH alpha, TSH beta, and free alpha-subunits released from the apoprotein by enzymatic deglycosylation. Oligosaccharides from each subunit displayed a distinct anion exchange HPLC profile due to a specific pattern of sialylation and sulfation. Six species were obtained from TSH alpha (with two glycosylation sites), including neutral oligosaccharides as well as those with one or two negative charges. For TSH beta (with one glycosylation site) at least eight oligosaccharide species were noted, representing nearly every permutation of sialylation and sulfation; approximately 30% contained three or more negative charges. Analysis of [3H]mannose-labeled oligosaccharides on Concanavalin-A-agarose showed 85% binding for those from TSH alpha, 70% for free alpha, and 50% for those from TSH beta. These data demonstrate that oligosaccharides from secreted TSH beta were more sialylated and sulfated, consistent with a more complex branching pattern, than those from TSH alpha. Oligosaccharides from free alpha-subunit were more sialylated than those from TSH alpha, and the net negative charge was intermediate between those of TSH alpha and TSH beta. Although great microheterogeneity is present even at the single glycosylation site on the beta-subunit of secreted TSH, a pattern of sialylation and sulfation could be discerned

Moessbauer spectroscopic studies of hemoglobin and its isolated subunits

International Nuclear Information System (INIS)

Hoy, G.R.; Cook, D.C.; Berger, R.L.; Friedman, F.K.

1986-01-01

Samples of 90% enriched 57Fe hemoglobin and its isolated subunits have been prepared. Moessbauer spectroscopic measurements have been made on three such samples. Sample one contained contributions of oxyhemoglobin, deoxyhemoglobin, and carbonmonoxyhemoglobin. This sample was studied from a temperature of 90 K down to 230 mK. Measurements were also made at 4.2 K using a small applied magnetic field of 1.0 T. In general, the measured quadrupole splittings and isomer shifts for each component agreed with previous measurements on single component samples in the literature, and thus demonstrated that chemically enriched hemoglobin has not been altered. The second and third samples were isolated alpha and beta subunits, respectively. We have found measurable Moessbauer spectral differences between the HbO 2 sites in the alpha subunit sample and the beta subunit sample. The measured Moessbauer spectral areas indicate that the iron ion has the largest mean-square displacement at the deoxy Hb sites as compared to that at the oxy- and carbonmonoxy Hb sites. The mean-square displacement at the HbO 2 sites is the smallest

A double mutation in exon 6 of the [beta]-hexosaminidase [alpha] subunit in a patient with the B1 variant of Tay-Sachs disease

Energy Technology Data Exchange (ETDEWEB)

Ainsworth, P.J. (Univ. of Western Ontario, Ontario (Canada) Child Health Research Institute, London, Ontario (Canada)); Coulter-Mackie, M.B. (Univ. of Western Ontario, Ontario (Canada) Child Health Research Institute, London, Ontario (Canada) Children' s Psychiatric Research Institute, London, Ontario (Canada))

1992-10-01

The B1 variant form of Tay-Sachs disease is enzymologically unique in that the causative mutation(s) appear to affect the active site in the [alpha] subunit of [beta]-hexosaminidase A without altering its ability to associate with the [beta] subunit. Most previously reported B1 variant mutations were found in exon 5 within codon 178. The coding sequence of the [alpha] subunit gene of a patient with the B1 variant form was examined with a combination of reverse transcription of mRNA to cDNA, PCR, and dideoxy sequencing. A double mutation in exon 6 has been identified: a G[sub 574][yields]C transversion causing a val[sub 192][yields]leu change and a G[sub 598][yields] A transition resulting in a val[sub 200][yields]met alteration. The amplified cDNAs were otherwise normal throughout their sequence. The 574 and 598 alterations have been confirmed by amplification directly from genomic DNA from the patient and her mother. Transient-expression studies of the two exon 6 mutations (singly or together) in COS-1 cells show that the G[sub 574][yields]C change is sufficient to cause the loss of enzyme activity. The biochemical phenotype of the 574 alteration in transfection studies is consistent with that expected for a B1 variant mutation. As such, this mutation differs from previously reported B1 variant mutations, all of which occur in exon 5. 31 refs., 2 figs., 2 tabs.

Amyloid beta precursor protein regulates male sexual behavior.

Science.gov (United States)

Park, Jin Ho; Bonthius, Paul J; Tsai, Houng-Wei; Bekiranov, Stefan; Rissman, Emilie F

2010-07-28

Sexual behavior is variable between individuals, ranging from celibacy to sexual addictions. Within normal populations of individual men, ranging from young to middle aged, testosterone levels do not correlate with libido. To study the genetic mechanisms that contribute to individual differences in male sexual behavior, we used hybrid B6D2F1 male mice, which are a cross between two common inbred strains (C57BL/6J and DBA/2J). Unlike most laboratory rodent species in which male sexual behavior is highly dependent upon gonadal steroids, sexual behavior in a large proportion of these hybrid male mice after castration is independent of gonadal steroid hormones and their receptors; thus, we have the ability to discover novel genes involved in this behavior. Gene expression arrays, validation of gene candidates, and transgenic mice that overexpress one of the genes of interest were used to reveal genes involved in maintenance of male sexual behavior. Several genes related to neuroprotection and neurodegeneration were differentially expressed in the hypothalamus of males that continued to mate after castration. Male mice overexpressing the human form of one of these candidate genes, amyloid beta precursor protein (APP), displayed enhanced sexual behavior before castration and maintained sexual activity for a longer duration after castration compared with controls. Our results reveal a novel and unexpected relationship between APP and male sexual behavior. We speculate that declining APP during normal aging in males may contribute to the loss of sexual function.

N-Benzylhydroxylamine addition to beta-aryl enoates. Enantioselective synthesis of beta-aryl-beta-amino acid precursors

Science.gov (United States)

Sibi; Liu

2000-10-19

Chiral Lewis acid catalyzed N-benzylhydroxylamine addition to pyrrolidinone-derived enoates afforded beta-aryl-beta-amino acid derivatives in high enantiomeric purity with moderate to very good chemical efficiency.

Cloning and Expression of Luteinizing Hormone Subunits in Chinese Hamster Ovary Cell Line

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Zeinab Soleimanifar

2016-10-01

Full Text Available Background: Luteinizing hormone (LH was secreted by the stimulating cells of the testes and ovaries in the anterior pituitary gland. The application of this hormone is in the treatment of men and women with infertility and amenorrhea respectively.Materials and Methods: In the present study the alpha and beta subunits of human LH gene were cloned into the pEGFP-N1 expression vector and produced the recombinant LH hormone in Chinese hamster ovary (CHO eukaryotic system.Results: Alpha and beta subunits of LH hormone were cloned between NheI and BamHI cut sites of pEGFP_N1 expression plasmid and confirmed by PCR.  Hormone expression was evaluated in CHO cell line by Western blotting using the specific antibody.Conclusion: Alpha and beta subunits of LH hormone were expressed in CHO cell line perfectly.

Arachidonic Acid-Induced Expression of the Organic Solute and Steroid Transporter-beta (Ost-beta) in a Cartilaginous Fish Cell Line

Science.gov (United States)

Hwang, Jae-Ho; Parton, Angela; Czechanski, Anne; Ballatori, Nazzareno; Barnes, David

2008-01-01

The organic solute and steroid transporter (OST/Ost) is a unique membrane transport protein heterodimer composed of subunits designated alpha and beta, that transports conjugated steroids and prostaglandin E2 across the plasma membrane. Ost was first identified in the liver of the cartilaginous fish Leucoraja erinacea, the little skate, and subsequently was found in many other species, including humans and rodents. The present study describes the isolation of a new cell line, LEE-1, derived from an early embryo of L. erinacea, and characterizes the expression of Ost in these cells. The mRNA size and amino acid sequence of Ost-beta in LEE-1 was identical to that previously reported for Ost-beta from skate liver, and the primary structure was identical to that of the spiny dogfish shark (Squalus acanthias) with the exception of a single amino acid. Ost-beta was found both on the plasma membrane and intracellularly in LEE-1 cells, consistent with its localization in other cell types. Interestingly, arachidonic acid, the precursor to eiconsanoids, strongly induced Ost-beta expression in LEE-1 cells and a lipid mixture containing arachidonic acid also induced Ost-alpha. Overall, the present study describes the isolation of a novel marine cell line, and shows that this cell line expresses relatively high levels of Ost when cultured in the presence of arachidonic acid. Although the function of this transport protein in embryo-derived cells is unknown, it may play a role in the disposition of eicosanoids or steroid-derived molecules. PMID:18407792

Familial Alzheimer's disease mutations in presenilin 1 do not alter levels of the secreted amyloid-beta protein precursor generated by beta-secretase cleavage.

Science.gov (United States)

Zhang, Can; Browne, Andrew; Kim, Doo Yeon; Tanzi, Rudolph E

2010-02-01

Alzheimer's disease (AD) is an insidious and progressive disease with a genetically complex and heterogenous etiology. More than 200 fully penetrant mutations in the amyloid beta-protein precursor (APP), presenilin 1 (or PSEN1), and presenilin 2 (PSEN2) have been linked to early-onset familial AD (FAD). 177 PSEN1 FAD mutations have been identified so far and account for more than approximately 80% of all FAD mutations. All PSEN1 FAD mutations can increase the Abeta42:Abeta40 ratio with seemingly different and incompletely understood mechanisms. A recent study has shown that the 286 amino acid N-terminal fragment of APP (N-APP), a proteolytic product of beta-secretase-derived secreted form of APP (sAPPbeta), could bind the death receptor, DR6, and lead to neurodegeneration. Here we asked whether PSEN1 FAD mutations lead to neurodegeneration by modulating sAPPbeta levels. All four different PSEN1 FAD mutations tested (in three mammalian cell lines) did not alter sAPPbeta levels. Therefore PS1 mutations do not appear to contribute to AD pathogenesis via altered production of sAPPbeta.

GABAA receptor subunit gene expression in human prefrontal cortex: comparison of schizophrenics and controls

Science.gov (United States)

Akbarian, S.; Huntsman, M. M.; Kim, J. J.; Tafazzoli, A.; Potkin, S. G.; Bunney, W. E. Jr; Jones, E. G.; Bloom, F. E. (Principal Investigator)

1995-01-01

The prefrontal cortex of schizophrenics is hypoactive and displays changes related to inhibitory, GABAergic neurons, and GABAergic synapses. These changes include decreased levels of glutamic acid decarboxylase (GAD), the enzyme for GABA synthesis, upregulation of muscimol binding, and downregulation of benzodiazepine binding to GABAA receptors. Studies in the visual cortex of nonhuman primates have demonstrated that gene expression for GAD and for several GABAA receptor subunit polypeptides is under control of neuronal activity, raising the possibility that similar mechanisms in the hypoactive prefrontal cortex of schizophrenics may explain the abnormalities in GAD and in GABAA receptor regulation. In the present study, which is the first of its type on human cerebral cortex, levels of mRNAs for six GABAA receptor subunits (alpha 1, alpha 2, alpha 5, beta 1, beta 2, gamma 2) and their laminar expression patterns were analyzed in the prefrontal cortex of schizophrenics and matched controls, using in situ hybridization histochemistry and densitometry. Three types of laminar expression pattern were observed: mRNAs for the alpha 1, beta 2, and gamma 2 subunits, which are the predominant receptor subunits expressed in the mature cortex, were expressed at comparatively high levels by cells of all six cortical layers, but most intensely by cells in lower layer III and layer IV. mRNAs for the alpha 2, alpha 5, and beta 1 subunits were expressed at lower levels; alpha 2 and beta 1 were expressed predominantly by cells in layers II, III, and IV; alpha 5 was expressed predominantly in layers IV, V, and VI. There were no significant changes in overall mRNA levels for any of the receptor subunits in the prefrontal cortex of schizophrenics, and the laminar expression pattern of all six receptor subunit mRNAs did not differ between schizophrenics and controls. Because gene expression for GABAA receptor subunits is not consistently altered in the prefrontal cortex of

Impact of rs361072 in the phosphoinositide 3-kinase p110beta gene on whole-body glucose metabolism and subunit protein expression in skeletal muscle

DEFF Research Database (Denmark)

Ribel-Madsen, Rasmus; Poulsen, Pernille; Holmkvist, Johan

2010-01-01

OBJECTIVE: Phosphoinositide 3-kinase (PI3K) is a major effector in insulin signaling. rs361072, located in the promoter of the gene (PIK3CB) for the p110beta subunit, has previously been found to be associated with homeostasis model assessment for insulin resistance (HOMA-IR) in obese subjects...... infusion. rs361072 did not associate with insulin-stimulated peripheral glucose disposal despite a decreased muscle p85alpha:p110beta protein ratio (P(add) = 0.03) in G allele carriers. No association with HOMA-IR or type 2 diabetes (odds ratio 1.07, P = 0.5) was identified, and obesity did not interact...

Tuning of the Na,K-ATPase by the beta subunit

Science.gov (United States)

Hilbers, Florian; Kopec, Wojciech; Isaksen, Toke Jost; Holm, Thomas Hellesøe; Lykke-Hartmann, Karin; Nissen, Poul; Khandelia, Himanshu; Poulsen, Hanne

2016-02-01

The vital gradients of Na+ and K+ across the plasma membrane of animal cells are maintained by the Na,K-ATPase, an αβ enzyme complex, whose α subunit carries out the ion transport and ATP hydrolysis. The specific roles of the β subunit isoforms are less clear, though β2 is essential for motor physiology in mammals. Here, we show that compared to β1 and β3, β2 stabilizes the Na+-occluded E1P state relative to the outward-open E2P state, and that the effect is mediated by its transmembrane domain. Molecular dynamics simulations further demonstrate that the tilt angle of the β transmembrane helix correlates with its functional effect, suggesting that the relative orientation of β modulates ion binding at the α subunit. β2 is primarily expressed in granule neurons and glomeruli in the cerebellum, and we propose that its unique functional characteristics are important to respond appropriately to the cerebellar Na+ and K+ gradients.

rRNA maturation in yeast cells depleted of large ribosomal subunit proteins.

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Gisela Pöll

Full Text Available The structural constituents of the large eukaryotic ribosomal subunit are 3 ribosomal RNAs, namely the 25S, 5.8S and 5S rRNA and about 46 ribosomal proteins (r-proteins. They assemble and mature in a highly dynamic process that involves more than 150 proteins and 70 small RNAs. Ribosome biogenesis starts in the nucleolus, continues in the nucleoplasm and is completed after nucleo-cytoplasmic translocation of the subunits in the cytoplasm. In this work we created 26 yeast strains, each of which conditionally expresses one of the large ribosomal subunit (LSU proteins. In vivo depletion of the analysed LSU r-proteins was lethal and led to destabilisation and degradation of the LSU and/or its precursors. Detailed steady state and metabolic pulse labelling analyses of rRNA precursors in these mutant strains showed that LSU r-proteins can be grouped according to their requirement for efficient progression of different steps of large ribosomal subunit maturation. Comparative analyses of the observed phenotypes and the nature of r-protein-rRNA interactions as predicted by current atomic LSU structure models led us to discuss working hypotheses on i how individual r-proteins control the productive processing of the major 5' end of 5.8S rRNA precursors by exonucleases Rat1p and Xrn1p, and ii the nature of structural characteristics of nascent LSUs that are required for cytoplasmic accumulation of nascent subunits but are nonessential for most of the nuclear LSU pre-rRNA processing events.

Copolymer semiconductors comprising thiazolothiazole or benzobisthiazole, or benzobisoxazole electron acceptor subunits, and electron donor subunits, and their uses in transistors and solar cells

Science.gov (United States)

Jenekhe, Samson A; Subramaniyan, Selvam; Ahmed, Eilaf; Xin, Hao; Kim, Felix Sunjoo

2014-10-28

The inventions disclosed, described, and/or claimed herein relate to copolymers comprising copolymers comprising electron accepting A subunits that comprise thiazolothiazole, benzobisthiazole, or benzobisoxazoles rings, and electron donating subunits that comprise certain heterocyclic groups. The copolymers are useful for manufacturing organic electronic devices, including transistors and solar cells. The invention also relates to certain synthetic precursors of the copolymers. Methods for making the copolymers and the derivative electronic devices are also described.

The Drosophila melanogaster DmCK2beta transcription unit encodes for functionally non-redundant protein isoforms.

Science.gov (United States)

Jauch, Eike; Wecklein, Heike; Stark, Felix; Jauch, Mandy; Raabe, Thomas

2006-06-07

Genes encoding for the two evolutionary highly conserved subunits of a heterotetrameric protein kinase CK2 holoenzyme are present in all examined eukaryotic genomes. Depending on the organism, multiple transcription units encoding for a catalytically active CK2alpha subunit and/or a regulatory CK2beta subunit may exist. The phosphotransferase activity of members of the protein kinase CK2alpha family is thought to be independent of second messengers but is modulated by interaction with CK2beta-like proteins. In the genome of Drosophila melanogaster, one gene encoding for a CK2alpha subunit and three genes encoding for CK2beta-like proteins are present. The X-linked DmCK2beta transcription unit encodes for several CK2beta protein isoforms due to alternative splicing of its primary transcript. We addressed the question whether CK2beta-like proteins are redundant in function. Our in vivo experiments show that variations of the very C-terminal tail of CK2beta isoforms encoded by the X-linked DmCK2beta transcription unit influence their functional properties. In addition, we find that CK2beta-like proteins encoded by the autosomal D. melanogaster genes CK2betates and CK2beta' cannot fully substitute for a loss of CK2beta isoforms encoded by DmCK2beta.

Metabolic Characterization of Intact Cells Reveals Intracellular Amyloid Beta but Not Its Precursor Protein to Reduce Mitochondrial Respiration

Science.gov (United States)

Schaefer, Patrick M.; von Einem, Bjoern; Walther, Paul; Calzia, Enrico; von Arnim, Christine A. F.

2016-01-01

One hallmark of Alzheimer´s disease are senile plaques consisting of amyloid beta (Aβ), which derives from the processing of the amyloid precursor protein (APP). Mitochondrial dysfunction has been linked to the pathogenesis of Alzheimer´s disease and both Aβ and APP have been reported to affect mitochondrial function in isolated systems. However, in intact cells, considering a physiological localization of APP and Aβ, it is pending what triggers the mitochondrial defect. Thus, the aim of this study was to dissect the impact of APP versus Aβ in inducing mitochondrial alterations with respect to their subcellular localization. We performed an overexpression of APP or beta-site amyloid precursor protein cleaving enzyme 1 (BACE1), increasing APP and Aβ levels or Aβ alone, respectively. Conducting a comprehensive metabolic characterization we demonstrate that only APP overexpression reduced mitochondrial respiration, despite lower extracellular Aβ levels compared to BACE overexpression. Surprisingly, this could be rescued by a gamma secretase inhibitor, oppositionally indicating an Aβ-mediated mitochondrial toxicity. Analyzing Aβ localization revealed that intracellular levels of Aβ and an increased spatial association of APP/Aβ with mitochondria are associated with reduced mitochondrial respiration. Thus, our data provide marked evidence for a prominent role of intracellular Aβ accumulation in Alzheimer´s disease associated mitochondrial dysfunction. Thereby it highlights the importance of the localization of APP processing and intracellular transport as a decisive factor for mitochondrial function, linking two prominent hallmarks of neurodegenerative diseases. PMID:28005987

Formation of thermally induced aggregates of the soya globulin beta-conglycinin.

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Mills, E N; Huang, L; Noel, T R; Gunning, A P; Morris, V J

2001-06-11

The effect of ionic strength (I) on the formation of thermally induced aggregates by the 7S globular storage protein of soya, beta-conglycinin, has been studied using atomic force microscopy. Aggregates were only apparent when I> or =0.1, and had a fibrous appearance, with a height (diameter) of 8-11 nm. At high ionic strength (I=1.0) the aggregates appeared to associate into clumps. When aggregate formation was studied at I=0.2, it was clear that aggregation only began at temperatures above the main thermal transition for the protein at 75 degrees C, as determined by differential scanning calorimetry. This coincided with a small change in secondary structure, as indicated by circular dichroism spectroscopy, suggesting that a degree of unfolding was necessary for aggregation to proceed. Despite prolonged heating the size of the aggregates did not increase indefinitely, suggesting that certain beta-conglycinin isoforms were able to act as chain terminators. At higher protein concentrations (1% w/v) the linear aggregates appeared to form large macroaggregates, which may be the precursors of protein gel formation. The ability of beta-conglycinin to form such distinctive aggregates is discussed in relation to the presence of acidic inserts in certain of the beta-conglycinin subunits, which may play an important role in limiting aggregate length.

MspI and PvuII polymorphisms in the Na,K-ATPase. beta. subunit gene ATP1B1

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Shull, M.M.; Pugh, D.G.; Lane, L.K.; Lingrel, J.B. (Univ. of Cincinnati College of Medicine, OH (USA))

1990-02-25

ATP1B HH1.2 is a 1.2 kb HindIII fragment from the 3{prime} portion of the human Na,K-ATPase {beta} subunit gene, ATP1B1. MspI identifies a two allele polymorphism (M1: 6.7 kb, M2: 5.3 kb). PvuII also detects a two-allele polymorphism (P1: 5.1 kb, P2: 4.7 kb). ATP1B1 has been assigned to chromosome 1q by somatic cell hybrid analysis. Codominant segregation of the MspI RFLP was observed in one two-generation family (5 individuals). Codominant segregation of the PvuII RFLP was observed in a two-generation (8 individuals) and a three-generation (12 individuals) family.

Neurine, an acetylcholine autolysis product, elevates secreted amyloid-beta protein precursor and amyloid-beta peptide levels, and lowers neuronal cell viability in culture: a role in Alzheimer's disease?

Science.gov (United States)

Tweedie, David; Brossi, Arnold; Chen, DeMoa; Ge, Yuan-Wen; Bailey, Jason; Yu, Qian-Sheng; Kamal, Mohammad A; Sambamurti, Kumar; Lahiri, Debomoy K; Greig, Nigel H

2006-09-01

Classical hallmarks of Alzheimer's disease (AD) are a synaptic loss, cholinergic neuron death, and abnormal protein deposition, particularly of toxic amyloid-beta peptide (Abeta) that is derived from amyloid-beta protein precursor (AbetaPP) by the action of beta- and gamma-secretases. The trigger(s) initiating the biochemical cascades that underpin these hallmarks have yet to be fully elucidated. The typical forebrain cholinergic cell demise associated with AD brain results in a loss of presynaptic cholinergic markers and acetylcholine (ACh). Neurine (vinyl-trimethyl-ammonium hydroxide) is a breakdown product of ACh, consequent to autolysis and is an organic poison found in cadavre brain. The time- and concentration-dependent actions of neurine were assessed in human neuroblastoma (NB, SK-N-SH) cells in culture by quantifying cell viability by lactate dehydrogenase (LDH) and MTS assay, and AbetaPP and Abeta levels by Western blot and ELISA. NB cells displayed evidence of toxicity to neurine at > or = 3 mg/ml, as demonstrated by elevated LDH levels in the culture media and a reduced cell viability shown by the MTS assay. Using subtoxic concentrations of neurine, elevations in AbetaPP and Abeta1-40 peptide levels were detected in conditioned media samples.

Role of the Rubisco small subunit. Final report for period May 1, 1997--April 30,2000

Energy Technology Data Exchange (ETDEWEB)

Spreitzer, Robert J.

2000-10-04

CO{sub 2} and O{sub 2} are mutually competitive at the active site of ribulose-1,5-biphosphate (RuBP) carboxylase/oxygenase (Rubisco). Rubisco contains two subunits, each present in eight copies. The 15-kD small subunit is coded by a family of nuclear RbcS genes. Until now, the role of the small subunit in Rubisco structure or catalytic efficiency is not known. Because of other work in eliminating the two RbcS genes in the green algo Chlamydomonas reinhardtii, it is now possible to address questions about the structure-function relationships of the eukaryotic small subunit. There are three specific aims in this project: (1) Alanine scanning mutagenesis is being used to dissect the importance of the {beta}A/{beta}B loop, a feature unique to the eukaryotic small subunit. (2) Random mutagenesis is being used to identify additional residues or regions of the small subunit that are important for holoenzyme assembly and function. (3) Attempts are being made to express foreign small subunits in Chlamydomonas to examine the contribution of small subunits to holoenzyme assembly, catalytic efficiency, and CO{sub 2}/O{sub 2} specificity.

Crystal Structure of Human [Beta]-Hexosaminidase B: Understanding the Molecular Basis of Sandhoff and Tay-Sachs Disease

Energy Technology Data Exchange (ETDEWEB)

Mark, Brian L.; Mahuran, Don J.; Cherney, Maia M.; Zhao, Dalian; Knapp, Spencer; James, Michael N.G.

2010-12-01

In humans, two major {beta}-hexosaminidase isoenzymes exist: Hex A and Hex B. Hex A is a heterodimer of subunits {alpha} and {beta} (60% identity), whereas Hex B is a homodimer of {beta}-subunits. Interest in human {beta}-hexosaminidase stems from its association with Tay-Sachs and Sandhoff disease; these are prototypical lysosomal storage disorders resulting from the abnormal accumulation of G{sub M2}-ganglioside (G{sub M2}). Hex A degrades G{sub M2} by removing a terminal N-acetyl-D-galactosamine ({beta}-GalNAc) residue, and this activity requires the G{sub M2}-activator, a protein which solubilizes the ganglioside for presentation to Hex A. We present here the crystal structure of human Hex B, alone (2.4 {angstrom}) and in complex with the mechanistic inhibitors GalNAc-isofagomine (2.2 {angstrom}) or NAG-thiazoline (2.5 {angstrom}). From these, and the known X-ray structure of the G{sub M2}-activator, we have modeled Hex A in complex with the activator and ganglioside. Together, our crystallographic and modeling data demonstrate how {alpha} and {beta}-subunits dimerize to form either Hex A or Hex B, how these isoenzymes hydrolyze diverse substrates, and how many documented point mutations cause Sandhoff disease ({beta}-subunit mutations) and Tay-Sachs disease ({alpha}-subunit mutations).

Adrenocorticotropin, beta-endorphin, and beta-lipotropin in normal thyroid and lung: possible implications for ectopic hormone secretion.

Science.gov (United States)

Clements, J A; Funder, J W; Tracy, K; Morgan, F J; Campbell, D J; Lewis, P; Hearn, M T

1982-12-01

The expression of the proopiomelanocortin (POMC) gene by normal lung and thyroid was examined by measurement of the content of ACTH, beta-lipotropin (beta LPH), and beta-endorphin (beta EP) in porcine lung and thyroid tissue. Acid extracts of normal porcine lung and thyroid tissue each contained appreciable amounts of immunoreactive (ir) ACTH, ir-beta LPH, and ir-beta EP. The content of ir-beta LPH in both tissues exceeded by severalfold, on a molar basis, the content of ir-ACTH and ir-beta EP, suggesting that the common precursor POMC was processed predominantly to peptides other than ir-ACTH and ir-beta EP. A porcine thyroid extract (Calcitare, porcine calcitonin, Armour) showed equivalent levels of beta EP-like immunoreactivity and bioactivity, measured by opiate radioreceptor assay; in contrast, ACTH-like bioactivity, measured by rat zona fasciculata steroidogenesis, was only 4% of ACTH-like immunoreactivity. On reversed phase high performance liquid chromatography, Calcitare showed multiple peaks of ACTH-like immunoreactivity, one of which coeluted with porcine ACTH-(1-39), and two much smaller peaks of beta EP-like immunoreactivity, of which the smaller coeluted with porcine beta EP. These data suggest that both lung and thyroid gland synthesize POMC, which in normal tissue is usually predominantly processed to species other than ACTH and beta EP. Ectopic secretion of ACTH and beta EP by lung and thyroid neoplasms may thus represent the loss of a system(s) normally responsible for processing the precursor beyond ACTH and beta EP.

Process for forming seamless tubing of zirconium or titanium alloys from welded precursors

International Nuclear Information System (INIS)

Sabol, G.P.; Barry, R.F.

1987-01-01

A process is described for forming seamless tubing of a material selected from zirconium, zirconium alloys, titanium, and titanium alloys, from welded precursor tubing of the material, having a heterogeneous structure resulting from the welding thereof. The process consists of: heating successive axial segments of the welded tubing, completely through the wall thereof, including the weld, to uniformly transform the heterogeneous, as welded, material into the beta phase; quenching the beta phase tubing segments, the heating and quenching effected sufficiently rapid enough to produce a fine sized beta grain structure completely throughout the precursor tubing, including the weld, and to prevent growth of beta grains within the material larger than 200 micrometers in diameter; and subsequently uniformly deforming the quenched precursor tubing by cold reduction steps to produce a seamless tubing of final size and shape

Elaboration of thorium uranium phosphate-diphosphate({beta}-TUPD) and {beta}-TUPD/monazite composite materials from crystallized precursors: sintering and study of the long term behavior of the ceramics; Elaboration de phosphate-diphosphate de thorium et d'uranium ({beta}-PDTU) et de materiaux composites {beta}-PDTU/Monazite a partir de precurseurs cristallises. Etudes du frittage et de la durabilite chimique

Energy Technology Data Exchange (ETDEWEB)

Clavier, N

2004-11-01

Thorium Phosphate-Diphosphate ({beta}-TPD) is actually considered as potential host matrix for the immobilization of radionuclides, and especially actinides, in the field of an underground repository. The studies reported in this work are based on the precipitation of the Thorium Phosphate Hydrogen-Phosphate Hydrate (TPHPH) as a precursor of {beta}-TPD. The crystal structure of TPHPH was solved then the reactions involved during its transformation into {beta}-TPD were established. It allows us to put in evidence a new monoclinic variety of TPD, called {alpha}-TPD, acting as intermediate of reaction. Moreover, the existence of a complete solid solution between TPHPH and UPHPH was demonstrated.The experimental conditions of sintering leading to an optimal densification of the pellets were determined. The relative density of the samples was always between 95 and 100% of the calculated value while a significant improvement of the homogeneity of the samples was noted. By this way, the process based on the precipitation of low-temperature crystallized precursors followed by their heat treatment at high temperature was applied to the preparation of {beta}-TUPD/Monazite based composites in the aim to incorporate simultaneously tri- and tetravalent actinides. The chemical durability of {beta}-TUPD sintered samples was evaluated. The normalized leaching rates determined in several experimental conditions revealed the good resistance of the solids to aqueous alteration. Moreover, the normalized dissolution rates exhibited a low dependence to temperature, pH as well as to several ions present in the leachate. For all the samples, thorium was quickly precipitated as a neo-formed phosphate phase identified to TPHPH. (author)

Mapping of the Mouse Actin Capping Protein Beta Subunit Gene

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Cooper John A

2000-07-01

Full Text Available Abstract Background Capping protein (CP, a heterodimer of α and β subunits, is found in all eukaryotes. CP binds to the barbed ends of actin filaments in vitro and controls actin assembly and cell motility in vivo. Vertebrates have three isoforms of CPβ produced by alternatively splicing from one gene; lower organisms have one gene and one isoform. Results We isolated genomic clones corresponding to the β subunit of mouse CP and identified its chromosomal location by interspecies backcross mapping. Conclusions The CPβ gene (Cappb1 mapped to Chromosome 4 between Cdc42 and D4Mit312. Three mouse mutations, snubnose, curly tail, and cribriform degeneration, map in the vicinity of the β gene.

Tuning of the Na,K-ATPase by the beta subunit

DEFF Research Database (Denmark)

Hilbers, Florian; Kopec, Wojciech; Isaksen, Toke Jost

2016-01-01

The vital gradients of Na(+) and K(+) across the plasma membrane of animal cells are maintained by the Na,K-ATPase, an αβ enzyme complex, whose α subunit carries out the ion transport and ATP hydrolysis. The specific roles of the β subunit isoforms are less clear, though β2 is essential for motor...... to the cerebellar Na(+) and K(+) gradients....... physiology in mammals. Here, we show that compared to β1 and β3, β2 stabilizes the Na(+)-occluded E1P state relative to the outward-open E2P state, and that the effect is mediated by its transmembrane domain. Molecular dynamics simulations further demonstrate that the tilt angle of the β transmembrane helix...

NMR solution structure of the mitochondrial F1beta presequence from Nicotiana plumbaginifolia.

Science.gov (United States)

Moberg, Per; Nilsson, Stefan; Ståhl, Annelie; Eriksson, Anna-Carin; Glaser, Elzbieta; Mäler, Lena

2004-03-05

We have isolated, characterized and determined the three-dimensional NMR solution structure of the presequence of ATPsynthase F1beta subunit from Nicotiana plumbaginifolia. A general method for purification of presequences is presented. The method is based on overexpression of a mutant precursor containing a methionine residue introduced at the processing site, followed by CNBr-cleavage and purification of the presequence on a cation-exchange column. The F1beta presequence, 53 amino acid residues long, retained its native properties as evidenced by inhibition of in vitro mitochondrial import and processing at micromolar concentrations. CD spectroscopy revealed that the F1beta presequence formed an alpha-helical structure in membrane mimetic environments such as SDS and DPC micelles (approximately 50% alpha-helix), and in acidic phospholipid bicelles (approximately 60% alpha-helix). The NMR solution structure of the F1beta presequence in SDS micelles was determined on the basis of 518 distance and 21 torsion angle constraints. The structure was found to contain two helices, an N-terminal amphipathic alpha-helix (residues 4-15) and a C-terminal alpha-helix (residues 43-53), separated by a largely unstructured 27 residue long internal domain. The N-terminal amphipathic alpha-helix forms the putative Tom20 receptor binding site, whereas the C-terminal alpha-helix is located upstream of the mitochondrial processing peptidase cleavage site.

Transcriptional regulators of Na, K-ATPase subunits

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Zhiqin eLi

2015-10-01

Full Text Available The Na,K-ATPase classically serves as an ion pump creating an electrochemical gradient across the plasma membrane that is essential for transepithelial transport, nutrient uptake and membrane potential. In addition, Na,K-ATPase also functions as a receptor, a signal transducer and a cell adhesion molecule. With such diverse roles, it is understandable that the Na,K-ATPase subunits, the catalytic alpha-subunit, the beta-subunit and the FXYD proteins, are controlled extensively during development and to accommodate physiological needs. The spatial and temporal expression of Na,K-ATPase is partially regulated at the transcriptional level. Numerous transcription factors, hormones, growth factors, lipids and extracellular stimuli modulate the transcription of the Na,K-ATPase subunits. Moreover, epigenetic mechanisms also contribute to the regulation of Na,K-ATPase expression. With the ever growing knowledge about diseases associated with the malfunction of Na,K-ATPase, this review aims at summarizing the best-characterized transcription regulators that modulate Na,K-ATPase subunit levels. As abnormal expression of Na,K-ATPase subunits have been observed in many carcinoma, we will also discuss transcription factors that are associated with epithelial-to-mesenchymal transition, a crucial step in the progression of many tumors to malignant disease.

Role of the Na(+)/K(+)-ATPase beta-subunit in peptide-mediated transdermal drug delivery.

Science.gov (United States)

Wang, Changli; Ruan, Renquan; Zhang, Li; Zhang, Yunjiao; Zhou, Wei; Lin, Jun; Ding, Weiping; Wen, Longping

2015-04-06

In this work, we discovered that the Na(+)/K(+)-ATPase beta-subunit (ATP1B1) on epidermal cells plays a key role in the peptide-mediated transdermal delivery of macromolecular drugs. First, using a yeast two-hybrid assay, we screened candidate proteins that have specific affinity for the short peptide TD1 (ACSSSPSKHCG) identified in our previous work. Then, we verified the specific binding of TD1 to ATP1B1 in yeast and mammalian cells by a pull-down ELISA and an immunoprecipitation assay. Finally, we confirmed that TD1 mainly interacted with the C-terminus of ATP1B1. Our results showed that the interaction between TD1 and ATP1B1 affected not only the expression and localization of ATP1B1, but also the epidermal structure. In addition, this interaction could be antagonized by the exogenous competitor ATP1B1 or be inhibited by ouabain, which results in the decreased delivery of macromolecular drugs across the skin. The discovery of a critical role of ATP1B1 in the peptide-mediated transdermal drug delivery is of great significance for the future development of new transdermal peptide enhancers.

Effects of cigarette smoke exposure on nicotinic acetylcholine receptor subunits {alpha}7 and {beta}2 in the sudden infant death syndrome (SIDS) brainstem

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Machaalani, Rita, E-mail: rita.machaalani@sydney.edu.au [Department of Medicine, The University of Sydney, NSW 2006 (Australia); Bosch Institute, The University of Sydney, NSW 2006 (Australia); The Children' s Hospital at Westmead, NSW 2145 (Australia); Say, Meichien [Department of Medicine, The University of Sydney, NSW 2006 (Australia); Bosch Institute, The University of Sydney, NSW 2006 (Australia); Waters, Karen A. [Department of Medicine, The University of Sydney, NSW 2006 (Australia); Bosch Institute, The University of Sydney, NSW 2006 (Australia); The Children' s Hospital at Westmead, NSW 2145 (Australia)

2011-12-15

It is postulated that nicotine, as the main neurotoxic constituent of cigarette smoke, influences SIDS risk through effects on nicotinic acetylcholine receptors (nAChRs) in brainstem nuclei that control respiration and arousal. This study compared {alpha}7 and {beta}2 nAChR subunit expression in eight nuclei of the caudal and rostral medulla and seven nuclei of the pons between SIDS (n = 46) and non-SIDS infants (n = 14). Evaluation for associations with known SIDS risk factors included comparison according to whether infants had a history of exposure to cigarette smoke in the home, and stratification for sleep position and gender. Compared to non-SIDS infants, SIDS infants had significantly decreased {alpha}7 in the caudal nucleus of the solitary tract (cNTS), gracile and cuneate nuclei, with decreased {beta}2 in the cNTS and increased {beta}2 in the facial. When considering only the SIDS cohort: 1-cigarette smoke exposure was associated with increased {alpha}7 in the vestibular nucleus and increased {beta}2 in the rostral dorsal motor nucleus of the vagus, rNTS and Cuneate, 2-there was a gender interaction for {alpha}7 in the gracile and cuneate, and {beta}2 in the cNTS and rostral arcuate nucleus, and 3-there was no effect of sleep position on {alpha}7, but prone sleep was associated with decreased {beta}2 in three nuclei of the pons. In conclusion, SIDS infants demonstrate differences in expression of {alpha}7 and {beta}2 nAChRs within brainstem nuclei that control respiration and arousal, which is independent on prior history of cigarette smoke exposure, especially for the NTS, with additional differences for smoke exposure ({beta}2), gender ({alpha}7 and {beta}2) and sleep position ({beta}2) evident. -- Highlights: Black-Right-Pointing-Pointer The 'normal' response to smoke exposure is decreased {alpha}7 and {beta}2 in certain nuclei. Black-Right-Pointing-Pointer SIDS infants have decreased {alpha}7 in cNTS, Grac and Cun. Black

Identification of a new adapter protein that may link the common beta subunit of the receptor for granulocyte/macrophage colony-stimulating factor, interleukin (IL)-3, and IL-5 to phosphatidylinositol 3-kinase.

Science.gov (United States)

Jücker, M; Feldman, R A

1995-11-17

Binding of human granulocyte/macrophage colony-stimulating factor (hGM-CSF) to its receptor induces the rapid activation of phosphatidylinositol-3 kinase (PI 3-kinase). As hGM-CSF receptor (hGMR) does not contain a consensus sequence for binding of PI 3-kinase, hGMR must use a distinct mechanism for its association with and activation of PI 3-kinase. Here, we describe the identification of a tyrosine-phosphorylated protein of 76-85 kDa (p80) that associates with the common beta subunit of hGMR and with the SH2 domains of the p85 subunit of PI 3-kinase in hGM-CSF-stimulated cells. Src/Yes and Lyn were tightly associated with the p80.PI 3-kinase complex, suggesting that p80 and other phosphotyrosyl proteins present in the complex were phosphorylated by Src family kinases. Tyrosine phosphorylation of p80 was only detected in hGM-CSF or human interleukin-3-stimulated cells, suggesting that activation of p80 might be specific for signaling via the common beta subunit. We postulate that p80 functions as an adapter protein that may participate in linking the hGM-CSF receptor to the PI 3-kinase signaling pathway.

Mining Protein Evolution for Insights into Mechanisms of Voltage-Dependent Sodium Channel Auxiliary Subunits.

Science.gov (United States)

Molinarolo, Steven; Granata, Daniele; Carnevale, Vincenzo; Ahern, Christopher A

2018-02-21

Voltage-gated sodium channel (VGSC) beta (β) subunits have been called the "overachieving" auxiliary ion channel subunit. Indeed, these subunits regulate the trafficking of the sodium channel complex at the plasma membrane and simultaneously tune the voltage-dependent properties of the pore-forming alpha-subunit. It is now known that VGSC β-subunits are capable of similar modulation of multiple isoforms of related voltage-gated potassium channels, suggesting that their abilities extend into the broader voltage-gated channels. The gene family for these single transmembrane immunoglobulin beta-fold proteins extends well beyond the traditional VGSC β1-β4 subunit designation, with deep roots into the cell adhesion protein family and myelin-related proteins - where inherited mutations result in a myriad of electrical signaling disorders. Yet, very little is known about how VGSC β-subunits support protein trafficking pathways, the basis for their modulation of voltage-dependent gating, and, ultimately, their role in shaping neuronal excitability. An evolutionary approach can be useful in yielding new clues to such functions as it provides an unbiased assessment of protein residues, folds, and functions. An approach is described here which indicates the greater emergence of the modern β-subunits roughly 400 million years ago in the early neurons of Bilateria and bony fish, and the unexpected presence of distant homologues in bacteriophages. Recent structural breakthroughs containing α and β eukaryotic sodium channels containing subunits suggest a novel role for a highly conserved polar contact that occurs within the transmembrane segments. Overall, a mixture of approaches will ultimately advance our understanding of the mechanism for β-subunit interactions with voltage-sensor containing ion channels and membrane proteins.

Nonsense mutations in ADTB3A cause complete deficiency of the beta3A subunit of adaptor complex-3 and severe Hermansky-Pudlak syndrome type 2.

Science.gov (United States)

Huizing, Marjan; Scher, Charles D; Strovel, Erin; Fitzpatrick, Diana L; Hartnell, Lisa M; Anikster, Yair; Gahl, William A

2002-02-01

Hermansky-Pudlak syndrome (HPS) is an autosomal recessive disease consisting of oculocutaneous albinism and a storage pool deficiency resulting from absent platelet dense bodies. The disorder is genetically heterogeneous. The majority of patients, including members of a large genetic isolate in northwest Puerto Rico, have mutations in HPS1. Another gene, ADTB3A, was shown to cause HPS-2 in two brothers having compound heterozygous mutations that allowed for residual production of the gene product, the beta3A subunit of adaptor complex-3 (AP-3). This heterotetrameric complex serves as a coat protein-mediating formation of intracellular vesicles, e.g. the melanosome and platelet dense body, from membranes of the trans-Golgi network. We determined the genomic organization of the human ADTB3A gene, with intron/exon boundaries, and describe a third patient with beta3A deficiency. This 5-y-old boy has two nonsense mutations, C1578T (R-->X) and G2028T (E-->X), which produce no ADTB3A mRNA and no beta3A protein. The associated mu3 subunit of AP-3 is also entirely absent. In fibroblasts, the cell biologic concomitant of this deficiency is robust and aberrant trafficking through the plasma membrane of LAMP-3, an integral lysosomal membrane protein normally carried directly to the lysosome. The clinical concomitant is a severe, G-CSF-responsive neutropenia in addition to oculocutaneous albinism and platelet storage pool deficiency. Our findings expand the molecular, cellular, and clinical spectrum of HPS-2 and call for an increased index of suspicion for this diagnosis among patients with features of albinism, bleeding, and neutropenia.

GSK3 inactivation is involved in mitochondrial complex IV defect in transforming growth factor (TGF) {beta}1-induced senescence

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Byun, Hae-Ok; Jung, Hyun-Jung; Seo, Yong-Hak; Lee, Young-Kyoung [Department of Biochemistry and Molecular Biology, Ajou University School of Medicine, Suwon 443-721 (Korea, Republic of); Department of Molecular Science and Technology, The Graduate School, Ajou University, Suwon 443-721 (Korea, Republic of); Hwang, Sung-Chul [Department of Pulmonary and Critical Care Medicine, Ajou University School of Medicine, Suwon 443-721 (Korea, Republic of); Seong Hwang, Eun [Department of Life Science, University of Seoul, Seoul 130-743 (Korea, Republic of); Yoon, Gyesoon, E-mail: ypeace@ajou.ac.kr [Department of Biochemistry and Molecular Biology, Ajou University School of Medicine, Suwon 443-721 (Korea, Republic of); Department of Molecular Science and Technology, The Graduate School, Ajou University, Suwon 443-721 (Korea, Republic of)

2012-09-10

Transforming growth factor {beta}1 (TGF {beta}1) induces Mv1Lu cell senescence by persistently producing mitochondrial reactive oxygen species (ROS) through decreased complex IV activity. Here, we investigated the molecular mechanism underlying the effect of TGF {beta}1 on mitochondrial complex IV activity. TGF {beta}1 progressively phosphorylated the negative regulatory sites of both glycogen synthase kinase 3 (GSK3) {alpha} and {beta}, corresponding well to the intracellular ROS generation profile. Pre-treatment of N-acetyl cysteine, an antioxidant, did not alter this GSK3 phosphorylation (inactivation), whereas pharmacological inhibition of GSK3 by SB415286 significantly increased mitochondrial ROS, implying that GSK3 phosphorylation is an upstream event of the ROS generation. GSK3 inhibition by SB415286 decreased complex IV activity and cellular O{sub 2} consumption rate and eventually induced senescence of Mv1Lu cell. Similar results were obtained with siRNA-mediated knockdown of GSK3. Moreover, we found that GSK3 not only exists in cytosol but also in mitochondria of Mv1Lu cell and the mitochondrial GSK3 binds complex IV subunit 6b which has no electron carrier and is topologically located in the mitochondrial intermembrane space. Involvement of subunit 6b in controlling complex IV activity and overall respiration rate was proved with siRNA-mediated knockdown of subunit 6b. Finally, TGF {beta}1 treatment decreased the binding of the subunit 6b to GSK3 and subunit 6b phosphorylation. Taken together, our results suggest that GSK3 inactivation is importantly involved in TGF {beta}1-induced complex IV defects through decreasing phosphorylation of the subunit 6b, thereby contributing to senescence-associated mitochondrial ROS generation.

Molecular basis of adult-onset and chronic G sub M2 gangliosidoses in patients of Ashkenazi Jewish origin: Substitution of serine for glycine at position 269 of the. alpha. -subunit of. beta. -hexosaminidase

Energy Technology Data Exchange (ETDEWEB)

Paw, B.H.; Kaback, M.M.; Neufeld, E.F. (Univ. of California, Los Angeles (USA))

1989-04-01

Chronic and adult-onset G{sub M2} gangliosidoses are neurological disorders caused by marked deficiency of the A isoenzyme of {beta}-hexosaminidase; they occur in the Ashkenazi Jewish population, though less frequently than classic (infantile) Tay-Sachs disease. Earlier biosynthetic studies had identified a defective {alpha}-subunit that failed to associate with the {beta}-subunit. The authors have now found a guanosine to adenosine transition at the 3{prime} end of exon 7, which causes substitution of serine for glycine at position 269 of the {alpha}-subunit. An RNase protection assay was used to localize the mutation to a segment of mRNA from fibroblasts of a patient with the adult-onset disorder. That segment of mRNA (after reverse transcription) and a corresponding segment of genomic DNA were amplified by the polymerase chain reaction and sequenced by the dideoxy method. The sequence analysis, together with an assay based on the loss of a ScrFI restriction site, showed that the patient was a compound heterozygote who had inherited the 269 (Gly {yields} Ser) mutation from his father and an allelic null mutation from his mother. The 269 (Gly {yields} Ser) mutation, in compound heterozygosity with a presumed null allele, was also found in fetal fibroblasts with an association-defective phenotype and in cells from five patients with chronic G{sub M2} gangliosidosis.

In vivo MRI evaluation of anabolic steroid precursor growth effects in a guinea pig model.

Science.gov (United States)

Tang, Haiying; Vasselli, Joseph R; Tong, Christopher; Heymsfield, Steven B; Wu, Ed X

2009-08-01

Anabolic steroids are widely used to increase skeletal muscle (SM) mass and improve physical performance. Some dietary supplements also include potent steroid precursors or active steroid analogs such as nandrolone. Our previous study reported the anabolic steroid effects on SM in a castrated guinea pig model with SM measured using a highly quantitative magnetic resonance imaging (MRI) protocol. The aim of the current study was to apply this animal model and in vivo MRI protocol to evaluate the growth effects of four widely used over-the-counter testosterone and nandrolone precursors: 4-androstene-3 17-dione (androstenedione), 4-androstene-3beta 17beta-diol (4-androsdiol), 19-nor-4-androstene-3beta-17beta-diol (bolandiol) and 19-nor-4-androstene-3 17-dione (19-norandrostenedione). The results showed that providing precursor to castrated male guinea pigs led to plasma steroid levels sufficient to maintain normal SM growth. The anabolic growth effects of these specific precursors on individual and total muscle volumes, sexual organs, and total adipose tissue over a 10-week treatment period, in comparison with those in the respective positive control testosterone and nandrolone groups, were documented quantitatively by MRI.

The structure of the protein phosphatase 2A PR65/A subunit reveals the conformation of its 15 tandemly repeated HEAT motifs

NARCIS (Netherlands)

Groves, M R; Hanlon, N; Turowski, P; Hemmings, B A; Barford, D

1999-01-01

The PR65/A subunit of protein phosphatase 2A serves as a scaffolding molecule to coordinate the assembly of the catalytic subunit and a variable regulatory B subunit, generating functionally diverse heterotrimers. Mutations of the beta isoform of PR65 are associated with lung and colon tumors. The

Topology characterization of a benzodiazepine-binding beta-rich domain of the GABAA receptor alpha1 subunit.

Science.gov (United States)

Xu, Zhiwen; Fang, Shisong; Shi, Haifeng; Li, Hoiming; Deng, Yiqun; Liao, Yinglei; Wu, Jiun-Ming; Zheng, Hui; Zhu, Huaimin; Chen, Hueih-Min; Tsang, Shui Ying; Xue, Hong

2005-10-01

Structural investigation of GABAA receptors has been limited by difficulties imposed by its trans-membrane-complex nature. In the present study, the topology of a membrane-proximal beta-rich (MPB) domain in the C139-L269 segment of the receptor alpha1 subunit was probed by mapping the benzodiazepine (BZ)-binding and epitopic sites, as well as fluorescence resonance energy transfer (FRET) analysis. Ala-scanning and semiconservative substitutions within this segment revealed the contribution of the phenyl rings of Y160 and Y210, the hydroxy group of S186 and the positive charge on R187 to BZ-binding. FRET with the bound BZ ligand indicated the proximity of Y160, S186, R187, and S206 to the BZ-binding site. On the other hand, epitope-mapping using the monoclonal antibodies (mAbs) against the MPB domain established a clustering of T172, R173, E174, Q196, and T197. Based on the lack of FRET between Trp substitutionally placed at R173 or V198 and bound BZ, this epitope-mapped cluster is located on a separate end of the folded protein from the BZ-binding site. Mutations of the five conserved Cys and Trp residues in the MPB domain gave rise to synergistic and rescuing effects on protein secondary structures and unfolding stability that point to a CCWCW-pentad, reminiscent to the CWC-triad "pin" of immunoglobulin (Ig)-like domains, important for the structural maintenance. These findings, together with secondary structure and fold predictions suggest an anti-parallel beta-strand topology with resemblance to Ig-like fold, having the BZ-binding and the epitopic residues being clustered at two different ends of the fold.

Novel subunit structure observed for noncooperative hemoglobin from Urechis caupo.

Science.gov (United States)

Kolatkar, P R; Meador, W E; Stanfield, R L; Hackert, M L

1988-03-05

Tetrameric hemoglobin from the "fat innkeeper" worm Urechis caupo possesses a novel subunit arrangement having an "inside out" quaternary structure in that the G/H helices are located on the outer surface of the tetramer. A 5-A resolution crystal structure reveals that although the individual subunits are beta-like, having a distinct D helix and the general myoglobin fold, the subunit contacts are very different from those previously observed for hemoglobins. Furthermore, the hemoglobin from U. caupo is also quite different from the unusual hemoglobin tetramer from clam which also has its G/H helices on the outer surface but with the hemes in close proximity through E-F helical contacts (Royer, W. E., Jr., Love, W. E., and Fenderson, F. F. (1985) Nature 316, 277-280).

Crystal structure of the C-terminal domain of the RAP74 subunit of human transcription factor IIF

Energy Technology Data Exchange (ETDEWEB)

Kamada, Katsuhiko; De Angelis, Jacqueline; Roeder, Robert G.; Burley, Stephen K. (Rockefeller)

2012-12-13

The x-ray structure of a C-terminal fragment of the RAP74 subunit of human transcription factor (TF) IIF has been determined at 1.02-{angstrom} resolution. The {alpha}/{beta} structure is strikingly similar to the globular domain of linker histone H5 and the DNA-binding domain of hepatocyte nuclear factor 3{gamma} (HNF-3{gamma}), making it a winged-helix protein. The surface electrostatic properties of this compact domain differ significantly from those of bona fide winged-helix transcription factors (HNF-3{gamma} and RFX1) and from the winged-helix domains found within the RAP30 subunit of TFIIF and the {beta} subunit of TFIIE. RAP74 has been shown to interact with the TFIIF-associated C-terminal domain phosphatase FCP1, and a putative phosphatase binding site has been identified within the RAP74 winged-helix domain.

17 beta-estradiol modifies nitric oxide-sensitive guanylyl cyclase expression and down-regulates its activity in rat anterior pituitary gland.

Science.gov (United States)

Cabilla, Jimena P; Díaz, María del Carmen; Machiavelli, Leticia I; Poliandri, Ariel H; Quinteros, Fernanda A; Lasaga, Mercedes; Duvilanski, Beatriz H

2006-09-01

Previous studies showed that 17 beta-estradiol (17 beta-E2) regulates the nitric oxide (NO)/soluble guanylyl cyclase (sGC)/cGMP pathway in many tissues. Evidence from our laboratory indicates that 17 beta-E2 disrupts the inhibitory effect of NO on prolactin release, decreasing sGC activity and affecting the cGMP pathway in anterior pituitary gland of adult ovariectomized and estrogenized rats. To ascertain the mechanisms by which 17 beta-E2 affects sGC activity, we investigated the in vivo and in vitro effects of 17 beta-E2 on sGC protein and mRNA expression in anterior pituitary gland from immature female rats. In the present work, we showed that 17 beta-E2 acute treatment exerted opposite effects on the two sGC subunits, increasing alpha1 and decreasing beta1 subunit protein and mRNA expression. This action on sGC protein expression was maximal 6-9 h after 17 beta-E2 administration. 17beta-E2 also caused the same effect on mRNA expression at earlier times. Concomitantly, 17 beta-E2 dramatically decreased sGC activity 6 and 9 h after injection. These effects were specific of 17 beta-E2, because they were not observed with the administration of other steroids such as progesterone and 17 alpha-estradiol. This inhibitory action of 17beta-E2 on sGC also required the activation of estrogen receptor (ER), because treatment with the pure ER antagonist ICI 182,780 completely blocked 17 beta-E2 action. 17 beta-E2 acute treatment caused the same effects on pituitary cells in culture. These results suggest that 17 beta-E2 exerts an acute inhibitory effect on sGC in anterior pituitary gland by down-regulating sGC beta 1 subunit and sGC activity in a specific, ER-dependent manner.

Crystal structure of the P pilus rod subunit PapA.

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Denis Verger

2007-05-01

Full Text Available P pili are important adhesive fibres involved in kidney infection by uropathogenic Escherichia coli strains. P pili are assembled by the conserved chaperone-usher pathway, which involves the PapD chaperone and the PapC usher. During pilus assembly, subunits are incorporated into the growing fiber via the donor-strand exchange (DSE mechanism, whereby the chaperone's G1 beta-strand that complements the incomplete immunoglobulin-fold of each subunit is displaced by the N-terminal extension (Nte of an incoming subunit. P pili comprise a helical rod, a tip fibrillum, and an adhesin at the distal end. PapA is the rod subunit and is assembled into a superhelical right-handed structure. Here, we have solved the structure of a ternary complex of PapD bound to PapA through donor-strand complementation, itself bound to another PapA subunit through DSE. This structure provides insight into the structural basis of the DSE reaction involving this important pilus subunit. Using gel filtration chromatography and electron microscopy on a number of PapA Nte mutants, we establish that PapA differs in its mode of assembly compared with other Pap subunits, involving a much larger Nte that encompasses not only the DSE region of the Nte but also the region N-terminal to it.

Common variants in the G protein beta3 subunit gene and thyroid disorders in a formerly iodine-deficient population.

Science.gov (United States)

Völzke, Henry; Bornhorst, Alexa; Rimmbach, Christian; Petersenn, Holger; Geissler, Ingrid; Nauck, Matthias; Wallaschofski, Henri; Kroemer, Heyo K; Rosskopf, Dieter

2009-10-01

Heterotrimeric G proteins are key mediators of signals from membrane receptors-including the thyroid-stimulating hormone (TSH) receptor-to cellular effectors. Gain-of-function mutations in the TSH receptor and the Galpha(S) subunit occur frequently in hyperfunctioning thyroid nodules and differentiated thyroid carcinomas, whereby the T allele of a common polymorphism (825C>T, rs5443) in the G protein beta3 subunit gene (GNB3) is associated with increased G protein-mediated signal transduction and a complex phenotype. The aim of this study was to investigate whether this common polymorphism affects key parameters of thyroid function and morphology and influences the pathogenesis of thyroid diseases in the general population. The population-based cross-sectional Study of Health in Pomerania is a general health survey with focus on thyroid diseases in northeast Germany, a formerly iodine-deficient area. Data from 3428 subjects (1800 men and 1628 women) were analyzed for an association of the GNB3 genotype with TSH, free triiodothyronine and thyroxine levels, urine iodine and thiocyanate excretion, and thyroid ultrasound morphology including thyroid volume, presence of goiter, and thyroid nodules. There was no association between GNB3 genotype status and the functional or morphological thyroid parameters investigated, neither in crude analyses nor upon multivariable analyses including known confounders of thyroid disorders. Based on the data from this large population-based survey, we conclude that the GNB3 825C>T polymorphism does not affect key parameters of thyroid function and morphology in the general population of a formerly iodine-deficient area.

Generation of the beta-amyloid peptide and the amyloid precursor protein C-terminal fragment gamma are potentiated by FE65L1.

Science.gov (United States)

Chang, Yang; Tesco, Giuseppina; Jeong, William J; Lindsley, Loren; Eckman, Elizabeth A; Eckman, Christopher B; Tanzi, Rudolph E; Guénette, Suzanne Y

2003-12-19

Members of the FE65 family of adaptor proteins, FE65, FE65L1, and FE65L2, bind the C-terminal region of the amyloid precursor protein (APP). Overexpression of FE65 and FE65L1 was previously reported to increase the levels of alpha-secretase-derived APP (APPs alpha). Increased beta-amyloid (A beta) generation was also observed in cells showing the FE65-dependent increase in APPs alpha. To understand the mechanism for the observed increase in both A beta and APPs alpha given that alpha-secretase cleavage of a single APP molecule precludes A beta generation, we examined the effects of FE65L1 overexpression on APP C-terminal fragments (APP CTFs). Our data show that FE65L1 potentiates gamma-secretase processing of APP CTFs, including the amyloidogenic CTF C99, accounting for the ability of FE65L1 to increase generation of APP C-terminal domain and A beta 40. The FE65L1 modulation of these processing events requires binding of FE65L1 to APP and APP CTFs and is not because of a direct effect on gamma-secretase activity, because Notch intracellular domain generation is not altered by FE65L1. Furthermore, enhanced APP CTF processing can be detected in early endosome vesicles but not in endoplasmic reticulum or Golgi membranes, suggesting that the effects of FE65L1 occur at or near the plasma membrane. Finally, although FE65L1 increases APP C-terminal domain production, it does not mediate the APP-dependent transcriptional activation observed with FE65.

Plasma beta HCG determination

International Nuclear Information System (INIS)

Amaral, L.B.D.; Pinto, J.C.M.; Linhares, E.; Linhares, Estevao

1981-01-01

There are three important indications for the early diagnosis of pregnancy through the determination of the beta sub-unit of chorionic gonadotrophin using radioimmunoassay: 1) some patient's or doctor's anxiety to discover the problem; 2) when it will be necessary to employ diagnostic or treatment procedures susceptible to affect the ovum; and 3) in the differential diagnosis of amenorrhoea, uterine hemorrhage and abdominal tumors. Other user's are the diagnosis of missed absortion, and the diagnosis and follow-up of chrorioncarcinoma. The AA. studied 200 determinations of plasma beta-HCG, considering the main difficulties occuring in the clinical use of this relevant laboratory tool in actual Obstetrics. (author) [pt

The prion protein as a receptor for amyloid-beta

NARCIS (Netherlands)

Kessels, Helmut W.; Nguyen, Louis N.; Nabavi, Sadegh; Malinow, Roberto

2010-01-01

Increased levels of brain amyloid-beta, a secreted peptide cleavage product of amyloid precursor protein (APP), is believed to be critical in the aetiology of Alzheimer's disease. Increased amyloid-beta can cause synaptic depression, reduce the number of spine protrusions (that is, sites of synaptic

Immunochemical analysis of Micrococcus lysodeikticus (luteus) F1-ATPase and its subunits.

Science.gov (United States)

Urban, C; Salton, M R

1983-08-31

The F1-ATPase from Micrococcus lysodeikticus has been purified to 95% protein homogeneity in this laboratory and as all other bacterial F1S, possesses five distinct subunits with molecular weights ranging from 60 000 to 10 000 (Huberman, M. and Salton, M.R.J. (1979) Biochim. Biophys. Acta 547, 230-240). In this communication, we demonstrate the immunochemical reactivities of antibodies to native and SDS-dissociated subunits with the native and dissociated F1-ATPase and show that: (1) the antibodies generated to the native or SDS-dissociated subunits react with the native molecule; (2) all of the subunits comprising the F1 are antigenically unique as determined by crossed immunoelectrophoresis and the Ouchterlony double-diffusion techniques; (3) antibodies to the SDS-denatured individual delta- and epsilon-subunits can be used to destabilize the interaction of these specific subunits with the rest of the native F1; and (4) all subunit antibodies as well as anti-native F1 were found to inhibit ATPase activity to varying degrees, the strongest inhibition being seen with antibodies to the total F1 and anti-alpha- and anti-beta-subunit antibodies. The interaction of specific subunit antibodies may provide a new and novel way to study further and characterize the catalytic portions of F1-ATPases and in general may offer an additional method for the examination of multimeric proteins.

Identification of Leptospira serovars by RFLP of the RNA polymerase beta subunit gene (rpoB

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Lenice Roteia Cardoso Jung

2015-06-01

Full Text Available Leptospires are usually classified by methods based on DNA-DNA hybridization and the conventional cross-agglutination absorption test, which uses polyclonal antibodies against lipopolysaccharides. In this study, the amplification of the rpoB gene, which encodes the beta-subunit of RNA polymerase, was used as an alternative tool to identify Leptospira. DNA extracts from sixty-eight serovars were obtained, and the hypervariable region located between 1990 and 2500-bp in the rpoB gene was amplified by polymerase chain reaction (PCR. The 600-bp amplicons of the rpoB gene were digested with the restriction endonucleases TaqI, Tru1I, Sau3AI and MslI, and the restriction fragments were separated by 6% polyacrylamide gel electrophoresis. Thirty-five fragment patters were obtained from the combined data of restriction fragment length polymorphism (PCR-RFLP analysis and used to infer the phylogenetic relationships among the Leptospira species and serovars. The species assignments obtained were in full agreement with the established taxonomic classifications. Twenty-two serovars were effectively identified based on differences in their molecular profiles. However, the other 46 serovars remained clustered in groups that included more than one serovar of different species. This study demonstrates the value of RFLP analysis of PCR-amplified rpoB as an initial method for identifying Leptospira species and serovars.

Identification of Leptospira serovars by RFLP of the RNA polymerase beta subunit gene (rpoB).

Science.gov (United States)

Jung, Lenice Roteia Cardoso; Bomfim, Maria Rosa Quaresma; Kroon, Erna Geessien; Nunes, Álvaro Cantini

2015-06-01

Leptospires are usually classified by methods based on DNA-DNA hybridization and the conventional cross-agglutination absorption test, which uses polyclonal antibodies against lipopolysaccharides. In this study, the amplification of the rpoB gene, which encodes the beta-subunit of RNA polymerase, was used as an alternative tool to identify Leptospira. DNA extracts from sixty-eight serovars were obtained, and the hypervariable region located between 1990 and 2500-bp in the rpoB gene was amplified by polymerase chain reaction (PCR). The 600-bp amplicons of the rpoB gene were digested with the restriction endonucleases TaqI, Tru1I, Sau3AI and MslI, and the restriction fragments were separated by 6% polyacrylamide gel electrophoresis. Thirty-five fragment patters were obtained from the combined data of restriction fragment length polymorphism (PCR-RFLP) analysis and used to infer the phylogenetic relationships among the Leptospira species and serovars. The species assignments obtained were in full agreement with the established taxonomic classifications. Twenty-two serovars were effectively identified based on differences in their molecular profiles. However, the other 46 serovars remained clustered in groups that included more than one serovar of different species. This study demonstrates the value of RFLP analysis of PCR-amplified rpoB as an initial method for identifying Leptospira species and serovars.

Increased Expression of Laminin Subunit Alpha 1 Chain by dCas9-VP160

OpenAIRE

Perrin, Arnaud; Rousseau, Jo?l; Tremblay, Jacques P.

2016-01-01

Laminin-111 protein complex links the extracellular matrix to integrin α7β1 in sarcolemma, thus replacing in dystrophic muscles links normally insured by the dystrophin complex. Laminin-111 injection in mdx mouse stabilized sarcolemma, restored serum creatine kinase to wild-type levels, and protected muscles from exercised-induced damages. These results suggested that increased laminin-111 is a potential therapy for DMD. Laminin subunit beta 1 and laminin subunit gamma 1 are expressed in adul...

β-Arrestin interacts with the beta/gamma subunits of trimeric G-proteins and dishevelled in the Wnt/Ca(2+ pathway in xenopus gastrulation.

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Katharina Seitz

Full Text Available β-Catenin independent, non-canonical Wnt signaling pathways play a major role in the regulation of morphogenetic movements in vertebrates. The term non-canonical Wnt signaling comprises multiple, intracellularly divergent, Wnt-activated and β-Catenin independent signaling cascades including the Wnt/Planar Cell Polarity and the Wnt/Ca(2+ cascades. Wnt/Planar Cell Polarity and Wnt/Ca(2+ pathways share common effector proteins, including the Wnt ligand, Frizzled receptors and Dishevelled, with each other and with additional branches of Wnt signaling. Along with the aforementioned proteins, β-Arrestin has been identified as an essential effector protein in the Wnt/β-Catenin and the Wnt/Planar Cell Polarity pathway. Our results demonstrate that β-Arrestin is required in the Wnt/Ca(2+ signaling cascade upstream of Protein Kinase C (PKC and Ca(2+/Calmodulin-dependent Protein Kinase II (CamKII. We have further characterized the role of β-Arrestin in this branch of non-canonical Wnt signaling by knock-down and rescue experiments in Xenopus embryo explants and analyzed protein-protein interactions in 293T cells. Functional interaction of β-Arrestin, the β subunit of trimeric G-proteins and Dishevelled is required to induce PKC activation and membrane translocation. In Xenopus gastrulation, β-Arrestin function in Wnt/Ca(2+ signaling is essential for convergent extension movements. We further show that β-Arrestin physically interacts with the β subunit of trimeric G-proteins and Dishevelled, and that the interaction between β-Arrestin and Dishevelled is promoted by the beta/gamma subunits of trimeric G-proteins, indicating the formation of a multiprotein signaling complex.

Synthesis of an A'B' Precursor to Angelmicin B: Product Diversification in the Suárez Lactol Fragmentation.

Science.gov (United States)

Li, Jialiang; Todaro, Louis; Mootoo, David R

2011-11-01

We describe a synthetic strategy for the angelimicin family of anthraquinoid natural products that involves converting a central highly oxygenated decalin intermediate to the AB and A'B' subunits. Herein, we report the synthesis of the bicyclic A'B' subunit that complements our earlier route to the tricyclic AB framework. The differentiating tact in the two syntheses focused on controlling the Suárez radical fragmentation of lactol precursors by modulating the substrate's structural rigidity. A more flexible lactol gave the tricyclic AB framework, whereas a more rigid substrate led to the bicyclic A'B' precursor, presumably through divergent pathways from the radical produced in the initial fragmentation step. These results establish a versatile advanced synthetic precursor for the angelimicins, and on a more general note, illustrate strategies for applying the Suárez fragmentation to diverse and complex molecular frameworks.

Differential expression of gill Na⁺,K⁺-ATPase alpha- and beta-subunits, Na⁺,K⁺,2Cl^- cotransporter and CFTR anion channel in juvenile anadromous and landlocked Atlantic salmon Salmo salar

DEFF Research Database (Denmark)

Nilsen, Tom O.; Ebbesson, Lars O. E.; Madsen, Steffen S.

2007-01-01

This study examines changes in gill Na(+),K(+)-ATPase (NKA) alpha- and beta-subunit isoforms, Na(+),K(+),2Cl(-) cotransporter (NKCC) and cystic fibrosis transmembrane conductance regulator (CFTR I and II) in anadromous and landlocked strains of Atlantic salmon during parr-smolt transformation, an...

Powerful beneficial effects of benfotiamine on cognitive impairment and beta-amyloid deposition in amyloid precursor protein/presenilin-1 transgenic mice.

Science.gov (United States)

Pan, Xiaoli; Gong, Neng; Zhao, Jing; Yu, Zhe; Gu, Fenghua; Chen, Jia; Sun, Xiaojing; Zhao, Lei; Yu, Meijing; Xu, Zhiru; Dong, Wenxin; Qin, Yan; Fei, Guoqiang; Zhong, Chunjiu; Xu, Tian-Le

2010-05-01

Reduction of glucose metabolism in brain is one of the main features of Alzheimer's disease. Thiamine (vitamin B1)-dependent processes are critical in glucose metabolism and have been found to be impaired in brains from patients with Alzheimer's disease. However, thiamine treatment exerts little beneficial effect in these patients. Here, we tested the effect of benfotiamine, a thiamine derivative with better bioavailability than thiamine, on cognitive impairment and pathology alterations in a mouse model of Alzheimer's disease, the amyloid precursor protein/presenilin-1 transgenic mouse. We show that after a chronic 8 week treatment, benfotiamine dose-dependently enhanced the spatial memory of amyloid precursor protein/presenilin-1 mice in the Morris water maze test. Furthermore, benfotiamine effectively reduced both amyloid plaque numbers and phosphorylated tau levels in cortical areas of the transgenic mice brains. Unexpectedly, these effects were not mimicked by another lipophilic thiamine derivative, fursultiamine, although both benfotiamine and fursultiamine were effective in increasing the levels of free thiamine in the brain. Most notably, benfotiamine, but not fursultiamine, significantly elevated the phosphorylation level of glycogen synthase kinase-3alpha and -3beta, and reduced their enzymatic activities in the amyloid precursor protein/presenilin-1 transgenic brain. Therefore, in the animal Alzheimer's disease model, benfotiamine appears to improve the cognitive function and reduce amyloid deposition via thiamine-independent mechanisms, which are likely to include the suppression of glycogen synthase kinase-3 activities. These results suggest that, unlike many other thiamine-related drugs, benfotiamine may be beneficial for clinical Alzheimer's disease treatment.

[Three regions of Rpb10 mini-subunit of nuclear RNA polymerases are strictly conserved in all eukaryotes].

Science.gov (United States)

Shpakovskiĭ, G V; Lebedenko, E N

1996-12-01

The rpb10+ cDNA from the fission yeast Schizosaccharomyces pombe was cloned using two independent approaches (PCR and genetic suppression). The cloned cDNA encoded the Rpb10 subunit common for all three RNA polymerases. Comparison of the deduced amino acid sequence of the Sz. pombe Rbp10 subunit (71 amino acid residues) with those of the homologous subunits of RNA polymerases I, II, and III from Saccharomyces cerevisiae and Home sapiens revealed that heptapeptides RCFT/SCGK (residues 6-12), RYCCRRM (residues 43-49), and HVDLIEK (residues 53-59) were evolutionarily the most conserved structural motifs of these subunits. It is shown that the Rbp10 subunit from Sz. pombe can substitute its homolog (ABC10 beta) in the baker's yeast S. cerevisiae.

The DFT-DVM theoretical study of the differences of quadrupole splitting and the iron electronic structure for the rough heme models for {alpha}- and {beta}-subunits in deoxyhemoglobin and for deoxymyoglobin

Energy Technology Data Exchange (ETDEWEB)

Yuryeva, E. I. [Institute of Solid State Chemistry of the Ural Branch of the Russian Academy of Sciences (Russian Federation); Oshtrakh, M. I., E-mail: oshtrakh@mail.utnet.ru [Ural State Technical University-UPI, Faculty of Physical Techniques and Devices for Quality Control (Russian Federation)

2008-01-15

Quantum chemical calculations of the iron electron structure and {sup 57}Fe quadrupole splitting were made by density functional theory and X{alpha} discrete variation method for the rough heme models for {alpha}- and {beta}-subunits in deoxyhemoglobin and for deoxymyoglobin accounting stereochemical differences of the active sites in native proteins. The calculations revealed differences of quadrupole splitting temperature dependences for three models indicating sensitivity of quadrupole splitting and Fe(II) electronic structure to small variations of iron stereochemistry.

High Affinity IgE-Fc Receptor alpha and gamma Subunit Interactions

International Nuclear Information System (INIS)

Rashid, A.; Housden, J. E. M.; Sabban, S.; Helm, B.

2014-01-01

Objective: To explore the relationships between the subunits (alpha, beta and gamma) of the high affinity IgE receptor (Fc and RI) and its ability to mediate transmembrane signaling. Study Design: Experimental study. Place and Duration of Study: Department of Molecular Biology and Biotechnology, University of Sheffield, UK, from 2008 to 2009. Methodology: The approach employed was to create a chimera (human alpha-gamma-gamma) using the extracellular (EC) domain of the human high affinity IgE receptor. The alpha subunit (huFc and RIalpha) of IgE receptor was spliced onto the rodent gamma TM and cytoplasmic domain (CD). This was transfected into the Rat Basophilic Leukemia cell line in order to assess the possibility of selectively activating cells transfected with this single pass construct for antigen induced mediator release. Results: The RBLs cell lines transfected with the huFc and RIalpha/gamma/gamma cDNA constructs were assessed for the cell surface expression of the huFc and RIalpha subunit and the response to the antigenic stimulus by looking for degranulation and intracellular Ca2+ mobilisation. The results obtained showed the absence of huFc and RIalpha subunit expression on the surface of transfected cells as seen by flowcytometric studies, beta-hexosaminidase assays and intracellular calcium mobilisation studies. Conclusion: In the present study the grounds for non-expression of huFc and RIalpha/gamma/gamma cDNA remains elusive but may be due to the fact that the human-rodent chimeric receptors are assembled differently than the endogenous rodent receptors as seen in study in which COS 7 cells were transfected with human/rat chimeric complexes. (author)

Sawtooth crashes at high beta on JET

Energy Technology Data Exchange (ETDEWEB)

Alper, B; Huysmans, G T.A.; Sips, A C.C. [Commission of the European Communities, Abingdon (United Kingdom). JET Joint Undertaking; Nave, M F.F. [Universidade Tecnica, Lisbon (Portugal). Inst. Superior Tecnico

1994-07-01

The sawtooth crashes on JET display features which depend on beta. The main observation is a transient bulging of flux surfaces (duration inferior to 30 microsec.), which is predominantly on the low field side and extends to larger radii as beta increases. This phenomenon reaches the plasma boundary when beta{sub N} exceeds 0.5 and in these cases is followed by an ELM within 50 microsec. These sawtooth/ELM events limit plasma performance. Modelling of mode coupling shows qualitative agreement between observations of the structure of the sawtooth precursor and the calculated internal kink mode at high beta. (authors). 6 refs., 5 figs.

Specific labeling of peptidoglycan precursors as a tool for bacterial cell wall studies

NARCIS (Netherlands)

van Dam, V.; Olrichs, N.K.; Breukink, E.J.

2009-01-01

Wall chart: The predominant component of the bacterial cell wall, peptidoglycan, consists of long alternating stretches of aminosugar subunits interlinked in a large three-dimensional network and is formed from precursors through several cytosolic and membrane-bound steps. The high tolerance of the

Small heat shock protein HspB8: its distribution in Alzheimer's disease brains and its inhibition of amyloid-beta protein aggregation and cerebrovascular amyloid-beta toxicity.

NARCIS (Netherlands)

Wilhelmus, M.M.M.; Boelens, W.C.; Otte-Holler, I.; Kamps, B.; Kusters, B.; Maat-Schieman, M.L.; Waal, R.M.W. de; Verbeek, M.M.

2006-01-01

Alzheimer's disease (AD) is characterized by pathological lesions, such as senile plaques (SPs) and cerebral amyloid angiopathy (CAA), both predominantly consisting of a proteolytic cleavage product of the amyloid-beta precursor protein (APP), the amyloid-beta peptide (Abeta). CAA is also the major

Structure of the T cell receptor in a Ti alpha V beta 2, alpha V beta 8-positive T cell line

DEFF Research Database (Denmark)

Hou, X; Dietrich, J; Kuhlmann, J

1994-01-01

not known; however, it has been suggested that each TcR contains two Ti dimers. To gain insight into the structure of the TcR we constructed a Ti alpha V beta 2, alpha V beta 8-positive T cell line which expressed the endogenous human TiV beta 8 and the transfected mouse TiV beta 2 both in association......The T cell receptor (TcR) is composed of at least six different polypeptide chains consisting of the clonotypic Ti heterodimer (Ti alpha beta or Ti gamma delta) and the noncovalently associated CD3 chains (CD3 gamma delta epsilon zeta). The exact number of subunits constituting the TcR is still...... with the endogenous Ti alpha and CD3 chains at the cell surface. Preclearing experiments with radioiodinated cell lysate prepared with digitonin lysis buffer demonstrated that depleting the lysate of Ti alpha V beta 8 by immunoprecipitation with anti V beta 8 monoclonal antibody (mAb) did not reduce the amount of Ti...

Compartmentalized beta subunit distribution determines characteristics and ethanol sensitivity of somatic, dendritic, and terminal large-conductance calcium-activated potassium channels in the rat central nervous system.

Science.gov (United States)

Wynne, P M; Puig, S I; Martin, G E; Treistman, S N

2009-06-01

Neurons are highly differentiated and polarized cells, whose various functions depend upon the compartmentalization of ion channels. The rat hypothalamic-neurohypophysial system (HNS), in which cell bodies and dendrites reside in the hypothalamus, physically separated from their nerve terminals in the neurohypophysis, provides a particularly powerful preparation in which to study the distribution and regional properties of ion channel proteins. Using electrophysiological and immunohistochemical techniques, we characterized the large-conductance calcium-activated potassium (BK) channel in each of the three primary compartments (soma, dendrite, and terminal) of HNS neurons. We found that dendritic BK channels, in common with somatic channels but in contrast to nerve terminal channels, are insensitive to iberiotoxin. Furthermore, analysis of dendritic BK channel gating kinetics indicates that they, like somatic channels, have fast activation kinetics, in contrast to the slow gating of terminal channels. Dendritic and somatic channels are also more sensitive to calcium and have a greater conductance than terminal channels. Finally, although terminal BK channels are highly potentiated by ethanol, somatic and dendritic channels are insensitive to the drug. The biophysical and pharmacological properties of somatic and dendritic versus nerve terminal channels are consistent with the characteristics of exogenously expressed alphabeta1 versus alphabeta4 channels, respectively. Therefore, one possible explanation for our findings is a selective distribution of auxiliary beta1 subunits to the somatic and dendritic compartments and beta4 to the terminal compartment. This hypothesis is supported immunohistochemically by the appearance of distinct punctate beta1 or beta4 channel clusters in the membrane of somatic and dendritic or nerve terminal compartments, respectively.

Expression of the alpha 6 beta 4 integrin by squamous cell carcinomas and basal cell carcinomas: possible relation to invasive potential?

DEFF Research Database (Denmark)

Rossen, K; Dahlstrøm, K K; Mercurio, A M

1994-01-01

We have studied the expression of alpha 6 beta 4 integrin, a carcinoma laminin receptor in ten squamous cell carcinomas (SCCs) and ten basal cell carcinomas (BCCs) of the skin in order to examine whether changes in alpha 6 beta 4 integrin expression may be related to invasive and metastatic...... potential. Monoclonal antibodies specific for each subunit were applied on cryosections, using a three step indirect peroxidase technique. In normal epidermis the basal cells expressed both the alpha 6 and the beta 4 subunits, and the expression was polarized against the basement membrane. In SCCs...

Localization in the Nucleolus and Coiled Bodies of Protein Subunits of the Ribonucleoprotein Ribonuclease P

Science.gov (United States)

Jarrous, Nayef; Wolenski, Joseph S.; Wesolowski, Donna; Lee, Christopher; Altman, Sidney

1999-01-01

The precise location of the tRNA processing ribonucleoprotein ribonuclease P (RNase P) and the mechanism of its intranuclear distribution have not been completely delineated. We show that three protein subunits of human RNase P (Rpp), Rpp14, Rpp29 and Rpp38, are found in the nucleolus and that each can localize a reporter protein to nucleoli of cells in tissue culture. In contrast to Rpp38, which is uniformly distributed in nucleoli, Rpp14 and Rpp29 are confined to the dense fibrillar component. Rpp29 and Rpp38 possess functional, yet distinct domains required for subnucleolar localization. The subunit Rpp14 lacks such a domain and appears to be dependent on a piggyback process to reach the nucleolus. Biochemical analysis suggests that catalytically active RNase P exists in the nucleolus. We also provide evidence that Rpp29 and Rpp38 reside in coiled bodies, organelles that are implicated in the biogenesis of several other small nuclear ribonucleoproteins required for processing of precursor mRNA. Because some protein subunits of RNase P are shared by the ribosomal RNA processing ribonucleoprotein RNase MRP, these two evolutionary related holoenzymes may share common intranuclear localization and assembly pathways to coordinate the processing of tRNA and rRNA precursors. PMID:10444065

The elusive indigo precursors in woad (Isatis tinctoria L.)--identification of the major indigo precursor, isatan A, and a structure revision of isatan B.

Science.gov (United States)

Oberthür, Christine; Schneider, Bernd; Graf, Heidemarie; Hamburger, Matthias

2004-01-01

A metabolite-profiling study of shock-frozen leaves of Isatis tinctoria L., an old indigo dye plant and medicinal herb, revealed a complex pattern of indigo-forming compounds with higher polarities than the known indigo precursors isatan B and indican. These highly unstable compounds underwent rapid post-harvest transformation and were not detected in air-dried leaves. The major indigo precursor, named isatan A (4), was isolated by rapid normal-phase and gel chromatography, along with isatan B (3). A full spectral data set of 3 showed that the previous structure assignment as 'indoxyl-5-ketogluconate' has to be revised to 1H-indol-3-yl beta-D-ribohex-3-ulopyranoside. Isatan A (4) was identified as 1H-indol-3-yl 6'-O-(carboxyacetyl)-beta-D-ribohex-3'-ulopyranoside. In aqueous solution, glycosides 3 and 4 occur as hydrates and undergo rapid hydrolysis under very mild acidic or basic conditions.

Complementation of Escherichia coli uncD mutant strains by a chimeric F1-beta subunit constructed from E. coli and spinach chloroplast F1-beta

NARCIS (Netherlands)

Burkovski, Andreas; Lill, H; Engelbrecht, Siegfried

1994-01-01

ATP-synthesizing F0F1-ATPases are complex enzymes consisting of at least eight different subunits. These subunits are conserved during evolution to a very variable degree ranging in pairwise comparison between, for example, Escherichia coli and spinach chloroplast from 20% to 66% identical residues.

Asymmetric expression of protein kinase CK2 subunits in human kidney tumors

DEFF Research Database (Denmark)

Stalter, G; Siemer, S; Becht, E

1994-01-01

of protein kinase CK2 alpha in tumors/normal tissue (T/N) was 1.58 and that of the protein kinase CK2 beta (T/N) was 2.65. The data suggest that the generally described increase in protein kinase CK2 activity in tumor cells may to some extent result from a deregulation in subunit biosynthesis or degradation...

Decreased agonist sensitivity of human GABA(A) receptors by an amino acid variant, isoleucine to valine, in the alpha1 subunit.

Science.gov (United States)

Westh-Hansen, S E; Rasmussen, P B; Hastrup, S; Nabekura, J; Noguchi, K; Akaike, N; Witt, M R; Nielsen, M

1997-06-25

Recombinant human GABA(A) receptors were investigated in vitro by coexpression of cDNAs coding for alpha1, beta2, and gamma2 subunits in the baculovirus/Sf-9 insect cell system. We report that a single amino acid exchange (isoleucine 121 to valine 121) in the N-terminal, extracellular part of the alpha1 subunit induces a marked decrease in agonist GABA(A) receptor ligand sensitivity. The potency of muscimol and GABA to inhibit the binding of the GABA(A) receptor antagonist [3H]SR 95531 (2-(3-carboxypropyl)-3-amino-6-(4-methoxyphenyl)pyridazinium bromide) was higher in receptor complexes of alpha1(ile 121) beta2gamma2 than in those of alpha1(val 121) beta2gamma2 (IC50 values were 32-fold and 26-fold lower for muscimol and GABA, respectively). The apparent affinity of the GABA(A) receptor antagonist bicuculline methiodide to inhibit the binding of [3H]SR 95531 did not differ between the two receptor complex variants. Electrophysiological measurements of GABA induced whole-cell Cl- currents showed a ten-fold decrease in the GABA(A) receptor sensitivity of alpha1 (val 121) beta2gamma2 as compared to alpha1(ile 121) beta2gamma2 receptor complexes. Thus, a relatively small change in the primary structure of the alpha1 subunit leads to a decrease selective for GABA(A) receptor sensitivity to agonist ligands, since no changes were observed in a GABA(A) receptor antagonist affinity and benzodiazepine receptor binding.

Purification and properties of a beta-galactosidase from carambola fruit with significant activity towards cell wall polysaccharides.

Science.gov (United States)

Balasubramaniam, Sumathi; Lee, Heng Chin; Lazan, Hamid; Othman, Roohaida; Ali, Zainon Mohd

2005-01-01

beta-Galactosidase (EC. 3.2.1.23) from ripe carambola (Averrhoa carambola L. cv. B10) fruit was fractionated through a combination of ion exchange and gel filtration chromatography into four isoforms, viz. beta-galactosidase I, II, III and IV. This beta-galactosidases had apparent native molecular masses of 84, 77, 58 and 130 kDa, respectively. beta-Galactosidase I, the predominant isoform, was purified to electrophoretic homogeneity; analysis of the protein by SDS-PAGE revealed two subunits with molecular masses of 48 and 36 kDa. N-terminal amino acid sequence of the respective polypeptides shared high similarities albeit at different domains, with the deduced amino acid sequence of certain plant beta-galactosidases, thus, explaining the observed low similarity between the two subunits. beta-Galactosidase I was probably a heterodimer that have glycoprotein properties and a pI value of 7.2, with one of the potential glycosylation sites appeared to reside within the 48-kDa-polypeptide. The purified beta-galactosidase I was substantially active in hydrolyzing (1-->4)beta-linked spruce and a mixture of (1-->3)beta- and (1-->6)beta-linked gum arabic galactans. This isoform also had the capability to solubilize and depolymerize structurally intact pectins as well as to modify alkaline-soluble hemicelluloses, reflecting in part changes that occur during ripening.

Generation of Transplantable Beta Cells for Patient-Specific Cell Therapy

Directory of Open Access Journals (Sweden)

Xiaojie Wang

2012-01-01

Full Text Available Islet cell transplantation offers a potential cure for type 1 diabetes, but it is challenged by insufficient donor tissue and side effects of current immunosuppressive drugs. Therefore, alternative sources of insulin-producing cells and isletfriendly immunosuppression are required to increase the efficiency and safety of this procedure. Beta cells can be transdifferentiated from precursors or another heterologous (non-beta-cell source. Recent advances in beta cell regeneration from somatic cells such as fibroblasts could circumvent the usage of immunosuppressive drugs. Therefore, generation of patient-specific beta cells provides the potential of an evolutionary treatment for patients with diabetes.

Radiosynthesis and ex vivo evaluation of [{sup 11}C]-SIB-1553A as a PET radiotracer for {beta}4 selective subtype nicotinic acetylcholine receptor

Energy Technology Data Exchange (ETDEWEB)

Sobrio, Franck [CEA, DSV, I2BM, CINAPS, LDMTEP, Caen, F-14074 (France); Universite de Caen Basse-Normandie, UMR CEA E2, Centre Cyceron, Caen, F-14074 (France)], E-mail: sobrio@cyceron.fr; Quentin, Thomas; Dhilly, Martine; Bourdier, Thomas; Tymciu, Sylvie; Debruyne, Daniele; Barre, Louisa [CEA, DSV, I2BM, CINAPS, LDMTEP, Caen, F-14074 (France); Universite de Caen Basse-Normandie, UMR CEA E2, Centre Cyceron, Caen, F-14074 (France)

2008-04-15

[{sup 11}C]-SIB-1553A (({+-})-4-[2-((N-[{sup 11}C]-methyl)-2-pyrrolidinyl)ethyl]thiophenol) was labelled with carbon-11 (t{sub 1/2}=20.4 min) and evaluated in vivo as potential radiotracer for noninvasive assessment of the {beta}4 subunit nicotinic acetylcholine neurotransmission system with positron emission tomography (PET). The labelling precursor was obtained within five steps from N-Boc-prolinal in 45-56% overall yields. The radiosynthesis of [{sup 11}C]-SIB-1553A was achieved by a selective N-[{sup 11}C]-methylation in 32 min with a radiochemical purity greater than 97%, 7.5-30 GBq/{mu}mol of specific radioactivity and 55-65% radiochemical yield (decay corrected, based on [{sup 11}C]methyl iodide). The ex vivo pharmacological profile of [{sup 11}C]-SIB-1553A was evaluated in rats with biodistribution studies in organs and in brain structures by autoradiography. The radiotracer uptake in the brain reached 0.49 %ID/g at 10 min and no brain radiometabolite was detected 40 min after intravenous injection. The quantification of radioactivity in various cerebral structures indicated a significantly higher radioactivity level at 15 min than at 30 min. Among the {beta}4 nAChR subunit-rich structures studied in the rat brain, only the thalamus at 15 and 30 min and the hippocampus at 30 min showed significantly higher uptake. Moreover, competition studies performed with SIB-1553A (15 min before the radiotracer injection) revealed only a low specific binding estimated to 7% of the total binding at 15 min and 13% at 30 min.

Dysfunctional C8 beta chain in patients with C8 deficiency.

Science.gov (United States)

Tschopp, J; Penea, F; Schifferli, J; Späth, P

1986-12-01

Two sera from unrelated individuals, each lacking C8 activity, were examined by Western blot analysis. Using antisera raised against whole C8, the two sera are shown to lack the C8 beta chain, indicating a C8 beta deficiency, which is frequently observed in cases of dysfunctional C8. In contrast, by means of a specific anti-C8-beta antiserum, a C8 beta-like polypeptide chain of apparently identical molecular weight compared to normal C8 beta was detected. Digestion of normal and dysfunctional C8 beta with Staphylococcus aureus V8 protease revealed distinct differences in the enzymatic digestion pattern. We conclude that the dysfunction in the C8 protein in these two patients resides in the dysfunctional C8 beta chain, and that this form of C8 deficiency is distinct from C8 deficiencies previously reported, in which one or both C8 subunits are lacking.

Guanine nucleotide-binding protein subunit beta-2-like 1, a new Annexin A7 interacting protein

International Nuclear Information System (INIS)

Du, Yue; Meng, Jinyi; Huang, Yuhong; Wu, Jun; Wang, Bo; Ibrahim, Mohammed M.; Tang, Jianwu

2014-01-01

Highlights: • RACK1 formed a complex with Annexin A7. • Depletion of RACK1 inhibited the proliferation, migration and invasion. • RACK1 RNAi abolished RACK1-Annexin A7 interaction. • RACK1-Annexin A7 may play a role in regulating the metastatic potentials. - Abstract: We report for the first time that Guanine nucleotide-binding protein subunit beta-2-like 1 (RACK1) formed a complex with Annexin A7. Hca-F and Hca-P are a pair of syngeneic mouse hepatocarcinoma cell lines established and maintained in our laboratory. Our previous study showed that both Annexin A7 and RACK1 were expressed higher in Hca-F (lymph node metastasis >70%) than Hca-P (lymph node metastasis <30%). Suppression of Annexin A7 expression in Hca-F cells induced decreased migration and invasion ability. In this study, knockdown of RACK1 by RNA interference (RNAi) had the same impact on metastasis potential of Hca-F cells as Annexin A7 down-regulation. Furthermore, by co-immunoprecipitation and double immunofluorescence confocal imaging, we found that RACK1 was in complex with Annexin A7 in control cells, but not in the RACK1-down-regulated cells, indicating the abolishment of RACK1-Annexin A7 interaction in Hca-F cells by RACK1 RNAi. Taken together, these results suggest that RACK1-Annexin A7 interaction may be one of the means by which RACK1 and Annexin A7 influence the metastasis potential of mouse hepatocarcinoma cells in vitro

Silencing of beta-carotene hydroxylase increases total carotenoid and beta-carotene levels in potato tubers

Directory of Open Access Journals (Sweden)

Pizzichini Daniele

2007-03-01

Full Text Available Abstract Background Beta-carotene is the main dietary precursor of vitamin A. Potato tubers contain low levels of carotenoids, composed mainly of the xanthophylls lutein (in the beta-epsilon branch and violaxanthin (in the beta-beta branch. None of these carotenoids have provitamin A activity. We have previously shown that tuber-specific silencing of the first step in the epsilon-beta branch, LCY-e, redirects metabolic flux towards beta-beta carotenoids, increases total carotenoids up to 2.5-fold and beta-carotene up to 14-fold. Results In this work, we silenced the non-heme beta-carotene hydroxylases CHY1 and CHY2 in the tuber. Real Time RT-PCR measurements confirmed the tuber-specific silencing of both genes . CHY silenced tubers showed more dramatic changes in carotenoid content than LCY-e silenced tubers, with beta-carotene increasing up to 38-fold and total carotenoids up to 4.5-fold. These changes were accompanied by a decrease in the immediate product of beta-carotene hydroxylation, zeaxanthin, but not of the downstream xanthophylls, viola- and neoxanthin. Changes in endogenous gene expression were extensive and partially overlapping with those of LCY-e silenced tubers: CrtISO, LCY-b and ZEP were induced in both cases, indicating that they may respond to the balance between individual carotenoid species. Conclusion Together with epsilon-cyclization of lycopene, beta-carotene hydroxylation is another regulatory step in potato tuber carotenogenesis. The data are consistent with a prevalent role of CHY2, which is highly expressed in tubers, in the control of this step. Combination of different engineering strategies holds good promise for the manipulation of tuber carotenoid content.

Eukaryotic RNA polymerase subunit RPB8 is a new relative of the OB family.

Science.gov (United States)

Krapp, S; Kelly, G; Reischl, J; Weinzierl, R O; Matthews, S

1998-02-01

RNA polymerase II subunit RPB8 is an essential subunit that is highly conserved throughout eukaryotic evolution and is present in all three types of nuclear RNA polymerases. We report the first high resolution structural insight into eukaryotic RNA polymerase architecture with the solution structure of RPB8 from Saccharomyces cerevisiae. It consists of an eight stranded, antiparallel beta-barrel, four short helical regions and a large, unstructured omega-loop. The strands are connected in classic Greek-key fashion. The overall topology is unusual and contains a striking C2 rotational symmetry. Furthermore, it is most likely a novel associate of the oligonucleotide/oligosaccharide (OB) binding protein class.

Organotypic vibrosections from whole brain adult Alzheimer mice (overexpressing amyloid-precursor-protein with the Swedish-Dutch-Iowa mutations as a model to study clearance of beta-amyloid plaques

Directory of Open Access Journals (Sweden)

Christian eHumpel

2015-04-01

Full Text Available Alzheimer´s disease is a severe neurodegenerative disorder of the brain, pathologically characterized by extracellular beta-amyloid plaques, intraneuronal Tau inclusions, inflammation, reactive glial cells, vascular pathology and neuronal cell death. The degradation and clearance of beta-amyloid plaques is an interesting therapeutic approach, and the proteases neprilysin (NEP, insulysin and matrix metalloproteinases (MMP are of particular interest. The aim of this project was to establish and characterize a simple in vitro model to study the degrading effects of these proteases. Organoytpic brain vibrosections (120 µm thick were sectioned from adult (9 month old wildtype and transgenic mice (expressing amyloid precursor protein (APP harboring the Swedish K670N/M671L, Dutch E693Q, and Iowa D694N mutations; APP_SDI and cultured for 2 weeks. Plaques were stained by immunohistochemistry for beta-amyloid and Thioflavin S. Our data show that plaques were evident in 2 week old cultures from 9 month old transgenic mice. These plaques were surrounded by reactive GFAP+ astroglia and Iba1+ microglia. Incubation of fresh slices for 2 weeks with 1-0.1-0.01 µg/ml of NEP, insulysin, MMP-2 or MMP-9 showed that NEP, insulysin and MMP-9 markedly degradeded beta-amyloid plaques but only at the highest concentration. Our data provide for the first time a potent and powerful living brain vibrosection model containing a high number of plaques, which allows to rapidly and simply study the degradation and clearance of beta-amyloid plaques in vitro.

The E1 beta-subunit of pyruvate dehydrogenase is surface-expressed in Lactobacillus plantarum and binds fibronectin.

Science.gov (United States)

Vastano, Valeria; Salzillo, Marzia; Siciliano, Rosa A; Muscariello, Lidia; Sacco, Margherita; Marasco, Rosangela

2014-01-01

Lactobacillus plantarum is among the species with a probiotic activity. Adhesion of probiotic bacteria to host tissues is an important principle for strain selection, because it represents a crucial step in the colonization process of either pathogens or commensals. Most bacterial adhesins are proteins, and a major target for them is fibronectin, an extracellular matrix glycoprotein. In this study we demonstrate that PDHB, a component of the pyruvate dehydrogenase complex, is a factor contributing to fibronectin-binding in L. plantarum LM3. By means of fibronectin overlay immunoblotting assay, we identified a L. plantarum LM3 surface protein with apparent molecular mass of 35 kDa. Mass spectrometric analysis shows that this protein is the pyruvate dehydrogenase E1 beta-subunit (PDHB). The corresponding pdhB gene is located in a 4-gene cluster encoding pyruvate dehydrogenase. In LM3-B1, carrying a null mutation in pdhB, the 35 kDa adhesin was not anymore detectable by immunoblotting assay. Nevertheless, the pdhB null mutation did not abolish pdhA, pdhC, and pdhD transcription in LM3-B1. By adhesion assays, we show that LM3-B1 cells bind to immobilized fibronectin less efficiently than wild type cells. Moreover, we show that pdhB expression is negatively regulated by the CcpA protein and is induced by bile. Copyright © 2013. Published by Elsevier GmbH.

Natural Modulators of Amyloid-Beta Precursor Protein Processing

Science.gov (United States)

Zhang, Can; Tanzi, Rudolph E.

2013-01-01

Alzheimer’s disease (AD) is a devastating neurodegenerative disease and the primary cause of dementia, with no cure currently available. The pathogenesis of AD is believed to be primarily driven by Aβ, the principal component of senile plaques. Aβ is an ~4 kDa peptide generated from the amyloid-β precursor protein (APP) through proteolytic secretases. Natural products, particularly those utilized in traditional Chinese medicine (TCM), have a long history alleviating common clinical disorders, including dementia. However, the cell/molecular pathways mediated by these natural products are largely unknown until recently when the underlying molecular mechanisms of the disorders begin to be elucidated. Here, the mechanisms with which natural products modulate the pathogenesis of AD are discussed, in particular, by focusing on their roles in the processing of APP. PMID:22998566

Regulation of glycogen synthase kinase-3{beta} (GSK-3{beta}) after ionizing radiation; Regulation der Glykogen Synthase Kinase-3{beta} (GSK-3{beta}) nach ionisierender Strahlung

Energy Technology Data Exchange (ETDEWEB)

Boehme, K.A.

2006-12-15

Glycogen Synthase Kinase-3{beta} (GSK-3{beta}) phosphorylates the Mdm2 protein in the central domain. This phosphorylation is absolutely required for p53 degradation. Ionizing radiation inactivates GSK-3{beta} by phosphorylation at serine 9 and in consequence prevents Mdm2 mediated p53 degradation. During the work for my PhD I identified Akt/PKB as the kinase that phosphorylates GSK-3{beta} at serine 9 after ionizing radiation. Ionizing radiation leads to phosphorylation of Akt/PKB at threonine 308 and serine 473. The PI3 Kinase inhibitor LY294002 completely abolished Akt/PKB serine 473 phosphorylation and prevented the induction of GSK-3{beta} serine 9 phosphorylation after ionizing radiation. Interestingly, the most significant activation of Akt/PKB after ionizing radiation occurred in the nucleus while cytoplasmic Akt/PKB was only weakly activated after radiation. By using siRNA, I showed that Akt1/PKBa, but not Akt2/PKB{beta}, is required for phosphorylation of GSK- 3{beta} at serine 9 after ionizing radiation. Phosphorylation and activation of Akt/PKB after ionizing radiation depends on the DNA dependent protein kinase (DNA-PK), a member of the PI3 Kinase family, that is activated by free DNA ends. Both, in cells from SCID mice and after knockdown of the catalytic subunit of DNA-PK by siRNA in osteosarcoma cells, phosphorylation of Akt/PKB at serine 473 and of GSK-3{beta} at serine 9 was completely abolished. Consistent with the principle that phosphorylation of GSK-3 at serine 9 contributes to p53 stabilization after radiation, the accumulation of p53 in response to ionizing radiation was largely prevented by downregulation of DNA-PK. From these results I conclude, that ionizing radiation induces a signaling cascade that leads to Akt1/PKBa activation mediated by DNA-PK dependent phosphorylation of serine 473. After activation Akt1/PKBa phosphorylates and inhibits GSK-3{beta} in the nucleus. The resulting hypophosphorylated form of Mdm2 protein is no longer

Synthetic peptides corresponding to human follicle-stimulating hormone (hFSH)-beta-(1-15) and hFSH-beta-(51-65) induce uptake of 45Ca++ by liposomes: evidence for calcium-conducting transmembrane channel formation

Energy Technology Data Exchange (ETDEWEB)

Grasso, P.; Santa-Coloma, T.A.; Reichert, L.E. Jr. (Department of Biochemistry, Albany Medical College, New York, NY (USA))

1991-06-01

We have previously described FSH receptor-mediated influx of 45Ca++ in cultured Sertoli cells from immature rats and receptor-enriched proteoliposomes via activation of voltage-sensitive and voltage-independent calcium channels. We have further shown that this effect of FSH does not require cholera toxin- or pertussis toxin-sensitive guanine nucleotide binding protein or activation of adenylate cyclase. In the present study, we have identified regions of human FSH-beta-subunit which appear to be involved in mediating calcium influx. We screened 11 overlapping peptide amides representing the entire primary structure of hFSH-beta-subunit for their effects on 45Ca++ flux in FSH receptor-enriched proteoliposomes. hFSH-beta-(1-15) and hFSH-beta-(51-65) induced uptake of 45Ca++ in a concentration-related manner. This effect of hFSH-beta-(1-15) and hFSH-beta-(51-65) was also observed in liposomes lacking incorporated FSH receptor. Reducing membrane fluidity by incubating liposomes (containing no receptor) with hFSH-beta-(1-15) or hFSH-beta-(51-65) at temperatures lower than the transition temperatures of their constituent phospholipids resulted in no significant (P greater than 0.05) difference in 45Ca++ uptake. The effectiveness of the calcium ionophore A23187, however, was abolished. Ruthenium red, a voltage-independent calcium channel antagonist, was able to completely block uptake of 45Ca++ induced by hFSH-beta-(1-15) and hFSH-beta-(51-65) whereas nifedipine, a calcium channel blocker specific for L-type voltage-sensitive calcium channels, was without effect. These results suggest that in addition to its effect on voltage-sensitive calcium channel activity, interaction of FSH with its receptor may induce formation of transmembrane aqueous channels which also facilitate influx of extracellular calcium.

Unsaturated free fatty acids increase benzodiazepine receptor agonist binding depending on the subunit composition of the GABAA receptor complex.

Science.gov (United States)

Witt, M R; Westh-Hansen, S E; Rasmussen, P B; Hastrup, S; Nielsen, M

1996-11-01

It has been shown previously that unsaturated free fatty acids (FFAs) strongly enhance the binding of agonist benzodiazepine receptor ligands and GABAA receptor ligands in the CNS in vitro. To investigate the selectivity of this effect, recombinant human GABAA/benzodiazepine receptor complexes formed by different subunit compositions (alpha x beta y gamma 2, x = 1, 2, 3, and 5; y = 1, 2, and 3) were expressed using the baculovirus-transfected Sf9 insect cell system. At 10(-4) M, unsaturated FFAs, particularly arachidonic (20:4) and docosahexaenoic (22:6) acids, strongly stimulated (> 200% of control values) the binding of [3H]flunitrazepam ([3H]FNM) to the alpha 3 beta 2 gamma 2 receptor combination in whole cell preparations. No effect or small increases in levels of unsaturated FFAs on [3H]FNM binding to alpha 1 beta x gamma 2 and alpha 2 beta x gamma 2 receptor combinations were observed, and weak effects (130% of control values) were detected using the alpha 5 beta 2 gamma 2 receptor combination. The saturated FFAs, stearic and palmitic acids, were without effect on [3H]FNM binding to any combination of receptor complexes. The hydroxylated unsaturated FFAs, ricinoleic and ricinelaidic acids, were shown to decrease the binding of [3H]FNM only if an alpha 1 beta 2 gamma 2 receptor combination was used. Given the heterogeneity of the GABAA/ benzodiazepine receptor subunit distribution in the CNS, the effects of FFAs on the benzodiazepine receptor can be assumed to vary at both cellular and regional levels.

The human chorionic gonadotropin-beta arginine68 to glutamic acid substitution fixes the conformation of the C-terminal peptide

DEFF Research Database (Denmark)

Charrel-Dennis, Marie; Terrazzini, Nadia; McBride, Jeffrey D

2005-01-01

Wild-type human chorionic gonadotropin (hCG) has been used as a contraceptive vaccine. However, extensive sequence homology with LH elicits production of cross-reactive antibodies. Substitution of arginine(68) of the beta-subunit (hCG(beta)) with glutamic acid (R68E) profoundly reduces the cross...

Role of the beta1-integrin cytoplasmic tail in mediating invasin-promoted internalization of Yersinia

DEFF Research Database (Denmark)

Gustavsson, Anna; Armulik, Annika; Brakebusch, Cord

2002-01-01

Invasin of Yersinia pseudotuberculosis binds to beta1-integrins on host cells and triggers internalization of the bacterium. To elucidate the mechanism behind the beta1-integrin-mediated internalization of Yersinia, a beta1-integrin-deficient cell line, GD25, transfected with wild-type beta1A, beta......1B or different mutants of the beta1A subunit was used. Both beta1A and beta1B bound to invasin-expressing bacteria, but only beta1A was able to mediate internalization of the bacteria. The cytoplasmic region of beta1A, differing from beta1B, contains two NPXY motifs surrounding a double threonine...... noted that cells affected in bacterial internalization exhibited reduced spreading capability when seeded onto invasin, suggesting a correlation between the internalization of invasin-expressing bacteria and invasin-induced spreading. Likewise, integrins defective in forming peripheral focal complex...

Complementary DNA and derived amino acid sequence of the α subunit of human complement protein C8: evidence for the existence of a separate α subunit messenger RNA

International Nuclear Information System (INIS)

Rao, A.G.; Howard, O.M.Z.; Ng, S.C.; Whitehead, A.S.; Colten, H.R.; Sodetz, J.M.

1987-01-01

The entire amino acid sequence of the α subunit (M/sub r/ 64,000) of the eight component of complement (C8) was determined by characterizing cDNA clones isolated from a human liver cDNA library. Two clones with overlapping inserts of net length 2.44 kilobases (kb) were isolated and found to contain the entire α coding region [1659 base pairs (bp)]. The 5' end consists of an untranslated region and a leader sequence of 30 amino acids. This sequence contains an apparent initiation Met, signal peptide, and propeptide which ends with an arginine-rich sequence that is characteristic of proteolytic processing sites found in the pro form of protein precursors. The 3' untranslated region contains two polyadenylation signals and a poly(A)sequence. RNA blot analysis of total cellular RNA from the human hepatoma cell line HepG2 revealed a message size of ∼2.5 kb. Features of the 5' and 3' sequences and the message size suggest that a separate mRNA codes for α and argues against the occurrence of a single-chain precursor form of the disulfide-linked α-λ subunit found in mature C8. Analysis of the derived amino acid sequence revealed several membrane surface seeking domains and a possible transmembrane domain. Analysis of the carbohydrate composition indicates 1 or 2 asparagine-linked but no O-linked oligosaccharide chains, a result consistent with predictions from the amino acid sequence. Most significantly, it exhibits a striking overall homology to human C9, with values of 24% on the basis of identity and 46% when conserved substitutions are allowed. As described in an accompanying report this homology also extends to the β subunit of C8

Helicobacter pylori VacA toxin/subunit p34: targeting of an anion channel to the inner mitochondrial membrane.

Directory of Open Access Journals (Sweden)

Grazyna Domańska

2010-04-01

Full Text Available The vacuolating toxin VacA, released by Helicobacter pylori, is an important virulence factor in the pathogenesis of gastritis and gastroduodenal ulcers. VacA contains two subunits: The p58 subunit mediates entry into target cells, and the p34 subunit mediates targeting to mitochondria and is essential for toxicity. In this study we found that targeting to mitochondria is dependent on a unique signal sequence of 32 uncharged amino acid residues at the p34 N-terminus. Mitochondrial import of p34 is mediated by the import receptor Tom20 and the import channel of the outer membrane TOM complex, leading to insertion of p34 into the mitochondrial inner membrane. p34 assembles in homo-hexamers of extraordinary high stability. CD spectra of the purified protein indicate a content of >40% beta-strands, similar to pore-forming beta-barrel proteins. p34 forms an anion channel with a conductivity of about 12 pS in 1.5 M KCl buffer. Oligomerization and channel formation are independent both of the 32 uncharged N-terminal residues and of the p58 subunit of the toxin. The conductivity is efficiently blocked by 5-nitro-2-(3-phenylpropylaminobenzoic acid (NPPB, a reagent known to inhibit VacA-mediated apoptosis. We conclude that p34 essentially acts as a small pore-forming toxin, targeted to the mitochondrial inner membrane by a special hydrophobic N-terminal signal.

Messenger RNAs from the Scutellum and Aleurone of Germinating Barley Encode (1-->3,1-->4)-beta-d-Glucanase, alpha-Amylase and Carboxypeptidase

DEFF Research Database (Denmark)

Mundy, John; Brandt, Anders; Fincher, Geoffrey B

1985-01-01

Polyclonal antibodies raised against barley (1-->3,1-->4)-beta-d-glucanase, alpha-amylase and carboxypeptidase were used to detect precursor polypeptides of these hydrolytic enzymes among the in vitro translation products of mRNA isolated from the scutellum and aleurone of germinating barley....... In the scutellum, mRNA encoding carboxypeptidase appeared to be relatively more abundant than that encoding alpha-amylase or (1-->3,1-->4)-beta-d-glucanase, while in the aleurone alpha-amylase and (1-->3,1-->4)-beta-d-glucanase mRNAs predominated. The apparent molecular weights of the precursors for (1......-->3,1-->4)-beta-d-glucanase, alpha-amylase, and carboxypeptidase were 33,000, 44,000, and 35,000, respectively. In each case these are slightly higher (1,500-5,000) than molecular weights of the mature enzymes. Molecular weights of precursors immunoprecipitated from aleurone and scutellum mRNA translation...

Glucose activates prenyltransferases in pancreatic islet {beta}-cells

Energy Technology Data Exchange (ETDEWEB)

Goalstone, Marc [Department of Medicine, University of Colorado, VA Medical Center, Denver, CO 80220 (United States); Kamath, Vasudeva [Department of Pharmaceutical Sciences, Wayne State University, VA Medical Center, Detroit, MI 48201 (United States); Kowluru, Anjaneyulu, E-mail: akowluru@med.wayne.edu [Department of Pharmaceutical Sciences, Wayne State University, VA Medical Center, Detroit, MI 48201 (United States)

2010-01-01

A growing body of evidence implicates small G-proteins [e.g., Cdc42 and Rac1] in glucose-stimulated insulin secretion [GSIS] in the islet {beta}-cell. These signaling proteins undergo post-translational modifications [e.g., prenylation] at their C-terminal cysteine residue and appear to be essential for the transport and fusion of insulin-containing secretory granules with the plasma membrane and the exocytotic secretion of insulin. However, potential regulation of the prenylating enzymes by physiological insulin secretogues [e.g., glucose] has not been investigated thus far. Herein, we report immunological localization, sub-cellular distribution and regulation of farnesyltransferases [FTases] and geranylgeranyltransferase [GGTase] by glucose in insulin-secreting INS 832/13 {beta}-cells and normal rat islets. Our findings suggest that an insulinotropic concentration of glucose [20 mM] markedly stimulated the expression of the {alpha}-subunits of FTase/GGTase-1, but not the {beta}-subunits of FTase or GGTase-1 without significantly affecting the predominantly cytosolic distribution of these holoenzymes in INS 832/13 cells and rodent islets. Under these conditions, glucose significantly stimulated [2.5- to 4.0-fold over basal] the activities of both FTase and GGTase-1 in both cell types. Together, these findings provide the first evidence to suggest that GSIS involves activation of the endogenous islet prenyltransferases by glucose, culminating in the activation of their respective G-protein substrates, which is necessary for cytoskeletal rearrangement, vesicular transport, fusion and secretion of insulin.

Association of the macrophage activating factor (MAF) precursor activity with polymorphism in vitamin D-binding protein.

Science.gov (United States)

Nagasawa, Hideko; Sasaki, Hideyuki; Uto, Yoshihiro; Kubo, Shinichi; Hori, Hitoshi

2004-01-01

Serum vitamin D-binding protein (Gc protein or DBP) is a highly expressed polymorphic protein, which is a precursor of the inflammation-primed macrophage activating factor, GcMAF, by a cascade of carbohydrate processing reactions. In order to elucidate the relationship between Gc polymorphism and GcMAF precursor activity, we estimated the phagocytic ability of three homotypes of Gc protein, Gc1F-1F, Gc1S-1S and Gc2-2, through processing of their carbohydrate moiety. We performed Gc typing of human serum samples by isoelectric focusing (IEF). Gc protein from human serum was purified by affinity chromatography with 25-hydroxyvitamin D3-sepharose. A phagocytosis assay of Gc proteins, modified using beta-glycosidase and sialidase, was carried out. The Gc1F-1F phenotype was revealed to possess Galbeta1-4GalNAc linkage by the analysis of GcMAF precursor activity using beta1-4 linkage-specific galactosidase from jack bean. The GcMAF precursor activity of the Gc1F-1F phenotype was highest among three Gc homotypes. The Gc polymorphism and carbohydrate diversity of Gc protein are significant for its pleiotropic effects.

Arf6 controls beta-amyloid production by regulating macropinocytosis of the Amyloid Precursor Protein to lysosomes.

Science.gov (United States)

Tang, Weihao; Tam, Joshua H K; Seah, Claudia; Chiu, Justin; Tyrer, Andrea; Cregan, Sean P; Meakin, Susan O; Pasternak, Stephen H

2015-07-14

Alzheimer's disease (AD) is characterized by the deposition of Beta-Amyloid (Aβ) peptides in the brain. Aβ peptides are generated by cleavage of the Amyloid Precursor Protein (APP) by the β - and γ - secretase enzymes. Although this process is tightly linked to the internalization of cell surface APP, the compartments responsible are not well defined. We have found that APP can be rapidly internalized from the cell surface to lysosomes, bypassing early and late endosomes. Here we show by confocal microscopy and electron microscopy that this pathway is mediated by macropinocytosis. APP internalization is enhanced by antibody binding/crosslinking of APP suggesting that APP may function as a receptor. Furthermore, a dominant negative mutant of Arf6 blocks direct transport of APP to lysosomes, but does not affect classical endocytosis to endosomes. Arf6 expression increases through the hippocampus with the development of Alzheimer's disease, being expressed mostly in the CA1 and CA2 regions in normal individuals but spreading through the CA3 and CA4 regions in individuals with pathologically diagnosed AD. Disruption of lysosomal transport of APP reduces both Aβ40 and Aβ42 production by more than 30 %. Our findings suggest that the lysosome is an important site for Aβ production and that altering APP trafficking represents a viable strategy to reduce Aβ production.

Identification of an indigo precursor from leaves of Isatis tinctoria (Woad).

Science.gov (United States)

Maugard, T; Enaud, E; Choisy, P; Legoy, M D

2001-11-01

Indole is presumably a product of indole-3-glycerol phosphate catabolism in Isatis tinctoria. It is oxidized into indoxyl and stored in young leaves as indigo precursor. Further oxidation and dimerization of indoxyl produces indigoid pigments. In this work, we describe an HPLC method dedicated to the identification and quantification of indigoid pigments (indigo, indirubin, isoindigo and isoindirubin) and indigo precursors produced in I. tinctoria (Woad). This work, carried out with two cultivars of I. tinctoria, has confirmed that the quantity of indigo precursors is dependent on the species and the harvest period. In addition we have shown for the first time that young leaves of I. tinctoria, harvested in June contained a new indigo precursor in addition to isatan B (indoxyl-5-ketogluconate) and indican (indoxyl-beta-D-glucoside). We suggest the name "isatan C" for this new indigo precursor in I. tinctoria. Its chemical characteristics point to an dioxindole ester with PM of 395. We have shown that isatan C reacts with isatan B increasing the red pigment production.

The p110beta isoform of phosphoinositide 3-kinase signals downstream of G protein-coupled receptors and is functionally redundant with p110gamma.

Science.gov (United States)

Guillermet-Guibert, Julie; Bjorklof, Katja; Salpekar, Ashreena; Gonella, Cristiano; Ramadani, Faruk; Bilancio, Antonio; Meek, Stephen; Smith, Andrew J H; Okkenhaug, Klaus; Vanhaesebroeck, Bart

2008-06-17

The p110 isoforms of phosphoinositide 3-kinase (PI3K) are acutely regulated by extracellular stimuli. The class IA PI3K catalytic subunits (p110alpha, p110beta, and p110delta) occur in complex with a Src homology 2 (SH2) domain-containing p85 regulatory subunit, which has been shown to link p110alpha and p110delta to Tyr kinase signaling pathways. The p84/p101 regulatory subunits of the p110gamma class IB PI3K lack SH2 domains and instead couple p110gamma to G protein-coupled receptors (GPCRs). Here, we show, using small-molecule inhibitors with selectivity for p110beta and cells derived from a p110beta-deficient mouse line, that p110beta is not a major effector of Tyr kinase signaling but couples to GPCRs. In macrophages, both p110beta and p110gamma contributed to Akt activation induced by the GPCR agonist complement 5a, but not by the Tyr kinase ligand colony-stimulating factor-1. In fibroblasts, which express p110beta but not p110gamma, p110beta mediated Akt activation by the GPCR ligands stromal cell-derived factor, sphingosine-1-phosphate, and lysophosphatidic acid but not by the Tyr kinase ligands PDGF, insulin, and insulin-like growth factor 1. Introduction of p110gamma in these cells reduced the contribution of p110beta to GPCR signaling. Taken together, these data show that p110beta and p110gamma can couple redundantly to the same GPCR agonists. p110beta, which shows a much broader tissue distribution than the leukocyte-restricted p110gamma, could thus provide a conduit for GPCR-linked PI3K signaling in the many cell types where p110gamma expression is low or absent.

Identification of beta-2 as a key cell adhesion molecule in PCa cell neurotropic behavior: a novel ex vivo and biophysical approach.

Science.gov (United States)

Jansson, Keith H; Castillo, Deborah G; Morris, Joseph W; Boggs, Mary E; Czymmek, Kirk J; Adams, Elizabeth L; Schramm, Lawrence P; Sikes, Robert A

2014-01-01

Prostate cancer (PCa) is believed to metastasize through the blood/lymphatics systems; however, PCa may utilize the extensive innervation of the prostate for glandular egress. The interaction of PCa and its nerve fibers is observed in 80% of PCa and is termed perineural invasion (PNI). PCa cells have been observed traveling through the endoneurium of nerves, although the underlying mechanisms have not been elucidated. Voltage sensitive sodium channels (VSSC) are multimeric transmembrane protein complexes comprised of a pore-forming α subunit and one or two auxiliary beta (β) subunits with inherent cell adhesion molecule (CAM) functions. The beta-2 isoform (gene SCN2B) interacts with several neural CAMs, while interacting putatively with other prominent neural CAMs. Furthermore, beta-2 exhibits elevated mRNA and protein levels in highly metastatic and castrate-resistant PCa. When overexpressed in weakly aggressive LNCaP cells (2BECFP), beta-2 alters LNCaP cell morphology and enhances LNCaP cell metastasis associated behavior in vitro. We hypothesize that PCa cells use beta-2 as a CAM during PNI and subsequent PCa metastasis. The objective of this study was to determine the effect of beta-2 expression on PCa cell neurotropic metastasis associated behavior. We overexpressed beta-2 as a fusion protein with enhanced cyan fluorescence protein (ECFP) in weakly aggressive LNCaP cells and observed neurotropic effects utilizing our novel ex vivo organotypic spinal cord co-culture model, and performed functional assays with neural matrices and atomic force microscopy. With increased beta-2 expression, PCa cells display a trend of enhanced association with nerve axons. On laminin, a neural CAM, overexpression of beta-2 enhances PCa cell migration, invasion, and growth. 2BECFP cells exhibit marked binding affinity to laminin relative to LNECFP controls, and recombinant beta-2 ectodomain elicits more binding events to laminin than BSA control. Functional overexpression of VSSC

Involvement of ribosomal protein L6 in assembly of functional 50S ribosomal subunit in Escherichia coli cells

International Nuclear Information System (INIS)

Shigeno, Yuta; Uchiumi, Toshio; Nomura, Takaomi

2016-01-01

Ribosomal protein L6, an essential component of the large (50S) subunit, primarily binds to helix 97 of 23S rRNA and locates near the sarcin/ricin loop of helix 95 that directly interacts with GTPase translation factors. Although L6 is believed to play important roles in factor-dependent ribosomal function, crucial biochemical evidence for this hypothesis has not been obtained. We constructed and characterized an Escherichia coli mutant bearing a chromosomal L6 gene (rplF) disruption and carrying a plasmid with an arabinose-inducible L6 gene. Although this ΔL6 mutant grew more slowly than its wild-type parent, it proliferated in the presence of arabinose. Interestingly, cell growth in the absence of arabinose was biphasic. Early growth lasted only a few generations (LI-phase) and was followed by a suspension of growth for several hours (S-phase). This suspension was followed by a second growth phase (LII-phase). Cells harvested at both LI- and S-phases contained ribosomes with reduced factor-dependent GTPase activity and accumulated 50S subunit precursors (45S particles). The 45S particles completely lacked L6. Complete 50S subunits containing L6 were observed in all growth phases regardless of the L6-depleted condition, implying that the ΔL6 mutant escaped death because of a leaky expression of L6 from the complementing plasmid. We conclude that L6 is essential for the assembly of functional 50S subunits at the late stage. We thus established conditions for the isolation of L6-depleted 50S subunits, which are essential to study the role of L6 in translation. - Highlights: • We constructed an in vivo functional assay system for Escherichia coli ribosomal protein L6. • Growth of an E. coli ΔL6 mutant was biphasic when L6 levels were depleted. • The ΔL6 mutant accumulated 50S ribosomal subunit precursors that sedimented at 45S. • L6 is a key player in the late stage of E. coli 50S subunit assembly.

Involvement of ribosomal protein L6 in assembly of functional 50S ribosomal subunit in Escherichia coli cells

Energy Technology Data Exchange (ETDEWEB)

Shigeno, Yuta [Division of Applied Biology, Faculty of Textile Science and Technology, Shinshu University, Ueda 386-8567 (Japan); Uchiumi, Toshio [Department of Biology, Faculty of Science, Niigata University, Niigata 950-2181 (Japan); Nomura, Takaomi, E-mail: nomurat@shinshu-u.ac.jp [Division of Applied Biology, Faculty of Textile Science and Technology, Shinshu University, Ueda 386-8567 (Japan)

2016-04-22

Ribosomal protein L6, an essential component of the large (50S) subunit, primarily binds to helix 97 of 23S rRNA and locates near the sarcin/ricin loop of helix 95 that directly interacts with GTPase translation factors. Although L6 is believed to play important roles in factor-dependent ribosomal function, crucial biochemical evidence for this hypothesis has not been obtained. We constructed and characterized an Escherichia coli mutant bearing a chromosomal L6 gene (rplF) disruption and carrying a plasmid with an arabinose-inducible L6 gene. Although this ΔL6 mutant grew more slowly than its wild-type parent, it proliferated in the presence of arabinose. Interestingly, cell growth in the absence of arabinose was biphasic. Early growth lasted only a few generations (LI-phase) and was followed by a suspension of growth for several hours (S-phase). This suspension was followed by a second growth phase (LII-phase). Cells harvested at both LI- and S-phases contained ribosomes with reduced factor-dependent GTPase activity and accumulated 50S subunit precursors (45S particles). The 45S particles completely lacked L6. Complete 50S subunits containing L6 were observed in all growth phases regardless of the L6-depleted condition, implying that the ΔL6 mutant escaped death because of a leaky expression of L6 from the complementing plasmid. We conclude that L6 is essential for the assembly of functional 50S subunits at the late stage. We thus established conditions for the isolation of L6-depleted 50S subunits, which are essential to study the role of L6 in translation. - Highlights: • We constructed an in vivo functional assay system for Escherichia coli ribosomal protein L6. • Growth of an E. coli ΔL6 mutant was biphasic when L6 levels were depleted. • The ΔL6 mutant accumulated 50S ribosomal subunit precursors that sedimented at 45S. • L6 is a key player in the late stage of E. coli 50S subunit assembly.

[Cloning of cDNA for RNA polymerase subunit from the fission yeast Schizosaccharomyces pombe by heterospecific complementation in Saccharomyces cerevisiae].

Science.gov (United States)

Shpakovskiĭ, G V; Lebedenko, E N; Thuriaux, P

1997-02-01

The rpb10 cDNA of the fission yeast Schizosaccharomyces pombe, encoding one of the five small subunits common to all three nuclear DNA-dependent RNA polymerases, was isolated from an expression cDNA library by two independent approaches: PCR-based screening and direct suppression by means of heterospecific complementation of a temperature-sensitive mutant defective in the corresponding gene of Saccharomyces cerevisiae. The cloned Sz. pombe cDNA encodes a protein Rpb10 of 71 amino acids with an M of 8,275 Da, sharing 51 amino acids (71% identity) with the subunit ABC10 beta of RNA polymerases I-III from S. cerevisiae. All eukaryotic members of this protein family have the same general organization featuring two highly conserved motifs (RCFT/SCGK and RYCCRRM) around an atypical zinc finger and an additional invariant HVDLIEK motif toward the C-terminal end. The last motif is only characteristics for homologs from eukaryotes. In keeping with this remarkable structural conservation, the Sz. pombe cDNA also fully complemented a S. cerevisiae deletion mutant lacking subunit ABC10 beta (null allele rpb10-delta 1::HIS3).

Effects of chronic ethanol treatment on the in vitro biosynthesis of pro-opiomelanocortin and its posttranslational processing to beta-endorphin in the intermediate lobe of the rat pituitary

Energy Technology Data Exchange (ETDEWEB)

Seizinger, B.R.; Hoellt, V.; Herz, A.

1984-09-01

Chronic treatment of rats with 15% (vol/vol) ethanol in tap water as their only source of liquid over a period of 3 weeks resulted in a strong decrease by almost 50% in tissue levels and in vitro release of immunoreactive beta-endorphin of the neurointermediate pituitary. Moreover, the in vitro incorporation of (3H)phenylalanine into peptides of the neurointermediate pituitary, immunoprecipitable with beta-endorphin antiserum, was found to be decreased by more than 30%. Analysis of beta-endorphin-related peptides on sodium dodecyl sulfate-polyacrylamide gel electrophoresis revealed that chronic ethanol treatment reduced the in vitro biosynthesis of the beta-endorphin precursor pro-opiomelanocortin. This ethanol-induced effect was combined with a retardation in the time course of the posttranslational processing of the precursor into beta-endorphin. Thus, chronic ethanol treatment may influence the activity of enzymes which process the opioid peptide precursor pro-opiomelanocortin, leading to a decreased formation of the final secretory product beta-endorphin.

Rapid PCR-mediated synthesis of competitor molecules for accurate quantification of beta(2) GABA(A) receptor subunit mRNA.

Science.gov (United States)

Vela, J; Vitorica, J; Ruano, D

2001-12-01

We describe a fast and easy method for the synthesis of competitor molecules based on non-specific conditions of PCR. RT-competitive PCR is a sensitive technique that allows quantification of very low quantities of mRNA molecules in small tissue samples. This technique is based on the competition established between the native and standard templates for nucleotides, primers or other factors during PCR. Thus, the most critical parameter is the use of good internal standards to generate a standard curve from which the amount of native sequences can be properly estimated. At the present time different types of internal standards and methods for their synthesis have been described. Normally, most of these methods are time-consuming and require the use of different sets of primers, different rounds of PCR or specific modifications, such as site-directed mutagenesis, that need subsequent analysis of the PCR products. Using our method, we obtained in a single round of PCR and with the same primer pair, competitor molecules that were successfully used in RT-competitive PCR experiments. The principal advantage of this method is high versatility and economy. Theoretically it is possible to synthesize a specific competitor molecule for each primer pair used. Finally, using this method we have been able to quantify the increase in the expression of the beta(2) GABA(A) receptor subunit mRNA that occurs during rat hippocampus development.

Lithium chloride increases the production of amyloid-beta peptide independently from its inhibition of glycogen synthase kinase 3.

Science.gov (United States)

Feyt, Christine; Kienlen-Campard, Pascal; Leroy, Karelle; N'Kuli, Francisca; Courtoy, Pierre J; Brion, Jean-Pierre; Octave, Jean-Noël

2005-09-30

Glycogen synthase kinase 3 (GSK3) is able to phosphorylate tau at many sites that are found to be phosphorylated in paired helical filaments in Alzheimer disease. Lithium chloride (LiCl) efficiently inhibits GSK3 and was recently reported to also decrease the production of amyloid-beta peptide (Abeta) from its precursor, the amyloid precursor protein. Therefore, lithium has been proposed as a combined therapeutic agent, inhibiting both the hyperphosphorylation of tau and the production of Abeta. Here, we demonstrate that the inhibition of GSK3 by LiCl induced the nuclear translocation of beta-catenin in Chinese hamster ovary cells and rat cultured neurons, in which a decrease in tau phosphorylation was observed. In both cellular models, a nontoxic concentration of LiCl increased the production of Abeta by increasing the beta-cleavage of amyloid precursor protein, generating more substrate for an unmodified gamma-secretase activity. SB415286, another GSK3 inhibitor, induced the nuclear translocation of beta-catenin and slightly decreased Abeta production. It is concluded that the LiCl-mediated increase in Abeta production is not related to GSK3 inhibition.

Synthesis of Precursors of the Agalacto (Exo) Fragment of the Quartromicins via an Auxiliary-Controlled Exo-Selective Diels-Alder Reaction

Science.gov (United States)

Qi, Jun; Roush, William R.

2008-01-01

A direct synthesis of the α-hydroxyaldehyde exo-5, a precursor of the exo-spirotetronate subunit o f the quartromicins, was achieved through an exo-selective Lewis acid-catalyzed Diels-Alder reaction of dienophile 12a and diene 1. PMID:16774259

Distinct roles of beta1 metal ion-dependent adhesion site (MIDAS), adjacent to MIDAS (ADMIDAS), and ligand-associated metal-binding site (LIMBS) cation-binding sites in ligand recognition by integrin alpha2beta1.

Science.gov (United States)

Valdramidou, Dimitra; Humphries, Martin J; Mould, A Paul

2008-11-21

Integrin-ligand interactions are regulated in a complex manner by divalent cations, and previous studies have identified ligand-competent, stimulatory, and inhibitory cation-binding sites. In collagen-binding integrins, such as alpha2beta1, ligand recognition takes place exclusively at the alpha subunit I domain. However, activation of the alphaI domain depends on its interaction with a structurally similar domain in the beta subunit known as the I-like or betaI domain. The top face of the betaI domain contains three cation-binding sites: the metal-ion dependent adhesion site (MIDAS), the ADMIDAS (adjacent to MIDAS), and LIMBS (ligand-associated metal-binding site). The role of these sites in controlling ligand binding to the alphaI domain has yet to be elucidated. Mutation of the MIDAS or LIMBS completely blocked collagen binding to alpha2beta1; in contrast mutation of the ADMIDAS reduced ligand recognition but this effect could be overcome by the activating monoclonal antibody TS2/16. Hence, the MIDAS and LIMBS appear to be essential for the interaction between alphaI and betaI, whereas occupancy of the ADMIDAS has an allosteric effect on the conformation of betaI. An activating mutation in the alpha2 I domain partially restored ligand binding to the MIDAS and LIMBS mutants. Analysis of the effects of Ca(2+), Mg(2+), and Mn(2+) on ligand binding to these mutants showed that the MIDAS is a ligand-competent site through which Mn(2+) stimulates ligand binding, whereas the LIMBS is a stimulatory Ca(2+)-binding site, occupancy of which increases the affinity of Mg(2+) for the MIDAS.

Global Proteome Analysis Identifies Active Immunoproteasome Subunits in Human Platelets*

Science.gov (United States)

Klockenbusch, Cordula; Walsh, Geraldine M.; Brown, Lyda M.; Hoffman, Michael D.; Ignatchenko, Vladimir; Kislinger, Thomas; Kast, Juergen

2014-01-01

The discovery of new functions for platelets, particularly in inflammation and immunity, has expanded the role of these anucleate cell fragments beyond their primary hemostatic function. Here, four in-depth human platelet proteomic data sets were generated to explore potential new functions for platelets based on their protein content and this led to the identification of 2559 high confidence proteins. During a more detailed analysis, consistently high expression of the proteasome was discovered, and the composition and function of this complex, whose role in platelets has not been thoroughly investigated, was examined. Data set mining resulted in identification of nearly all members of the 26S proteasome in one or more data sets, except the β5 subunit. However, β5i, a component of the immunoproteasome, was identified. Biochemical analyses confirmed the presence of all catalytically active subunits of the standard 20S proteasome and immunoproteasome in human platelets, including β5, which was predominantly found in its precursor form. It was demonstrated that these components were assembled into the proteasome complex and that standard proteasome as well as immunoproteasome subunits were constitutively active in platelets. These findings suggest potential new roles for platelets in the immune system. For example, the immunoproteasome may be involved in major histocompatibility complex I (MHC I) peptide generation, as the MHC I machinery was also identified in our data sets. PMID:25146974

A transmembrane polar interaction is involved in the functional regulation of integrin alpha L beta 2.

Science.gov (United States)

Vararattanavech, Ardcharaporn; Chng, Choon-Peng; Parthasarathy, Krupakar; Tang, Xiao-Yan; Torres, Jaume; Tan, Suet-Mien

2010-05-14

Integrins are heterodimeric transmembrane (TM) receptors formed by noncovalent associations of alpha and beta subunits. Each subunit contains a single alpha-helical TM domain. Inside-out activation of an integrin involves the separation of its cytoplasmic tails, leading to disruption of alphabeta TM packing. The leukocyte integrin alpha L beta 2 is required for leukocyte adhesion, migration, proliferation, cytotoxic function, and antigen presentation. In this study, we show by mutagenesis experiments that the packing of alpha L beta 2 TMs is consistent with that of the integrin alpha IIb beta 3 TMs. However, molecular dynamics simulations of alpha L beta 2 TMs in lipids predicted a polar interaction involving the side chains of alpha L Ser1071 and beta2 Thr686 in the outer-membrane association clasp (OMC). This is supported by carbonyl vibrational shifts observed in isotope-labeled alpha L beta 2 TM peptides that were incorporated into lipid bilayers. Molecular dynamics studies simulating the separation of alpha L beta 2 tails showed the presence of polar interaction during the initial perturbation of the inner-membrane association clasp. When the TMs underwent further separation, the polar interaction was disrupted. OMC polar interaction is important in regulating the functions of beta2 integrins because mutations that disrupt the OMC polar interaction generated constitutively activated alpha L beta 2, alpha M beta 2, and alpha X beta 2 in 293T transfectants. We also show that the expression of mutant beta2 Thr686Gly in beta2-deficient T cells rescued cell adhesion to intercellular adhesion molecule 1, but the cells showed overt elongated morphologies in response to chemokine stromal-cell-derived factor 1 alpha treatment as compared to wild-type beta2-expressing cells. These two TM polar residues are totally conserved in other members of the beta2 integrins in humans and across different species. Our results provide an example of the stabilizing effect of polar

Soluble beta-amyloid precursor protein is related to disease progression in amyotrophic lateral sclerosis.

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Petra Steinacker

Full Text Available BACKGROUND: Biomarkers of disease progression in amyotrophic lateral sclerosis (ALS could support the identification of beneficial drugs in clinical trials. We aimed to test whether soluble fragments of beta-amyloid precursor protein (sAPPα and sAPPß correlated with clinical subtypes of ALS and were of prognostic value. METHODOLOGY/PRINCIPAL FINDINGS: In a cross-sectional study including patients with ALS (N = 68 with clinical follow-up data over 6 months, Parkinson's disease (PD, N = 20, and age-matched controls (N = 40, cerebrospinal fluid (CSF levels of sAPPα a, sAPPß and neurofilaments (NfH(SMI35 were measured by multiplex assay, Progranulin by ELISA. CSF sAPPα and sAPPß levels were lower in ALS with a rapidly-progressive disease course (p = 0.03, and p = 0.02 and with longer disease duration (p = 0.01 and p = 0.01, respectively. CSF NfH(SMI35 was elevated in ALS compared to PD and controls, with highest concentrations found in patients with rapid disease progression (p<0.01. High CSF NfH(SMI3 was linked to low CSF sAPPα and sAPPß (p = 0.001, and p = 0.007, respectively. The ratios CSF NfH(SMI35/CSF sAPPα,-ß were elevated in patients with fast progression of disease (p = 0.002 each. CSF Progranulin decreased with ongoing disease (p = 0.04. CONCLUSIONS: This study provides new CSF candidate markers associated with progression of disease in ALS. The data suggest that a deficiency of cellular neuroprotective mechanisms (decrease of sAPP is linked to progressive neuro-axonal damage (increase of NfH(SMI35 and to progression of disease.

Molecular cloning and phylogenetic analysis of integrins alpha v beta 1 and alpha v beta 6 of one-humped camel (Camelus dromedarius)

DEFF Research Database (Denmark)

Du, Junzheng; Larska, Magdalena Larska; Chang, Huiyun

2010-01-01

Bactrian camels can relatively easily be infected with FMDV, but dromedary camels remain resistant even to high doses of the virus. To understand the different susceptibility between the two camel species from the standpoint of viral receptors, this work reports the sequences of the dromedary camel...... into the Artiodactyla group, together with those of Bactrian camel, pig, sheep, and cattle that are susceptible to FMDV infection. Compared with the Bactrian camel integrins, 4, 10, and 8 amino acid changes were found in the dromedary camel alpha v, beta 1, and beta 6 subunits, respectively. This study...... will be of importance in understanding the differences of integrins as FMDV receptors among dromedary camel and other species. Crown...

Purification and characterization of selenocysteine beta-lyase from Citrobacter freundii

International Nuclear Information System (INIS)

Chocat, P.; Esaki, N.; Tanizawa, K.; Nakamura, K.; Tanaka, H.; Soda, K.

1985-01-01

The purification and characterization of bacterial selenocysteine beta-lyase, an enzyme which specifically catalyzes the cleavage of L-selenocysteine to L-alanine and Se0, are presented. The enzyme, purified to near homogeneity from Citrobacter freundii, is monomeric with a molecular weight of ca. 64,000 and contains 1 mol of pyridoxal 5'-phosphate as a cofactor per mol of enzyme. L-Selenocysteine is the sole substrate. L-Cysteine is a competitive inhibitor of the enzyme. The enzyme also catalyzes the alpha, beta elimination of beta-chloro-L-alanine to form NH 3 , pyruvate, and Cl- and is irreversibly inactivated during the reaction. The physicochemical properties, e.g., amino acid composition and subunit structure, of the bacterial enzyme are fairly different from those of the pig liver enzyme. However, the catalytic properties of both enzymes, e.g., substrate specificity and inactivation by the substrate or a mechanism-based inactivator, beta-chloro-L-alanine, are very similar

Expression Profile of the Integrin Receptor Subunits in the Guinea Pig Sclera.

Science.gov (United States)

Wang, Kevin K; Metlapally, Ravikanth; Wildsoet, Christine F

2017-06-01

The ocular dimensional changes in myopia reflect increased scleral remodeling, and in high myopia, loss of scleral integrity leads to biomechanical weakening and continued scleral creep. As integrins, a type of cell surface receptors, have been linked to scleral remodeling, they represent potential targets for myopia therapies. As a first step, this study aimed to characterize the integrin subunits at the messenger RNA level in the sclera of the guinea pig, a more recently added but increasingly used animal model for myopia research. Primers for α and β integrin subunits were designed using NCBI/UCSC Genome Browser and Primer3 software tools. Total RNA was extracted from normal scleral tissue and isolated cultured scleral fibroblasts, as well as liver and lung, as reference tissues, all from guinea pig. cDNA was produced by reverse transcription, PCR was used to amplify products of predetermined sizes, and products were sequenced using standard methods. Guinea pig scleral tissue expressed all known integrin alpha subunits except αD and αE. The latter integrin subunits were also not expressed by cultured guinea pig scleral fibroblasts; however, their expression was confirmed in guinea pig liver. In addition, isolated cultured fibroblasts did not express integrin subunits αL, αM, and αX. This difference between results for cultured cells and intact sclera presumably reflects the presence in the latter of additional cell types. Both guinea pig scleral tissue and isolated scleral fibroblasts expressed all known integrin beta subunits. All results were verified through sequencing. The possible contributions of integrins to scleral remodeling make them plausible targets for myopia prevention. Data from this study will help guide future ex vivo and in vitro studies directed at understanding the relationship between scleral integrins and ocular growth regulation in the guinea pig model for myopia.

FXYD proteins reverse inhibition of the Na+-K+ pump mediated by glutathionylation of its beta1 subunit.

Science.gov (United States)

Bibert, Stéphanie; Liu, Chia-Chi; Figtree, Gemma A; Garcia, Alvaro; Hamilton, Elisha J; Marassi, Francesca M; Sweadner, Kathleen J; Cornelius, Flemming; Geering, Käthi; Rasmussen, Helge H

2011-05-27

The seven members of the FXYD protein family associate with the Na(+)-K(+) pump and modulate its activity. We investigated whether conserved cysteines in FXYD proteins are susceptible to glutathionylation and whether such reactivity affects Na(+)-K(+) pump function in cardiac myocytes and Xenopus oocytes. Glutathionylation was detected by immunoblotting streptavidin precipitate from biotin-GSH loaded cells or by a GSH antibody. Incubation of myocytes with recombinant FXYD proteins resulted in competitive displacement of native FXYD1. Myocyte and Xenopus oocyte pump currents were measured with whole-cell and two-electrode voltage clamp techniques, respectively. Native FXYD1 in myocytes and FXYD1 expressed in oocytes were susceptible to glutathionylation. Mutagenesis identified the specific cysteine in the cytoplasmic terminal that was reactive. Its reactivity was dependent on flanking basic amino acids. We have reported that Na(+)-K(+) pump β(1) subunit glutathionylation induced by oxidative signals causes pump inhibition in a previous study. In the present study, we found that β(1) subunit glutathionylation and pump inhibition could be reversed by exposing myocytes to exogenous wild-type FXYD3. A cysteine-free FXYD3 derivative had no effect. Similar results were obtained with wild-type and mutant FXYD proteins expressed in oocytes. Glutathionylation of the β(1) subunit was increased in myocardium from FXYD1(-/-) mice. In conclusion, there is a dependence of Na(+)-K(+) pump regulation on reactivity of two specifically identified cysteines on separate components of the multimeric Na(+)-K(+) pump complex. By facilitating deglutathionylation of the β(1) subunit, FXYD proteins reverse oxidative inhibition of the Na(+)-K(+) pump and play a dynamic role in its regulation.

Transforming growth factor-beta1 stimulates the production of insulin-like growth factor-I and insulin-like growth factor-binding protein-3 in human bone marrow stromal osteoblast progenitors

DEFF Research Database (Denmark)

Kveiborg, Marie; Flyvbjerg, Allan; Eriksen, E F

2001-01-01

While transforming growth factor-beta1 (TGF-beta1) regulates proliferation and differentiation of human osteoblast precursor cells, the mechanisms underlying these effects are not known. Several hormones and locally acting growth factors regulate osteoblast functions through changes in the insulin......-like growth factors (IGFs) and IGF-binding proteins (IGFBPs). Thus, we studied the effects of TGF-beta1 on IGFs and IGFBPs in human marrow stromal (hMS) osteoblast precursor cells. TGF-beta1 increased the steady-state mRNA level of IGF-I up to 8.5+/-0.6-fold (P...

Increasing protein stability by improving beta-turns.

Science.gov (United States)

Fu, Hailong; Grimsley, Gerald R; Razvi, Abbas; Scholtz, J Martin; Pace, C Nick

2009-11-15

Our goal was to gain a better understanding of how protein stability can be increased by improving beta-turns. We studied 22 beta-turns in nine proteins with 66-370 residues by replacing other residues with proline and glycine and measuring the stability. These two residues are statistically preferred in some beta-turn positions. We studied: Cold shock protein B (CspB), Histidine-containing phosphocarrier protein, Ubiquitin, Ribonucleases Sa2, Sa3, T1, and HI, Tryptophan synthetase alpha-subunit, and Maltose binding protein. Of the 15 single proline mutations, 11 increased stability (Average = 0.8 +/- 0.3; Range = 0.3-1.5 kcal/mol), and the stabilizing effect of double proline mutants was additive. On the basis of this and our previous work, we conclude that proteins can generally be stabilized by replacing nonproline residues with proline residues at the i + 1 position of Type I and II beta-turns and at the i position in Type II beta-turns. Other turn positions can sometimes be used if the phi angle is near -60 degrees for the residue replaced. It is important that the side chain of the residue replaced is less than 50% buried. Identical substitutions in beta-turns in related proteins give similar results. Proline substitutions increase stability mainly by decreasing the entropy of the denatured state. In contrast, the large, diverse group of proteins considered here had almost no residues in beta-turns that could be replaced by Gly to increase protein stability. Improving beta-turns by substituting Pro residues is a generally useful way of increasing protein stability. 2009 Wiley-Liss, Inc.

Crystal structure of Agaricus bisporus mushroom tyrosinase: identity of the tetramer subunits and interaction with tropolone.

Science.gov (United States)

Ismaya, Wangsa T; Rozeboom, Henriëtte J; Weijn, Amrah; Mes, Jurriaan J; Fusetti, Fabrizia; Wichers, Harry J; Dijkstra, Bauke W

2011-06-21

Tyrosinase catalyzes the conversion of phenolic compounds into their quinone derivatives, which are precursors for the formation of melanin, a ubiquitous pigment in living organisms. Because of its importance for browning reactions in the food industry, the tyrosinase from the mushroom Agaricus bisporus has been investigated in depth. In previous studies the tyrosinase enzyme complex was shown to be a H(2)L(2) tetramer, but no clues were obtained of the identities of the subunits, their mode of association, and the 3D structure of the complex. Here we unravel this tetramer at the molecular level. Its 2.3 Å resolution crystal structure is the first structure of the full fungal tyrosinase complex. The complex comprises two H subunits of ∼392 residues and two L subunits of ∼150 residues. The H subunit originates from the ppo3 gene and has a fold similar to other tyrosinases, but it is ∼100 residues larger. The L subunit appeared to be the product of orf239342 and has a lectin-like fold. The H subunit contains a binuclear copper-binding site in the deoxy-state, in which three histidine residues coordinate each copper ion. The side chains of these histidines have their orientation fixed by hydrogen bonds or, in the case of His85, by a thioether bridge with the side chain of Cys83. The specific tyrosinase inhibitor tropolone forms a pre-Michaelis complex with the enzyme. It binds near the binuclear copper site without directly coordinating the copper ions. The function of the ORF239342 subunits is not known. Carbohydrate binding sites identified in other lectins are not conserved in ORF239342, and the subunits are over 25 Å away from the active site, making a role in activity unlikely. The structures explain how calcium ions stabilize the tetrameric state of the enzyme.

beta. -Amyloid gene dosage in Alzheimer's disease

Energy Technology Data Exchange (ETDEWEB)

Murdoch, G H; Manuelidis, L; Kim, J H; Manuelidis, E E

1988-01-11

The 4-5 kd amyloid ..beta..-peptide is a major constituent of the characteristic amyloid plaque of Alzheimer's disease. It has been reported that some cases of sporatic Alzheimer's disease are associated with at least a partial duplication of chromosome 21 containing the gene corresponding to the 695 residue precursor of this peptide. To contribute to an understanding of the frequency to such a duplication event in the overall Alzheimer's population, the authors have determined the gene dosage of the ..beta..-amyloid gene in this collection of cases. All cases had a clinical diagnosis of Alzheimer's confirmed neuropathologically. Each Alzheimer's case had an apparent normal diploid ..beta..-amyloid gene dosage, while control Down's cases had the expected triploid dosage. Thus partial duplication of chromosome 21 may be a rare finding in Alzheimer's disease. Similar conclusions were just reported in several studies of the Harvard Alzheimer collection.

GABA-A receptor beta3 and alpha5 subunit gene cluster on chromosome 15q11-q13 and bipolar disorder: a genetic association study.

Science.gov (United States)

Papadimitriou, G N; Dikeos, D G; Karadima, G; Avramopoulos, D; Daskalopoulou, E G; Stefanis, C N

2001-05-08

There is accumulated evidence that the genes coding for the receptor of gamma aminobutyric acid (GABA), the most important inhibitory neurotransmitter in the CNS, may be involved in the pathogenesis of affective disorders. In a previous study, we have found a genetic association between the GABA-A receptor alpha5 subunit gene locus (GABRA5) on chromosome 15q11-of 13 and bipolar affective disorder. The aim of the present study was to examine the same subjects to see if there exists a genetic association between bipolar affective disorder and the GABA receptor beta3 subunit gene (GABRB3), which is located within 100 kb from GABRA5. The sample consisted of 48 bipolar patients compared to 44 controls (blood donors). All subjects were Greek, unrelated, and personally interviewed. Diagnosis was based on DSM-IV and ICD-10 criteria. The marker used was a dinucleotide (CA) repeat polymorphism with 12 alleles 179 to 201 bp long; genotyping was successful in all patients and 43 controls. The distribution of GABRB3 genotypes among the controls did not deviate significantly from the Hardy-Weinberg equilibrium. No differences in allelic frequencies between bipolar patients and controls were found for GABRB3, while this locus and GABRA5 did not seem to be in significant linkage disequilibrium. In conclusion, the GABRB3 CA-repeat polymorphism we investigated does not present the observed association between bipolar affective illness and GABRA5. This could be due to higher mutation rate in the GABRB3 CA-repeat polymorphism, but it might also signify that GABRA5 is the gene actually associated with the disease. Copyright 2001 Wiley-Liss, Inc.

Characterization of arrangement and expression of the beta-2 microglobulin locus in the sandbar and nurse shark.

Science.gov (United States)

Chen, Hao; Kshirsagar, Sarika; Jensen, Ingvill; Lau, Kevin; Simonson, Caitlin; Schluter, Samuel F

2010-02-01

Beta 2 microglobulin (beta2m) is an essential subunit of major histocompatibility complex (MHC) type I molecules. In this report, beta2m cDNAs were identified and sequenced from sandbar shark spleen cDNA library. Sandbar shark beta2m gene encodes one amino acid less than most teleost beta2m genes, and 3 amino acids less than mammal beta2m genes. Although sandbar shark beta2m protein contains one beta sheet less than that of human in the predicted protein structure, the overall structure of beta2m proteins is conserved during evolution. Germline gene for the beta2m in sandbar and nurse shark is present as a single locus. It contains three exons and two introns. CpG sites are evenly distributed in the shark beta2m loci. Several DNA repeat elements were also identified in the shark beta2m loci. Sequence analysis suggests that the beta2m locus is not linked to the MHC I loci in the shark genome.

Global proteome analysis identifies active immunoproteasome subunits in human platelets.

Science.gov (United States)

Klockenbusch, Cordula; Walsh, Geraldine M; Brown, Lyda M; Hoffman, Michael D; Ignatchenko, Vladimir; Kislinger, Thomas; Kast, Juergen

2014-12-01

The discovery of new functions for platelets, particularly in inflammation and immunity, has expanded the role of these anucleate cell fragments beyond their primary hemostatic function. Here, four in-depth human platelet proteomic data sets were generated to explore potential new functions for platelets based on their protein content and this led to the identification of 2559 high confidence proteins. During a more detailed analysis, consistently high expression of the proteasome was discovered, and the composition and function of this complex, whose role in platelets has not been thoroughly investigated, was examined. Data set mining resulted in identification of nearly all members of the 26S proteasome in one or more data sets, except the β5 subunit. However, β5i, a component of the immunoproteasome, was identified. Biochemical analyses confirmed the presence of all catalytically active subunits of the standard 20S proteasome and immunoproteasome in human platelets, including β5, which was predominantly found in its precursor form. It was demonstrated that these components were assembled into the proteasome complex and that standard proteasome as well as immunoproteasome subunits were constitutively active in platelets. These findings suggest potential new roles for platelets in the immune system. For example, the immunoproteasome may be involved in major histocompatibility complex I (MHC I) peptide generation, as the MHC I machinery was also identified in our data sets. © 2014 by The American Society for Biochemistry and Molecular Biology, Inc.

The effect of polylysine on casein-kinase-2 activity is influenced by both the structure of the protein/peptide substrates and the subunit composition of the enzyme

DEFF Research Database (Denmark)

Meggio, F; Boldyreff, B; Marin, O

1992-01-01

, moreover, is variably accounted for by changes in Vmax and/or Km, depending on the structure of the peptide substrate. Maximum stimulation with all protein/peptide substrates tested requires the presence of the beta subunit, since the recombinant alpha subunit is much less responsive than CK2 holoenzyme......The mechanism by which polybasic peptides stimulate the activity of casein kinase 2 (CK2) has been studied by comparing the effect of polylysine on the phosphorylation of a variety of protein and peptide substrates by the native CK2 holoenzyme and by its recombinant catalytic alpha subunit, either...

Affinity crosslinking of /sup 125/I-human beta-endorphin to cell lines possessing either mu or delta type opioid binding sites

Energy Technology Data Exchange (ETDEWEB)

Keren, O.; Gioannini, T.L.; Hiller, J.M.; Simon, E.J.

1986-01-01

Affinity crosslinking of human /sup 125/I-beta-Endorphin to cell lines possessing either mu or delta binding sites was carried out. Autoradiography of SDS-PAGE gels from these crosslinked cell lines revealed that these two sites contain major peptide subunits that differ in molecular size. This confirms our earlier finding in mammalian brain which demonstrated separate and distinct subunits for mu and delta opioid receptors.

HPLC Analysis of [Alpha]- and [Beta]-Acids in Hops

Science.gov (United States)

Danenhower, Travis M.; Force, Leyna J.; Petersen, Kenneth J.; Betts, Thomas A.; Baker, Gary A.

2008-01-01

Hops have been used for centuries to impart aroma and bitterness to beer. The cones of the female hop plant contain both essential oils, which include many of the fragrant components of hops, and a collection of compounds known as [alpha]- and [beta]-acids that are the precursors to bittering agents. In order for brewers to predict the ultimate…

Characterization of the beta amyloid precursor protein-like gene in the central nervous system of the crab Chasmagnathus. Expression during memory consolidation.

Science.gov (United States)

Fustiñana, Maria Sol; Ariel, Pablo; Federman, Noel; Freudenthal, Ramiro; Romano, Arturo

2010-09-01

Human β-amyloid, the main component in the neuritic plaques found in patients with Alzheimer's disease, is generated by cleavage of the β-amyloid precursor protein. Beyond the role in pathology, members of this protein family are synaptic proteins and have been associated with synaptogenesis, neuronal plasticity and memory, both in vertebrates and in invertebrates. Consolidation is necessary to convert a short-term labile memory to a long-term and stable form. During consolidation, gene expression and de novo protein synthesis are regulated in order to produce key proteins for the maintenance of plastic changes produced during the acquisition of new information. Here we partially cloned and sequenced the beta-amyloid precursor protein like gene homologue in the crab Chasmagnathus (cappl), showing a 37% of identity with the fruit fly Drosophila melanogaster homologue and 23% with Homo sapiens but with much higher degree of sequence similarity in certain regions. We observed a wide distribution of cappl mRNA in the nervous system as well as in muscle and gills. The protein localized in all tissues analyzed with the exception of muscle. Immunofluorescence revealed localization of cAPPL in associative and sensory brain areas. We studied gene and protein expression during long-term memory consolidation using a well characterized memory model: the context-signal associative memory in this crab species. mRNA levels varied at different time points during long-term memory consolidation and correlated with cAPPL protein levels cAPPL mRNA and protein is widely distributed in the central nervous system of the crab and the time course of expression suggests a role of cAPPL during long-term memory formation.

Precursor directed synthesis - ``molecular'' mechanisms in the Soft Chemistry approaches and their use for template-free synthesis of metal, metal oxide and metal chalcogenide nanoparticles and nanostructures

Science.gov (United States)

Seisenbaeva, Gulaim A.; Kessler, Vadim G.

2014-05-01

This review provides an insight into the common reaction mechanisms in Soft Chemistry processes involved in nucleation, growth and aggregation of metal, metal oxide and chalcogenide nanoparticles starting from metal-organic precursors such as metal alkoxides, beta-diketonates, carboxylates and their chalcogene analogues and demonstrates how mastering the precursor chemistry permits us to control the chemical and phase composition, crystallinity, morphology, porosity and surface characteristics of produced nanomaterials.This review provides an insight into the common reaction mechanisms in Soft Chemistry processes involved in nucleation, growth and aggregation of metal, metal oxide and chalcogenide nanoparticles starting from metal-organic precursors such as metal alkoxides, beta-diketonates, carboxylates and their chalcogene analogues and demonstrates how mastering the precursor chemistry permits us to control the chemical and phase composition, crystallinity, morphology, porosity and surface characteristics of produced nanomaterials. To Professor David Avnir on his 65th birthday.

Quantification of syntrophic fatty acid-beta-oxidizing bacteria in a mesophilic biogas reactor by oligonucleotide probe hybridization

DEFF Research Database (Denmark)

Hansen, K.W.; Ahring, Birgitte Kiær; Raskin, L.

1999-01-01

Small-subunit rRNA sequences were obtained for two saturated fatty acid-beta-oxidizing syntrophic bacteria, Syntrophomonas sapovorans and Syntrophomonas wolfei LYE, and sequence analysis confirmed their classification as members of the family Syntrophomonadaceae. S, wolfei LYE was closely related...... fatty acid-beta-oxidizing syntrophic bacteria in methanogenic environments, the microbial community structure of a sample from a full-scale biogas plant was determined. Hybridization results with probes for syntrophic bacteria-and methanogens were compared to specific methanogenic activities...

beta2-adaptin is constitutively de-phosphorylated by serine/threonine protein phosphatase PP2A and phosphorylated by a staurosporine-sensitive kinase

DEFF Research Database (Denmark)

Lauritsen, Jens Peter Holst; Menné, C; Kastrup, J

2000-01-01

Clathrin-mediated endocytosis includes cycles of assembly and disassembly of the clathrin-coated vesicle constituents. How these cycles are regulated is still not fully known but previous studies have indicated that phosphorylation of coat subunits may play a role. Here we describe that beta2-ada...... the hypothesis that phosphorylation/de-phosphorylation of coat proteins plays a regulatory role in the assembly/disassembly cycle of clathrin-coated vesicles.......Clathrin-mediated endocytosis includes cycles of assembly and disassembly of the clathrin-coated vesicle constituents. How these cycles are regulated is still not fully known but previous studies have indicated that phosphorylation of coat subunits may play a role. Here we describe that beta2......-adaptin undergoes cycles of phosphorylation/de-phosphorylation in intact cells. Thus, beta2-adaptin was constitutively de-phosphorylated by serine/threonine protein phosphatase 2A and phosphorylated by a staurosporine-sensitive kinase in vivo. Confocal laser scanning microscopy demonstrated...

Differential expression of Mediator complex subunit MED15 in testicular germ cell tumors.

Science.gov (United States)

Klümper, Niklas; Syring, Isabella; Offermann, Anne; Shaikhibrahim, Zaki; Vogel, Wenzel; Müller, Stefan C; Ellinger, Jörg; Strauß, Arne; Radzun, Heinz Joachim; Ströbel, Philipp; Brägelmann, Johannes; Perner, Sven; Bremmer, Felix

2015-09-17

Testicular germ cell tumors (TGCT) are the most common cancer entities in young men with increasing incidence observed in the last decades. For therapeutic management it is important, that TGCT are divided into several histological subtypes. MED15 is part of the multiprotein Mediator complex which presents an integrative hub for transcriptional regulation and is known to be deregulated in several malignancies, such as prostate cancer and bladder cancer role, whereas the role of the Mediator complex in TGCT has not been investigated so far. Aim of the study was to investigate the implication of MED15 in TGCT development and its stratification into histological subtypes. Immunohistochemical staining (IHC) against Mediator complex subunit MED15 was conducted on a TGCT cohort containing tumor-free testis (n = 35), intratubular germ cell neoplasia unclassified (IGCNU, n = 14), seminomas (SEM, n = 107) and non-seminomatous germ cell tumors (NSGCT, n = 42), further subdivided into embryonic carcinomas (EC, n = 30), yolk sac tumors (YST, n = 5), chorionic carcinomas (CC, n = 5) and teratomas (TER, n = 2). Quantification of MED15 protein expression was performed through IHC followed by semi-quantitative image analysis using the Definiens software. In tumor-free seminiferous tubules, MED15 protein expression was absent or only low expressed in spermatogonia. Interestingly, the precursor lesions IGCNU exhibited heterogeneous but partly very strong MED15 expression. SEM weakly express the Mediator complex subunit MED15, whereas NSGCT and especially EC show significantly enhanced expression compared to tumor-free testis. In conclusion, MED15 is differentially expressed in tumor-free testis and TGCT. While MED15 is absent or low in tumor-free testis and SEM, NSGCT highly express MED15, hinting at the diagnostic potential of this marker to distinguish between SEM and NSGCT. Further, the precursor lesion IGCNU showed increased nuclear MED15

Reduced amyloidogenic processing of the amyloid beta-protein precursor by the small-molecule Differentiation Inducing Factor-1.

Science.gov (United States)

Myre, Michael A; Washicosky, Kevin; Moir, Robert D; Tesco, Giuseppina; Tanzi, Rudolph E; Wasco, Wilma

2009-04-01

The detection of cell cycle proteins in Alzheimer's disease (AD) brains may represent an early event leading to neurodegeneration. To identify cell cycle modifiers with anti-Abeta properties, we assessed the effect of Differentiation-Inducing Factor-1 (DIF-1), a unique, small-molecule from Dictyostelium discoideum, on the proteolysis of the amyloid beta-protein precursor (APP) in a variety of different cell types. We show that DIF-1 slows cell cycle progression through G0/G1 that correlates with a reduction in cyclin D1 protein levels. Western blot analysis of DIF-treated cells and conditioned medium revealed decreases in the levels of secreted APP, mature APP, and C-terminal fragments. Assessment of conditioned media by sandwich ELISA showed reduced levels of Abeta40 and Abeta42, also demonstrating that treatment with DIF-1 effectively decreases the ratio of Abeta42 to Abeta40. In addition, DIF-1 significantly diminished APP phosphorylation at residue T668. Interestingly, site-directed mutagenesis of APP residue Thr668 to alanine or glutamic acid abolished the effect of DIF-1 on APP proteolysis and restored secreted levels of Abeta. Finally, DIF-1 prevented the accumulation of APP C-terminal fragments induced by the proteasome inhibitor lactacystin, and calpain inhibitor N-acetyl-leucyl-leucyl-norleucinal (ALLN). Our findings suggest that DIF-1 affects G0/G1-associated amyloidogenic processing of APP by a gamma-secretase-, proteasome- and calpain-insensitive pathway, and that this effect requires the presence of residue Thr668.

Characterization of amyloid beta peptides from brain extracts of transgenic mice overexpressing the London mutant of human amyloid precursor protein.

Science.gov (United States)

Pype, Stefan; Moechars, Dieder; Dillen, Lieve; Mercken, Marc

2003-02-01

Alzheimer's disease (AD) is marked by the presence of neurofibrillary tangles and amyloid plaques in the brain of patients. To study plaque formation, we report on further quantitative and qualitative analysis of human and mouse amyloid beta peptides (Abeta) from brain extracts of transgenic mice overexpressing the London mutant of human amyloid precursor protein (APP). Using enzyme-linked immunosorbant assays (ELISAs) specific for either human or rodent Abeta, we found that the peptides from both species aggregated to form plaques. The ratios of deposited Abeta1-42/1-40 were in the order of 2-3 for human and 8-9 for mouse peptides, indicating preferential deposition of Abeta42. We also determined the identity and relative levels of other Abeta variants present in protein extracts from soluble and insoluble brain fractions. This was done by combined immunoprecipitation and mass spectrometry (IP/MS). The most prominent peptides truncated either at the carboxyl- or the amino-terminus were Abeta1-38 and Abeta11-42, respectively, and the latter was strongly enriched in the extracts of deposited peptides. Taken together, our data indicate that plaques of APP-London transgenic mice consist of aggregates of multiple human and mouse Abeta variants, and the human variants that we identified were previously detected in brain extracts of AD patients.

Biochemical studies of mouse brain tubulin: colchicine binding (DEAE-cellulose filter) assay and subunits ( α and β) biosynthesis and degradation (in newborn brain)

Energy Technology Data Exchange (ETDEWEB)

Tse, Cek-Fyne [Univ. of Rochester, NY (United States)

1978-01-01

A DEAE-cellulose filter assay, measuring (³H)colchicine bound to colchicine binding protein (CBP) absorbed on filter discs, has been modified to include lM sucrose in the incubation medium for complexing colchicine to CBP in samples before applying the samples to filter discs (single point assay). Due to the much greater stability of colchicine binding capacity in the presence of lM sucrose, multiple time-point assays and least squares linear regression analysis were not necessary for accurate determination of CBP in hybrid mouse brain at different stages of development. The highest concentrations of CBP were observed in the 160,000g supernatant and pellet of newborn brain homogenate. Further studies of the modified filter assay documented that the assay has an overall counting efficiency of 27.3%, that DEAE-cellulose filters bind and retain all tubulin in the assay samples, and that one molecule of colchicine binds approximately one molecule of tubulin dimer. Therefore, millimoles of colchicine bound per milligram total protein can be used to calculate tubulin content. With this technique tubulin content of brain supernatant was found to be 11.9% for newborn, and 7.15% for 11 month old mice. Quantitative densitometry was also used to measure mouse brain supernatant actin content for these two stages. In vivo synthesis and degradation rates of tubulin ..cap alpha.. and ..beta.. subunits of two day mouse brain 100,000g supernatant were studied after intracerebral injection of (³H)leucine. Quantitative changes of the ratio of tritium specific activities of tubulin ..cap alpha.. and ..beta.. subunits with time were determined. The pattern of change was biphasic. During the first phase the ratio decreased; during the second phase the ratio increased continuously. An interpretation consistent with all the data in this study is that the ..cap alpha.. subunit is synthesized at a more rapid rate than the ..beta.. subunit. (ERB)

Structural domains required for channel function of the mouse transient receptor potential protein homologue TRP1beta.

Science.gov (United States)

Engelke, Michael; Friedrich, Olaf; Budde, Petra; Schäfer, Christina; Niemann, Ursula; Zitt, Christof; Jüngling, Eberhard; Rocks, Oliver; Lückhoff, Andreas; Frey, Jürgen

2002-07-17

Transient receptor potential proteins (TRP) are supposed to participate in the formation of store-operated Ca(2+) influx channels by co-assembly. However, little is known which domains facilitate the interaction of subunits. Contribution of the N-terminal coiled-coil domain and ankyrin-like repeats and the putative pore region of the mouse TRP1beta (mTRP1beta) variant to the formation of functional cation channels were analyzed following overexpression in HEK293 (human embryonic kidney) cells. MTRP1beta expressing cells exhibited enhanced Ca(2+) influx and enhanced whole-cell membrane currents compared to mTRP1beta deletion mutants. Using a yeast two-hybrid assay only the coiled-coil domain facilitated homodimerization of the N-terminus. These results suggest that the N-terminus of mTRP1beta is required for structural organization thus forming functional channels.

Characterization of the beta amyloid precursor protein-like gene in the central nervous system of the crab Chasmagnathus. Expression during memory consolidation

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Fustiñana Maria

2010-09-01

Full Text Available Abstract Background Human β-amyloid, the main component in the neuritic plaques found in patients with Alzheimer's disease, is generated by cleavage of the β-amyloid precursor protein. Beyond the role in pathology, members of this protein family are synaptic proteins and have been associated with synaptogenesis, neuronal plasticity and memory, both in vertebrates and in invertebrates. Consolidation is necessary to convert a short-term labile memory to a long-term and stable form. During consolidation, gene expression and de novo protein synthesis are regulated in order to produce key proteins for the maintenance of plastic changes produced during the acquisition of new information. Results Here we partially cloned and sequenced the beta-amyloid precursor protein like gene homologue in the crab Chasmagnathus (cappl, showing a 37% of identity with the fruit fly Drosophila melanogaster homologue and 23% with Homo sapiens but with much higher degree of sequence similarity in certain regions. We observed a wide distribution of cappl mRNA in the nervous system as well as in muscle and gills. The protein localized in all tissues analyzed with the exception of muscle. Immunofluorescence revealed localization of cAPPL in associative and sensory brain areas. We studied gene and protein expression during long-term memory consolidation using a well characterized memory model: the context-signal associative memory in this crab species. mRNA levels varied at different time points during long-term memory consolidation and correlated with cAPPL protein levels Conclusions cAPPL mRNA and protein is widely distributed in the central nervous system of the crab and the time course of expression suggests a role of cAPPL during long-term memory formation.

Evaluation of Zinc-alpha-2-Glycoprotein and Proteasome Subunit beta-Type 6 Expression in Prostate Cancer Using Tissue Microarray Technology.

LENUS (Irish Health Repository)

2010-07-23

Prostate cancer (CaP) is a significant cause of illness and death in males. Current detection strategies do not reliably detect the disease at an early stage and cannot distinguish aggressive versus nonaggressive CaP leading to potential overtreatment of the disease and associated morbidity. Zinc-alpha-2-glycoprotein (ZAG) and proteasome subunit beta-Type 6 (PSMB-6) were found to be up-regulated in the serum of CaP patients with higher grade tumors after 2-dimensional difference gel electrophoresis analysis. The aim of this study was to investigate if ZAG and PSMB-6 were also overexpressed in prostatic tumor tissue of CaP patients. Immunohistochemical analysis was performed on CaP tissue microarrays with samples from 199 patients. Confirmatory gene expression profiling for ZAG and PSMB-6 were performed on 4 cases using Laser Capture Microdissection and TaqMan real-time polymerase chain reaction. ZAG expression in CaP epithelial cells was inversely associated with Gleason grade (benign prostatic hyperplasia>G3>G4\\/G5). PSMB-6 was not expressed in either tumor or benign epithelium. However, strong PSMB-6 expression was noted in stromal and inflammatory cells. Our results indicate ZAG as a possible predictive marker of Gleason grade. The inverse association between grade and tissue expression with a rising serum protein level is similar to that seen with prostate-specific antigen. In addition, the results for both ZAG and PSMB-6 highlight the challenges in trying to associate the protein levels in serum with tissue expression.

The Mechanism of $\\beta$-Delayed Two-Proton Emission

CERN Multimedia

2002-01-01

The nucleus $^{31}$Ar seems to be the most prolific ${\\beta}$-2p precursor known to date and is at the same time the one with the largest production yields at ISOLDE, where the most sensitive experiments can be done. Our purpose with this experiment is to study the ${\\beta}$-2p branches in detail, search for ${\\beta}$-3p events, place them in the decay scheme and obtain information on the decay mechanism for ${\\beta}$-2p via the energy distribution and the angular correlation between the two protons. As a by product we shall also resolve existing inconsistencies in the level scheme.\\\\ \\\\ The nucleus $^{31}$Ar, produced in a cold plasma ion source unit by the impact of a 1 GeV proton beam of 0.5 Hz frequency, had an average yield over one week of 1.5 $^{31}$Ar atoms/s. The beam passed through the central hole of an annular Si detector ($\\Omega$ = 4.3~\\%) and stopped in a thin carbon foil tilted 45$^o$ with respect to the beam direction. A 70~\\% coaxial HPGe-detector ($\\Omega$~=~7.4~\\%) was located opposite to ...

Electrochemical and spectroscopic characterisation of amphetamine-like drugs: Application to the screening of 3,4-methylenedioxymethamphetamine (MDMA) and its synthetic precursors

OpenAIRE

Milhazes, Nuno; Martins, Pedro; Uriarte, Eugenio; Garrido, Jorge; Calheiros, Rita; Marques, M. Paula M.; Borges, Fernanda

2007-01-01

A complete physicochemical characterisation of MDMA and its synthetic precursors MDA, 3,4-methylenedioxybenzaldehyde (piperonal) and 3,4-methylenedioxy-beta-methyl-beta-nitrostyrene was carried out through voltammetric assays and Raman spectroscopy combined with theoretical (DFT) calculations. The former provided important analytical redox data, concluding that the oxidative mechanism of the N-demethylation of MDMA involves the removal of an electron from the amino-nitrogen atom, leading to t...

Endotoxin/lipopolysaccharide activates NF-kappa B and enhances tumor cell adhesion and invasion through a beta 1 integrin-dependent mechanism.

LENUS (Irish Health Repository)

Wang, Jiang Huai

2012-02-03

Beta(1) integrins play a crucial role in supporting tumor cell attachment to and invasion into the extracellular matrix. Endotoxin\\/LPS introduced by surgery has been shown to enhance tumor metastasis in a murine model. Here we show the direct effect of LPS on tumor cell adhesion and invasion in extracellular matrix proteins through a beta(1) integrin-dependent pathway. The human colorectal tumor cell lines SW480 and SW620 constitutively expressed high levels of the beta(1) subunit, whereas various low levels of alpha(1), alpha(2), alpha(4), and alpha(6) expression were detected. SW480 and SW620 did not express membrane-bound CD14; however, LPS in the presence of soluble CD14 (sCD14) significantly up-regulated beta(1) integrin expression; enhanced tumor cell attachment to fibronectin, collagen I, and laminin; and strongly promoted tumor cell invasion through the Matrigel. Anti-beta(1) blocking mAbs (4B4 and 6S6) abrogated LPS- plus sCD14-induced tumor cell adhesion and invasion. Furthermore, LPS, when combined with sCD14, resulted in NF-kappaB activation in both SW480 and SW620 cells. Inhibition of the NF-kappaB pathway significantly attenuated LPS-induced up-regulation of beta(1) integrin expression and prevented tumor cell adhesion and invasion. These results provide direct evidence that although SW480 and SW620 cells do not express membrane-bound CD14, LPS in the presence of sCD14 can activate NF-kappaB, up-regulate beta(1) integrin expression, and subsequently promote tumor cell adhesion and invasion. Moreover, LPS-induced tumor cell attachment to and invasion through extracellular matrix proteins is beta(1) subunit-dependent.

Resveratrol and Amyloid-Beta: Mechanistic Insights

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Yongming Jia

2017-10-01

Full Text Available The amyloid-beta (Aβ hypothesis that dyshomeostasis between Aβ production and clearance is a very early, key molecular factor in the etiology of Alzheimer’s disease (AD has been proposed and examined in the AD research field. Scientists have focused on seeking natural products or drugs to influence the dynamic equilibrium of Aβ, targeting production and clearance of Aβ. There is emerging evidence that resveratrol (Res, a naturally occurring polyphenol mainly found in grapes and red wine, acts on AD in numerous in vivo and in vitro models. Res decreases the amyloidogenic cleavage of the amyloid precursor protein (APP, enhances clearance of amyloid beta-peptides, and reduces Aβ aggregation. Moreover, Res also protects neuronal functions through its antioxidant properties. This review discusses the action of Res on Aβ production, clearance and aggregation and multiple potential mechanisms, providing evidence of the useful of Res for AD treatment.

Isolation, characterization, and aggregation of a structured bacterial matrix precursor.

Science.gov (United States)

Chai, Liraz; Romero, Diego; Kayatekin, Can; Akabayov, Barak; Vlamakis, Hera; Losick, Richard; Kolter, Roberto

2013-06-14

Biofilms are surface-associated groups of microbial cells that are embedded in an extracellular matrix (ECM). The ECM is a network of biopolymers, mainly polysaccharides, proteins, and nucleic acids. ECM proteins serve a variety of structural roles and often form amyloid-like fibers. Despite the extensive study of the formation of amyloid fibers from their constituent subunits in humans, much less is known about the assembly of bacterial functional amyloid-like precursors into fibers. Using dynamic light scattering, atomic force microscopy, circular dichroism, and infrared spectroscopy, we show that our unique purification method of a Bacillus subtilis major matrix protein component results in stable oligomers that retain their native α-helical structure. The stability of these oligomers enabled us to control the external conditions that triggered their aggregation. In particular, we show that stretched fibers are formed on a hydrophobic surface, whereas plaque-like aggregates are formed in solution under acidic pH conditions. TasA is also shown to change conformation upon aggregation and gain some β-sheet structure. Our studies of the aggregation of a bacterial matrix protein from its subunits shed new light on assembly processes of the ECM within bacterial biofilms.

Mutation in rod PDE6 linked to congenital stationary night blindness impairs the enzyme inhibition by its gamma-subunit.

Science.gov (United States)

Muradov, Khakim G; Granovsky, Alexey E; Artemyev, Nikolai O

2003-03-25

Photoreceptor cGMP phosphodiesterase (PDE6) is the effector enzyme in the vertebrate visual transduction cascade. The activity of rod PDE6 catalytic alpha- and beta-subunits is blocked in the dark by two inhibitory Pgamma-subunits. The inhibition is released upon light-stimulation of photoreceptor cells. Mutation H258N in PDE6beta has been linked to congenital stationary night blindness (CSNB) in a large Danish family (Rambusch pedigree) (Gal, A., Orth, U., Baehr, W., Schwinger, E., and Rosenberg, T. (1994) Nat. Genet. 7, 64-67.) We have analyzed the consequences of this mutation for PDE6 function using a Pgamma-sensitive PDE6alpha'/PDE5 chimera, Chi16. Biochemical analysis of the H257N mutant, an equivalent of PDE6betaH258N, demonstrates that this substitution does not alter the ability of chimeric PDE to dimerize or the enzyme's catalytic properties. The sensitivity of H257N to a competitive inhibitor zaprinast was also unaffected. However, the mutant displayed a significant impairment in the inhibitory interaction with Pgamma, which was apparent from a approximately 20-fold increase in the K(i) value (46 nM) and incomplete maximal inhibition. The inhibitory defect of H257N is not due to perturbation of noncatalytic cGMP binding to the PDE6alpha' GAF domains. The noncatalytic cGMP-binding characteristics of the H257N mutant were similar to those of the parent PDE6alpha'/PDE5 chimera. Since rod PDE6 in the Rambusch CSNB is a catalytic heterodimer of the wild-type PDE6alpha and mutant PDE6beta, Chi16 and H257N were coexpressed, and a heterodimeric PDE, Chi16/H257N, was isolated. It displayed two Pgamma inhibitory sites with the K(i) values of 5 and 57 nM. Our results support the hypothesis that mutation H258N in PDE6beta causes CSNB through incomplete inhibition of PDE6 activity by Pgamma, which leads to desensitization of rod photoreceptors.

Inhibition of iodine-125-labeled human follitropin binding to testicular receptor by epidermal growth factor and synthetic peptides

International Nuclear Information System (INIS)

Sluss, P.M.; Krystek, S.R. Jr.; Andersen, T.T.; Melson, B.E.; Huston, J.S.; Ridge, R.; Reichert, L.E. Jr.

1986-01-01

Two tetrapeptide sequence homologies between mouse epidermal growth factor precursor (mEGFP) and human follitropin (FSH) were revealed by a computer program that identifies identical residues among polypeptide sequences. The two tetrapeptides, Lys-Thr-Cys-Thr (KTCT) and Thr-Arg-Asp-Leu (TRDL), are present in the hormone-specific beta subunit of FSH from all species studied. These tetrapeptides are not present in the alpha subunit, which is common to all pituitary glycoprotein hormones. Both tetrapeptides are also found in mEGFP, and one tetrapeptide, TRDL, is located within the 53-residue form of mEGF purified from mouse submaxillary glands. Computer-generated hydropathy profiles predicted that both tetrapeptides are located in hydrophilic portions of the FSH beta subunit and that TRDL is in a hydrophilic portion of commercially available mEGF. Therefore, the tetrapeptides might be accessible to receptor binding sites for FSH. We report that mEGF inhibits binding of 125 I-labeled human FSH to receptors in testis by 50% (I50) at a concentration of 1.8 X 10(-5) M. No binding inhibition was observed by GnRH or arginine-vasopressin at 10(-4) M, neither of which contain the tetrapeptide sequences. FSH beta subunit, which contains both tetrapeptides, also inhibited binding (I50 = 9 X 10(-8) M) of 125 I-labeled human FSH to testis receptor. Thus, it appears that FSH beta subunit and mEGF are capable of inhibiting binding of FSH to testicular FSH receptors, presumably through interactions that include the homologous tetrapeptides. This presumption was supported by the observation that the synthetic tetrapeptides (KTCT or TRDL) were also active in inhibiting binding of 125 I-labeled human FSH to testis receptor

Matrix proteins of Nipah and Hendra viruses interact with beta subunits of AP-3 complexes.

Science.gov (United States)

Sun, Weina; McCrory, Thomas S; Khaw, Wei Young; Petzing, Stephanie; Myers, Terrell; Schmitt, Anthony P

2014-11-01

Paramyxoviruses and other negative-strand RNA viruses encode matrix proteins that coordinate the virus assembly process. The matrix proteins link the viral glycoproteins and the viral ribonucleoproteins at virus assembly sites and often recruit host machinery that facilitates the budding process. Using a co-affinity purification strategy, we have identified the beta subunit of the AP-3 adapter protein complex, AP3B1, as a binding partner for the M proteins of the zoonotic paramyxoviruses Nipah virus and Hendra virus. Binding function was localized to the serine-rich and acidic Hinge domain of AP3B1, and a 29-amino-acid Hinge-derived polypeptide was sufficient for M protein binding in coimmunoprecipitation assays. Virus-like particle (VLP) production assays were used to assess the relationship between AP3B1 binding and M protein function. We found that for both Nipah virus and Hendra virus, M protein expression in the absence of any other viral proteins led to the efficient production of VLPs in transfected cells, and this VLP production was potently inhibited upon overexpression of short M-binding polypeptides derived from the Hinge region of AP3B1. Both human and bat (Pteropus alecto) AP3B1-derived polypeptides were highly effective at inhibiting the production of VLPs. VLP production was also impaired through small interfering RNA (siRNA)-mediated depletion of AP3B1 from cells. These findings suggest that AP-3-directed trafficking processes are important for henipavirus particle production and identify a new host protein-virus protein binding interface that could become a useful target in future efforts to develop small molecule inhibitors to combat paramyxoviral infections. Henipaviruses cause deadly infections in humans, with a mortality rate of about 40%. Hendra virus outbreaks in Australia, all involving horses and some involving transmission to humans, have been a continuing problem. Nipah virus caused a large outbreak in Malaysia in 1998, killing 109 people

New Insights in the Amyloid-Beta Interaction with Mitochondria

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Carlos Spuch

2012-01-01

Full Text Available Biochemical and morphological alterations of mitochondria may play an important role in the pathogenesis of Alzheimer’s disease (AD. Particularly, mitochondrial dysfunction is a hallmark of amyloid-beta-induced neuronal toxicity in Alzheimer’s disease. The recent emphasis on the intracellular biology of amyloid-beta and its precursor protein (APP has led researchers to consider the possibility that mitochondria-associated and mitochondrial amyloid-beta may directly cause neurotoxicity. Both proteins are known to localize to mitochondrial membranes, block the transport of nuclear-encoded mitochondrial proteins to mitochondria, interact with mitochondrial proteins, disrupt the electron transport chain, increase reactive oxygen species production, cause mitochondrial damage, and prevent neurons from functioning normally. In this paper, we will outline current knowledge of the intracellular localization of amyloid-beta. Moreover, we summarize evidence from AD postmortem brain as well as animal AD models showing that amyloid-beta triggers mitochondrial dysfunction through a number of pathways such as impairment of oxidative phosphorylation, elevation of reactive oxygen species production, alteration of mitochondrial dynamics, and interaction with mitochondrial proteins. Thus, this paper supports the Alzheimer cascade mitochondrial hypothesis such as the most important early events in this disease, and probably one of the future strategies on the therapy of this neurodegenerative disease.

Crotacetin, a novel snake venom C-type lectin, is homolog of convulxin

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G. Rádis-Baptista

2005-12-01

Full Text Available Snake venom (sv C-type lectins encompass a group of hemorrhagic toxins, which are able to interfere with hemostasis. They share significant similarity in their primary structures with C-type lectins of other animals, and also present a conserved carbohydrate recognition domain (CRD. A very well studied sv C-type lectin is the heterodimeric toxin, convulxin (CVX, from the venoms of South American rattlesnakes, Crotalus durissus terrificus and C. d. cascavella. It consists of two subunits, alfa (CVXalpha , 13.9 kDa and beta (CVXbeta , 12.6 kDa, joined by inter and intra-chain disulfide bounds, and is arranged in a tetrameric alpha4beta4 conformation. Convulxin is able to activate platelet and induce their aggregation by acting via p62/GPVI collagen receptor. Several cDNA precursors, homolog of CVX subunits, were cloned by PCR homology screening. As determined by computational analysis, one of them, named crotacetin beta subunit, was predicted as a polypeptide with a tridimensional conformation very similar to other subunits of convulxin-like snake toxins. Crotacetin was purified from C. durissus venoms by gel permeation and reverse phase high performance liquid chromatography. The heterodimeric crotacetin is expressed in the venoms of several C. durissus subspecies, but it is prevalent in the venom of C. durissus cascavella. As inferred from homology modeling, crotacetin induces platelet aggregation but noticeably exhibits antimicrobial activity against Gram-positive and Gram-negative bacteria.

Specific recognition of the C-terminal end of A beta 42 by a high affinity monoclonal antibody

DEFF Research Database (Denmark)

Axelsen, Trine Veje; Holm, Arne; Birkelund, Svend

2009-01-01

The neurotoxic peptide A beta(42) is derived from the amyloid precursor protein by proteolytic cleavage and is deposited in the brain of patients suffering from Alzheimer's disease (AD). In this study we generate a high affinity monoclonal antibody that targets the C-terminal end of A beta(42......) with high specificity. By this is meant that the paratope of the antibody must enclose the C-terminal end of A beta(42) including the carboxy-group of amino acid 42, and not just recognize a linear epitope in the C-terminal part of A beta. This has been accomplished by using a unique antigen construct made...... by the Ligand Presenting Assembly technology (LPA technology). This strategy results in dimeric presentation of the free C-terminal end of A beta(42). The generated Mab A beta1.1 is indeed specific for the C-terminal end of A beta(42) to which it binds with high affinity. Mab A beta1.1 recognizes the epitope...

Role of the Rubisco Small Subunit

Energy Technology Data Exchange (ETDEWEB)

Spreitzer, Robert Joseph [Univ. of Nebraska, Lincoln, NE (United States)

2016-11-05

Ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco) catalyzes the rate-limiting step of CO₂ fixation in photosynthesis. However, it is a slow enzyme, and O₂ competes with CO₂ at the active site. Oxygenation initiates the photorespiratory pathway, which also results in the loss of CO₂. If carboxylation could be increased or oxygenation decreased, an increase in net CO₂ fixation would be realized. Because Rubisco provides the primary means by which carbon enters all life on earth, there is much interest in engineering Rubisco to increase the production of food and renewable energy. Rubisco is located in the chloroplasts of plants, and it is comprised of two subunits. Much is known about the chloroplast-gene-encoded large subunit (rbcL gene), which contains the active site, but much less is known about the role of the nuclear-gene-encoded small subunit in Rubisco function (rbcS gene). Both subunits are coded by multiple genes in plants, which makes genetic engineering difficult. In the eukaryotic, green alga Chlamydomonas reinhardtii, it has been possible to eliminate all the Rubisco genes. These Rubisco-less mutants can be maintained by providing acetate as an alternative carbon source. In this project, focus has been placed on determining whether the small subunit might be a better genetic-engineering target for improving Rubisco. Analysis of a variable-loop structure (βA-βB loop) of the small subunit by genetic selection, directed mutagenesis, and construction of chimeras has shown that the small subunit can influence CO₂/O₂ specificity. X-ray crystal structures of engineered chimeric-loop enzymes have indicated that additional residues and regions of the small subunit may also contribute to Rubisco function. Structural dynamics of the small-subunit carboxyl terminus was also investigated. Alanine-scanning mutagenesis of the most-conserved small-subunit residues has identified a

Intrasteric control of AMPK via the gamma1 subunit AMP allosteric regulatory site.

Science.gov (United States)

Adams, Julian; Chen, Zhi-Ping; Van Denderen, Bryce J W; Morton, Craig J; Parker, Michael W; Witters, Lee A; Stapleton, David; Kemp, Bruce E

2004-01-01

AMP-activated protein kinase (AMPK) is a alphabetagamma heterotrimer that is activated in response to both hormones and intracellular metabolic stress signals. AMPK is regulated by phosphorylation on the alpha subunit and by AMP allosteric control previously thought to be mediated by both alpha and gamma subunits. Here we present evidence that adjacent gamma subunit pairs of CBS repeat sequences (after Cystathionine Beta Synthase) form an AMP binding site related to, but distinct from the classical AMP binding site in phosphorylase, that can also bind ATP. The AMP binding site of the gamma(1) CBS1/CBS2 pair, modeled on the structures of the CBS sequences present in the inosine monophosphate dehydrogenase crystal structure, contains three arginine residues 70, 152, and 171 and His151. The yeast gamma homolog, snf4 contains a His151Gly substitution, and when this is introduced into gamma(1), AMP allosteric control is substantially lost and explains why the yeast snf1p/snf4p complex is insensitive to AMP. Arg70 in gamma(1) corresponds to the site of mutation in human gamma(2) and pig gamma(3) genes previously identified to cause an unusual cardiac phenotype and glycogen storage disease, respectively. Mutation of any of AMP binding site Arg residues to Gln substantially abolishes AMP allosteric control in expressed AMPK holoenzyme. The Arg/Gln mutations also suppress the previously described inhibitory properties of ATP and render the enzyme constitutively active. We propose that ATP acts as an intrasteric inhibitor by bridging the alpha and gamma subunits and that AMP functions to derepress AMPK activity.

Phylogenetic characterization of the ubiquitous electron transfer flavoprotein families ETF-alpha and ETF-beta.

Science.gov (United States)

Tsai, M H; Saier, M H

1995-06-01

Electron transfer flavoproteins (ETF) are alpha beta-heterodimers found in eukaryotic mitochondria and bacteria. We have identified currently sequenced protein members of the ETF-alpha and ETF-beta families. Members of these two families include (a) the ETF subunits of mammals and bacteria, (b) homologous pairs of proteins (FixB/FixA) that are essential for nitrogen fixation in some bacteria, and (c) a pair of carnitine-inducible proteins encoded by two open reading frames in Escherichia coli (YaaQ and YaaR). These three groups of proteins comprise three clusters on both the ETF-alpha and ETF-beta phylogenetic trees, separated from each other by comparable phylogenetic distances. This fact suggests that these two protein families evolved with similar overall rates of evolutionary divergence. Relative regions of sequence conservation are evaluated, and signature sequences for both families are derived.

Role of cytochrome B in the processing of the subunits of complex III in the yeast mitochondria

International Nuclear Information System (INIS)

Sen, K.G.

1986-01-01

The work described in this dissertation deals with the effect of cytochrome b on the biogenesis and assembly of the subunits of complex III in the mitochondrial membrane of the yeast Saccharomyces cerevisiae. The cytochrome b-mutants (Box mutants of S. cerevisiae form an excellent system to study such a role of cytochome B. The amounts of cytochrome c 1 in the mitochrondria, as determined both spectroscopically and immunologically, were not affected by the absence of cytochrome b. Pulse labelling of the cells with ( 35 S) methionine in the presence of CCCP showed the accumulation of the precursors to the core protein I and the iron-sulfur protein in similar amounts in the mutant Box 6-2 and the wild type cells. Synthesis of the iron sulfur protein and the cytochrome c 1 by in vitro translation of mRNA isolated from wild type and mutant Box 6-2 in a rabbit reticulocyte lysate system, also confirmed that the synthesis of the nuclear encoded subunits was not affected in the mutants. Pulse labeling of the cells in the absence of CCCP and subsequent chase with cold methionine, however, showed much less of the mature subunits of core protein I and the iron-sulfur protein in the mitochrondria of the mutant cells relative to the wild type. These results indicate that cytochrome b is necessary for the proper processing of certain subunits of complex III

Silencing p110{beta} prevents rapid depletion of nuclear pAkt

Energy Technology Data Exchange (ETDEWEB)

Ye, Zhi-wei; Ghalali, Aram; Hoegberg, Johan [Institute of Environmental Medicine, Karolinska Institutet, S-17177 Stockholm (Sweden); Stenius, Ulla, E-mail: ulla.stenius@ki.se [Institute of Environmental Medicine, Karolinska Institutet, S-17177 Stockholm (Sweden)

2011-12-02

Highlights: Black-Right-Pointing-Pointer p110{beta} was essential for the statin- and ATP-induced depletion of nuclear pAkt and an associated inhibition of growth. Black-Right-Pointing-Pointer p110{beta} knock-out inhibited statin-induced changes in binding between FKBP51, pAkt and PTEN. Black-Right-Pointing-Pointer Data supports the hypothesis that nuclear pAkt is important for anti-cancer effects of statins. -- Abstract: The p110{beta} subunit in the class IA PI3K family may act as an oncogene and is critical for prostate tumor development in PTEN knockout mice. We tested the possible involvement of p110{beta} in a recently described rapid depletion of phosphorylated Akt (pAkt) in the nucleus. Previous work showed that this down-regulation is induced by extracellular ATP or by statins and is mediated by the purinergic receptor P2X7. Here, we used p110{beta} knock out mouse embryonic fibroblasts (MEFs) and siRNA-treated cancer cells. We found that p110{beta} is essential for ATP- or statin-induced nuclear pAkt depletion in MEFs and in several cancer cell lines including prostate cancer cells. ATP, statin or the selective P2X7 agonist BzATP also inhibited cell growth, and this inhibition was not seen in p110{beta} knock out cells. We also found that p110{beta} was necessary for statin-induced changes in binding between FKBP51, pAkt and PTEN. Our data show that p110{beta} is essential for the ATP- and statin-induced effects and support a role of nuclear pAkt in cancer development. They also provide support for a chemopreventive effect of statins mediated by depletion of nuclear pAkt.

Fetzima (levomilnacipran), a drug for major depressive disorder as a dual inhibitor for human serotonin transporters and beta-site amyloid precursor protein cleaving enzyme-1.

Science.gov (United States)

Rizvi, Syed Mohd Danish; Shaikh, Sibhghatulla; Khan, Mahiuddin; Biswas, Deboshree; Hameed, Nida; Shakil, Shazi

2014-01-01

Pharmacological management of Major Depressive Disorder includes the use of serotonin reuptake inhibitors which targets serotonin transporters (SERT) to increase the synaptic concentrations of serotonin. Beta-site amyloid precursor protein cleaving enzyme-1 (BACE-1) is responsible for amyloid β plaque formation. Hence it is an interesting target for Alzheimer's disease (AD) therapy. This study describes molecular interactions of a new Food and Drug Administration approved antidepressant drug named 'Fetzima' with BACE-1 and SERT. Fetzima is chemically known as levomilnacipran. The study has explored a possible link between the treatment of Depression and AD. 'Autodock 4.2' was used for docking study. The free energy of binding (ΔG) values for 'levomilnacipran-SERT' interaction and 'levomilnacipran-BACE1' interaction were found to be -7.47 and -8.25 kcal/mol, respectively. Levomilnacipran was found to interact with S438, known to be the most important amino acid residue of serotonin binding site of SERT during 'levomilnacipran-SERT' interaction. In the case of 'levomilnacipran-BACE1' interaction, levomilnacipran interacted with two very crucial aspartic acid residues of BACE-1, namely, D32 and D228. These residues are accountable for the cleavage of amyloid precursor protein and the subsequent formation of amyloid β plaques in AD brain. Hence, Fetzima (levomilnacipran) might act as a potent dual inhibitor of SERT and BACE-1 and expected to form the basis of a future dual therapy against depression and AD. It is an established fact that development of AD is associated with Major Depressive Disorder. Therefore, the design of new BACE-1 inhibitors based on antidepressant drug scaffolds would be particularly beneficial.

Synthesis of multi-walled carbon nanotubes/{beta}-FeOOH nanocomposites with high adsorption capacity

Energy Technology Data Exchange (ETDEWEB)

Song Haojie, E-mail: shj6922@163.com [School of Materials Science and Engineering, Jiangsu University (China); Liu Lei [Pharmaceutic College of Henan University (China); Jia Xiaohua; Min Chunying [School of Materials Science and Engineering, Jiangsu University (China)

2012-12-15

A hybrid nanostructure of multi-walled carbon nanotubes (CNTs) and {beta}-ferric oxyhydroxide ({beta}-FeOOH) nanoparticles is synthesized by ultrasonic-assisted in situ hydrolysis of the precursor ferric chloride and CNTs. Characterization by X-ray diffraction, scanning electron microscopy , and transmission electron microscopy establishes the nanohybrid structure of the synthesized sample. The results revealed that the surface of CNTs was uniformly assembled by numerous {beta}-FeOOH nanoparticles and had an average diameter of 3 nm. The formation route of anchoring {beta}-FeOOH nanoparticles onto CNTs was proposed as the intercalation and adsorption of iron ions onto the wall of CNTs, followed by the nucleation and growth of {beta}-FeOOH nanoparticles. The values of remanent magnetization (M{sub r}) and coercivity (H{sub c}) of the as-synthesized CNTs/{beta}-FeOOH nanocomposites were 0.1131 emu g, and 490.824 Oe, respectively. Furthermore, CNTs/{beta}-FeOOH nanocomposites showed a very high adsorption capacity of Congo red and thus these nanocomposites can be used as good adsorbents and can be used for the removal of the dye of Congo red from the waste water system.

Additional beta-delayed protons from the T/sub z/ = -3/2 nuclei 21Mg, 25Si, 29S, and 41Ti

International Nuclear Information System (INIS)

Zhou, Z.Y.; Schloemer, E.C.; Cable, M.D.; Ahmed, M.; Reiff, J.E.; Cerny, J.

1985-01-01

Beta-delayed proton emission has proven to be a useful tool for understanding nuclei far from stability. Most of the beta decay strength is concentrated at and below the isobaric analog state (IAS) of the parent nucleus (precursor) ground state. Identification of beta strength to states above the IAS is important not only because it provides additional tests of nuclear models of beta decay, but also because proton emission from such beta daughter nuclei frequently constitutes an important component of the background in searches for more exotic nuclei. Therefore the authors have re-measured the beta-delayed proton spectra of four T/sub z/ = -3/2 nuclei: 21 Mg, 25 Si, 29 S, and 41 Ti. Fourteen previously unreported proton groups were observed and beta decay branching ratios were derived for each

A novel beta-adrenoceptor ligand for positron emission tomography : Evaluation in experimental animals

NARCIS (Netherlands)

van Waarde, A; Elsinga, PH; Doze, P; Heldoorn, M; Jaeggi, KA; Vaalburg, W

1998-01-01

Myocardial and pulmonary beta-adrenoceptors can be imaged and quantified with the antagonist (S)-4-[3[(1,1-dimethylethyl)amino]-2-hydroxypropoxy]-1,3-dihydro-2H-benzimidazol-2-[C-11]-one (S-[C-11]CGP-12177). The synthesis of this ligand (based on the reaction of a precursor with [C-11]phosgene) is

Interactions of the integrin subunit beta1A with protein kinase B/Akt, p130Cas and paxillin contribute to regulation of radiation survival

DEFF Research Database (Denmark)

Seidler, Julia; Durzok, Rita; Brakebusch, Cord

2005-01-01

25beta1B cells, which express mutant beta1B-integrins, were compared in terms of radiation survival and beta1-integrin signaling. MATERIALS AND METHODS: Cells grown on fibronectin, collagen-III, laminin, vitronectin, anti-beta1-integrin-IgG (beta1-IgG) or poly-l-lysine were irradiated with 0-6Gy...... and phosphorylation were analyzed by Western blot technique. RESULTS: Adhesion of GD25beta1A cells to extracellular matrix proteins or beta1-IgG resulted in growth factor-independent radiation survival. In contrast, serum starved GD25beta1B cells showed a significant (Pradiation survival on all...... phosphorylation. Phosphorylated p130Cas and paxillin subsequently prevented activation of cell death-regulating JNK. CONCLUSIONS: The data show that beta1-integrin-mediated signaling through the cytoplasmic integrin domains is critical for efficient pro-survival regulation after irradiation. Profound knowledge...

Quantification of syntrophic fatty acid-{beta}-oxidizing bacteria in a mesophilic biogas reactor by oligonucleotide probe hybridization

Energy Technology Data Exchange (ETDEWEB)

Hansen, K.H.; Ahring, B.K.; Raskin, L.

1999-11-01

Small-subunit rRNA sequences were obtained for two saturated fatty acid-{beta}-oxidizing syntrophic bacteria, Syntrophomonas sapovorans and Syntrophomonas wolfei LYB, and sequence analysis confirmed their classification as members of the family Syntrophomonadaceae. S.wolfei LYB was closely related to S.wolfei subsp. solfei, but S. sapovorans did not cluster with the other members of the genus Syntrophomonas. Five oligonucleotide probes targeting the small-subunit rRNA of different groups within the family Syntrophomonadaceae, which contains all currently known saturated fatty acid-{beta}-oxidizing syntrophic bacteria, were developed and characterized. The probes were designed to be specific at the family, genus, and species levels and were characterized by temperature-of-dissociation and specificity studies. To demonstrate the usefulness of the probes for the detection and quantification of saturated fatty acid-{beta}-oxidizing syntrophic bacteria in methanogenic environments, the microbial community structure of a sample from a full-scale biogas plant was determined. Hybridization results with probes for syntrophic bacteria and methanogens were compared to specific methanogenic activities and microbial numbers determined with most-probable-number estimates. Most of the methanogenic rRNA was comprised of Methanomicrobiales rRNA, suggesting that members of this order served as the main hydrogen-utilizing microorganisms. Between 0.2 and 1% of the rRNA was attributed to the Syntrophomonadaceae, or which the majority was accounted for by the genus Syntrophomonas.

Rare earth [beta]-diketonate and carboxylate metal complexes as precursors for MOCVD of oxide films

Energy Technology Data Exchange (ETDEWEB)

Kuzmina, N.P. (Dept. of Chemistry, Moscow State Univ. (Russian Federation)); Martynenko, L.I. (Dept. of Chemistry, Moscow State Univ. (Russian Federation)); Tu, Z.A. (Dept. of Chemistry, Moscow State Univ. (Russian Federation)); Kaul, A.R. (Dept. of Chemistry, Moscow State Univ. (Russian Federation)); Girichev, G.V. (Dept. of Chemistry, Moscow State Univ. (Russian Federation)); Giricheva, N.I. (Dept. of Chemistry, Moscow State Univ. (Russian Federation)); Rykov, A.N. (Dept. of Chemistry, Moscow State Univ. (Russian Federation)); Korenev, Y.M. (Dept. of Chemistry, Moscow State Univ. (Russian Federation))

1993-08-01

Volatile and thermostable complexes of lanthanide acetylacetonates and pivalates were obtained and investigated by different methods. These compounds were used for lanthanide oxide containing film producing and for fabrication of silica optical fibers doped by lanthanide oxide. The properties of these and already known volatile precursors are compared. (orig.).

The Golgi-Localized γ-Ear-Containing ARF-Binding (GGA Proteins Alter Amyloid-β Precursor Protein (APP Processing through Interaction of Their GAE Domain with the Beta-Site APP Cleaving Enzyme 1 (BACE1.

Directory of Open Access Journals (Sweden)

Bjoern von Einem

Full Text Available Proteolytic processing of amyloid-β precursor protein (APP by beta-site APP cleaving enzyme 1 (BACE1 is the initial step in the production of amyloid beta (Aβ, which accumulates in senile plaques in Alzheimer's disease (AD. Essential for this cleavage is the transport and sorting of both proteins through endosomal/Golgi compartments. Golgi-localized γ-ear-containing ARF-binding (GGA proteins have striking cargo-sorting functions in these pathways. Recently, GGA1 and GGA3 were shown to interact with BACE1, to be expressed in neurons, and to be decreased in AD brain, whereas little is known about GGA2. Since GGA1 impacts Aβ generation by confining APP to the Golgi and perinuclear compartments, we tested whether all GGAs modulate BACE1 and APP transport and processing. We observed decreased levels of secreted APP alpha (sAPPα, sAPPβ, and Aβ upon GGA overexpression, which could be reverted by knockdown. GGA-BACE1 co-immunoprecipitation was impaired upon GGA-GAE but not VHS domain deletion. Autoinhibition of the GGA1-VHS domain was irrelevant for BACE1 interaction. Our data suggest that all three GGAs affect APP processing via the GGA-GAE domain.

Thermoluminescent dosimetry of beta radiations of 90 Sr/ 90 Y using amorphous ZrO2

International Nuclear Information System (INIS)

Rivera M, T.; Olvera T, L.; Azorin N, J.; Barrera R, M.; Soto E, A.M.

2005-01-01

In this work the results of studying the thermoluminescent properties (Tl) of the zirconium oxide in its amorphous state (ZrO 2 -a) before beta radiations of 90 Sr/ 90 Y are presented. The amorphous powders of the zirconium oxide were synthesized by means of the sol-gel technique. The sol-gel process using alkoxides like precursors, is an efficient method to prepare a matrix of zirconium oxide by hydrolysis - condensation of the precursor to form chains of Zr-H 3 and Zr-O 2 . One of the advantages of this technique is the obtention of gels at low temperatures with very high purity and homogeneity. The powders were characterized by means of thermal analysis and by X-ray diffraction. The powders of ZrO 2 -a, previously irradiated with beta particles of 90 Sr/ 90 Y, presented a thermoluminescent curve with two peaks at 150 and 257 C. The dissipation of the information of the one ZrO 2 -a was of 40% the first 2 hours remaining constant the information for the following 30 days. The reproducibility of the information was of ± 2.5% in standard deviation. The studied characteristics allow to propose to the amorphous zirconium oxide as thermoluminescent dosemeter for the detection of beta radiation. (Author)

Human beta-cell precursors mature into functional insulin-producing cells in an immunoisolation device: implications for diabetes cell therapies.

Science.gov (United States)

Lee, Seung-Hee; Hao, Ergeng; Savinov, Alexei Y; Geron, Ifat; Strongin, Alex Y; Itkin-Ansari, Pamela

2009-04-15

Islet transplantation is limited by the need for chronic immunosuppression and the paucity of donor tissue. As new sources of human beta-cells are developed (e.g., stem cell-derived tissue), transplanting them in a durable device could obviate the need for immunosuppression, while also protecting the patient from any risk of tumorigenicity. Here, we studied (1) the survival and function of encapsulated human beta-cells and their progenitors and (2) the engraftment of encapsulated murine beta-cells in allo- and autoimmune settings. Human islets and human fetal pancreatic islet-like cell clusters were encapsulated in polytetrafluorethylene devices (TheraCyte) and transplanted into immunodeficient mice. Graft survival and function was measured by immunohistochemistry, circulating human C-peptide levels, and blood glucose levels. Bioluminescent imaging was used to monitor encapsulated neonatal murine islets. Encapsulated human islet-like cell clusters survived, replicated, and acquired a level of glucose responsive insulin secretion sufficient to ameliorate hyperglycemia in diabetic mice. Bioluminescent imaging of encapsulated murine neonatal islets revealed a dynamic process of cell death followed by regrowth, resulting in robust long-term allograft survival. Further, in the non-obese diabetic (NOD) mouse model of type I diabetes, encapsulated primary beta-cells ameliorated diabetes without stimulating a detectable T-cell response. We demonstrate for the first time that human beta-cells function is compatible with encapsulation in a durable, immunoprotective device. Moreover, our study suggests that encapsulation of beta-cells before terminal differentiation will be a successful approach for new cell-based therapies for diabetes, such as those derived from stem cells.

Beta-lipotropin is the major component of the plasma opioid response to surgical stress in humans

Energy Technology Data Exchange (ETDEWEB)

Porro, C.A.; Facchinetti, F.; Bertellini, E.; Petraglia, F.; Stacca, R.; Barbieri, G.C.; Genazzani, A.R.

1987-12-07

There is growing experimental evidence that beta-endorphin immunoreactivity is raised by surgical stress in patients undergoing general anesthesia. As the assay methods employed to date did not allow to fully discriminate between beta-endorphin and its immediate precursor, beta-lipotropin, the authors have investigated in the present study plasma levels of these two peptides by separating them by chromatography on plasma extracts prior to radioimmunoassay. Beta-lipotropin, but not beta-endorphin, plasma levels were found to be significantly elevated during surgery in the general anesthesia group, while no change was found in either peptide concentration in the spinal one. Cortisol plasma levels also increased significantly 90 minutes after the beginning of surgery. Although the sampling time they adopted may have prevented them from detecting an early peak of beta-endorphin during the first 30 minutes of surgery, the major component of the pituitary opioid response to surgical stress appears to be related to beta-lipotropin. This is in agreement with results of experimental work on various kinds of stress in animals and humans and seems to rule out a role for plasma beta-endorphin in post-operative analgesia. 38 references, 1 figure, 1 table.

Defining carbohydrate specificity of Ricinus communis agglutinin as Gal beta 1-->4GlcNAc (II) > Gal beta 1-->3GlcNAc (I) > Gal alpha 1-->3Gal (B) > Gal beta 1-->3GalNAc (T).

Science.gov (United States)

Wu, J H; Herp, A; Wu, A M

1993-03-01

To define carbohydrate specificity of Ricinus communis agglutinin (RCA1), the combining site of RCA1 was further characterized by quantitative precipitin (QPA) and precipitin-inhibition assays (QPIA). Among the oligosaccharides tested for QPIA, Gal beta 1-->4GlcNAc (II, human blood group type II precursor sequence) was found to be 7.1 times more active than Gal beta 1-->3GalNAc (T, Thomsen-Friedenreich sequence) and about 1.7 times more active than the other three disaccharides tested--Gal beta 1-->4Man, Gal beta 1-->3DAra and Gal beta 1-->6GalNAc. Gal alpha 1-->4Gal, the receptor of the uropathogenic E. coli ligand was 3.6 times less active than the II sequence. These results indicate that the beta 1-->4 linkage of the terminal Gal to subterminal GlcNAc is important as this beta 1-->4GlcNAc sequence is at least 1.6 times more active than other types of disaccharides. Among the glycoproteins examined for QPA, native and desialized bovine submandibular glycoproteins, native and desialized human plasma alpha 1-acid glycoproteins, as well as crude hog stomach mucin and its three mild acid hydrolyzed products reacted well with the lectin. These glycoproteins precipitated over 75% of the lectin nitrogen added indicating that RCA1 has the ability to recognize Gal beta 1-->4/3GlcNAc and/or the related residues at the non-reducing ends and at positions in the interior of the chains. However, Tn (GalNAc alpha 1-->Ser/Thr sequence) rich glycoproteins such as desialized ovine submandibular glycoprotein and desialized armadillo salivary glycoprotein, in which over 90% of the carbohydrate side chains are Tn determinants with none or only a trace of I/II or T determinants, precipitated poorly with RCA1. From the present and previous results obtained, the carbohydrate specificity of RCA1 can be constructed and summarized in decreasing order by lectin determinants as follows: II (Gal beta 1-->4GlcNAc) > I (Gal beta 1-->3GlcNAc) > E (Gal alpha 1-->4Gal) and B (Gal alpha 1-->3Gal

$\\beta$-delayed fission in proton-rich nuclei in the lead region

CERN Document Server

AUTHOR|(CDS)2085005; Huyse, Mark; Popescu, Lucia

Nuclear fission is the breakup of an atomic nucleus into two (sometimes three) fragments, thereby releasing a large amount of energy. Soon after its discovery in the late 1930’s, the gross properties of the fission phenomenon were explained by macroscopic nuclear models. Certain features however, such as asymmetric fission-fragment mass distributions in the actinide region, require the inclusion of microscopic effects. This interplay of the microscopic motion of individual nucleons on this macroscopic process is, until today, not yet fully understood. The phenomenon of fission has therefore been of recurring interest for both theoretical and experimental studies. This thesis work focuses on the $\\beta$-delayed fission ($\\beta$DF) process, an excellent tool to study low-energy fission of exotic nuclei, which was discovered in 1966 in the actinide region. In this two-step process, a precursor nucleus first undergoes $\\beta$-decay to an excited level in the daughter nucleus, which may subsequently fission. Rec...

Electrochemical and spectroscopic characterisation of amphetamine-like drugs: Application to the screening of 3,4-methylenedioxymethamphetamine (MDMA) and its synthetic precursors

Energy Technology Data Exchange (ETDEWEB)

Milhazes, Nuno [CEQOFFUP, Faculdade de Farmacia, Universidade do Porto (Portugal); Departamento de Quimica Organica, Faculdade de Farmacia, Universidade do Porto (Portugal); Instituto Superior de Ciencias da Saude-Norte, Gandra, Paredes (Portugal); Martins, Pedro [Departamento de Quimica Organica, Facultade de Farmacia, Universidad de Santiago de Compostela (Spain); Uriarte, Eugenio [Departamento de Quimica Organica, Facultade de Farmacia, Universidad de Santiago de Compostela (Spain); Garrido, Jorge [Unidade I and D ' Quimica-Fisica Molecular' (Portugal); Departamento de Engenharia Quimica, ISEP, Instituto Politecnico do Porto (Portugal); Calheiros, Rita [Unidade I and D ' Quimica-Fisica Molecular' (Portugal); Marques, M. Paula M. [Unidade I and D ' Quimica-Fisica Molecular' (Portugal); Departamento de Bioquimica, Faculdade de Ciencias e Tecnologia, Universidade de Coimbra (Portugal); Borges, Fernanda [Departamento de Quimica Organica, Faculdade de Farmacia, Universidade do Porto (Portugal) and Unidade I and D ' Quimica-Fisica Molecular' (Portugal)]. E-mail: fborges@ff.up.pt

2007-07-23

A complete physicochemical characterisation of MDMA and its synthetic precursors MDA, 3,4-methylenedioxybenzaldehyde (piperonal) and 3,4-methylenedioxy-{beta}-methyl-{beta}-nitrostyrene was carried out through voltammetric assays and Raman spectroscopy combined with theoretical (DFT) calculations. The former provided important analytical redox data, concluding that the oxidative mechanism of the N-demethylation of MDMA involves the removal of an electron from the amino-nitrogen atom, leading to the formation of a primary amine and an aldehyde. The vibrational spectroscopic experiments enable to afford a rapid and reliable detection of this type of compounds, since they yield characteristic spectral patterns that lead to an unequivocal identification. Moreover, the rational synthesis of the drug of abuse 3,4-methylenedioxymethamphetamine (MDMA or 'ecstasy') from one of its most relevant precursors 3,4-methylene-dioxyamphetamine (MDA), is reported. In addition, several approaches for the N-methylation of MDA, a limiting synthetic step, were attempted and the overall yields compared.

Electrochemical and spectroscopic characterisation of amphetamine-like drugs: application to the screening of 3,4-methylenedioxymethamphetamine (MDMA) and its synthetic precursors.

Science.gov (United States)

Milhazes, Nuno; Martins, Pedro; Uriarte, Eugenio; Garrido, Jorge; Calheiros, Rita; Marques, M Paula M; Borges, Fernanda

2007-07-23

A complete physicochemical characterisation of MDMA and its synthetic precursors MDA, 3,4-methylenedioxybenzaldehyde (piperonal) and 3,4-methylenedioxy-beta-methyl-beta-nitrostyrene was carried out through voltammetric assays and Raman spectroscopy combined with theoretical (DFT) calculations. The former provided important analytical redox data, concluding that the oxidative mechanism of the N-demethylation of MDMA involves the removal of an electron from the amino-nitrogen atom, leading to the formation of a primary amine and an aldehyde. The vibrational spectroscopic experiments enable to afford a rapid and reliable detection of this type of compounds, since they yield characteristic spectral patterns that lead to an unequivocal identification. Moreover, the rational synthesis of the drug of abuse 3,4-methylenedioxymethamphetamine (MDMA or "ecstasy") from one of its most relevant precursors 3,4-methylene-dioxyamphetamine (MDA), is reported. In addition, several approaches for the N-methylation of MDA, a limiting synthetic step, were attempted and the overall yields compared.

X11beta rescues memory and long-term potentiation deficits in Alzheimer's disease APPswe Tg2576 mice.

LENUS (Irish Health Repository)

Mitchell, Jacqueline C

2009-12-01

Increased production and deposition of amyloid beta-protein (Abeta) are believed to be key pathogenic events in Alzheimer\\'s disease. As such, routes for lowering cerebral Abeta levels represent potential therapeutic targets for Alzheimer\\'s disease. X11beta is a neuronal adaptor protein that binds to the intracellular domain of the amyloid precursor protein (APP). Overexpression of X11beta inhibits Abeta production in a number of experimental systems. However, whether these changes to APP processing and Abeta production induced by X11beta overexpression also induce beneficial effects to memory and synaptic plasticity are not known. We report here that X11beta-mediated reduction in cerebral Abeta is associated with normalization of both cognition and in vivo long-term potentiation in aged APPswe Tg2576 transgenic mice that model the amyloid pathology of Alzheimer\\'s disease. Overexpression of X11beta itself has no detectable adverse effects upon mouse behaviour. These findings support the notion that modulation of X11beta function represents a therapeutic target for Abeta-mediated neuronal dysfunction in Alzheimer\\'s disease.

Structure of poly (. beta. -alanine) polymerized in the solid state. Koso jugo shita. beta. -alanine no kozo

Energy Technology Data Exchange (ETDEWEB)

Sakabe, Hiroshi; Nakamura, Hiroyoshi; Kimura, Hirokazu; Konishi, Takashi [Kyoto Inst. of Tech., Kyoto (Japan). Faculty of Textile Science

1989-12-05

The structure of poly({beta}-alanine) polymerized in the solid state was studied. This polymerization was carried out on a single crystal of {beta}-alanine at 170 centigrade for 40 h in an evacuated tube. The crystal structure of the polymer was assigned to I-type crystal of Nylon 3. The polymer chains were oriented vertical to the crystal side and different to monomer crystal orientation. This may be caused by the molecular layer slipping along the cleavage plane of monomer crystal. A scanning electron microscope(SEM) showed the band structure of hundreds nm width of same orientation, but X ray showed only unoriented rings, so that they are estimated to be the structure of fine fibril like assembly or necklace like continuous chain structure of grains. Near the surface, whiskers which were thought to be oligomer of low degree of polymerization, were observed. The SEM of end view of the etched surface did not show the laminated structure but showed the network structure of about 1 mu-m which is thought to be fibril precursor. 12 refs., 10 figs.

Purification, characterization and gene cloning of thermostable O-acetyl-L-serine sulfhydrylase forming {beta}-cyano-L-alanine

Energy Technology Data Exchange (ETDEWEB)

Omura, Hironori; Yoshida, Toyokazu; Nagasawa, Toru [Gifu Univ. (Japan). Dept. of Biomolecular Science; Kuroda, Masako [Ikeda Food Research Co., Ltd., Fukuyama, Hiroshima (Japan); Kobayashi, Michihiko; Shimizu, Sakayu [Kyoto Univ. (Japan). Agricultural Sciences

2003-10-01

A thermophilic and cyanide ion-tolerant bacterium, Bacillus stearothermophilus CN3 isolated from a hot spring in Japan, was found to produce thermostable {beta}-cyano-L-alanine synthase. The enzyme catalyzes the synthesis of {beta}-cyano-L-alanine from O-acetyl-L-serine and cyanide ions. The purified enzyme has a molecular mass of approximately 70 kDa and consists of two identical sub-units. It was stable in the pH range of 6.0 to 10.0 and up to 70degC. The enzyme also catalyzes the synthesis of various {beta}-substituted-L-alanine derivatives from O-acetyl-L-serine and nucleophilic reagents. The gene encoding the {beta}-cyano-L-alanine synthase was isolated from B. stearothermophilus CN3. Sequence homology analysis revealed that the {beta}-cyano-L-alanine synthase of the bacterium is O-acetyl-L-serine sulfhydrylase. A recombinant plasmid, constructed by ligation of the cloned gene and an expression vector, pKK223-3, was introduced into E. coli JM109. The transformed E. coli cells overexpressed {beta}-cyano-L-alanine synthase. Heat stable {beta}-cyano-L-alanine synthase can be applied to the synthesis of [4-{sup 11}C]L-2,4-diaminobutyric acid as a tracer for positron emission tomography. (author)

beta-Arrestin Interacts with the Beta/Gamma Subunits of Trimeric G-Proteins and Dishevelled in the Wnt/Ca2+ Pathway in Xenopus Gastrulation

Czech Academy of Sciences Publication Activity Database

Seitz, K.; Dursch, V.; Harnoš, J.; Bryja, Vítězslav; Gentzel, M.; Schambony, A.

2014-01-01

Roč. 9, č. 1 (2014) E-ISSN 1932-6203 R&D Projects: GA ČR(CZ) GC204/09/J030 Grant - others:GA ČR(CZ) GA204/09/0498 Institutional support: RVO:68081707 Keywords : CONVERGENT EXTENSION MOVEMENTS * WNT SIGNALING PATHWAYS * WNT/BETA-CATENIN Subject RIV: BO - Biophysics Impact factor: 3.234, year: 2014

Precursor directed synthesis--"molecular" mechanisms in the Soft Chemistry approaches and their use for template-free synthesis of metal, metal oxide and metal chalcogenide nanoparticles and nanostructures.

Science.gov (United States)

Seisenbaeva, Gulaim A; Kessler, Vadim G

2014-06-21

This review provides an insight into the common reaction mechanisms in Soft Chemistry processes involved in nucleation, growth and aggregation of metal, metal oxide and chalcogenide nanoparticles starting from metal-organic precursors such as metal alkoxides, beta-diketonates, carboxylates and their chalcogene analogues and demonstrates how mastering the precursor chemistry permits us to control the chemical and phase composition, crystallinity, morphology, porosity and surface characteristics of produced nanomaterials.

Mapping of the mouse actin capping protein {alpha} subunit genes and pseudogenes

Energy Technology Data Exchange (ETDEWEB)

Hart, M.C.; Korshunova, Y.O.; Cooper, J.A. [Washington Univ. School of Medicine, St. Louis, MO (United States)

1997-02-01

Capping protein (CP), a heterodimer of {alpha} and {beta} subunits, is found in all eukaryotes. CP binds to the barbed ends of actin filaments in vitro and controls actin assembly and cell motility in vivo. Vertebrates have three {alpha} isoforms ({alpha}1, {alpha}2, {alpha}3) produced from different genes, whereas lower organisms have only one gene and one isoform. We isolated genomic clones corresponding to the a subunits of mouse CP and found three {alpha}1 genes, two of which are pseudogenes, and a single gene for both {alpha}2 and {alpha}3. Their chromosomal locations were identified by interspecies backcross mapping. The {alpha}1 gene (Cappa1) mapped to Chromosome 3 between D3Mit11 and D3Mit13. The {alpha}1 pseudogenes (Cappa1-ps1 and Cappa1-ps2) mapped to Chromosomes 1 and 9, respectively. The {alpha}2 gene (Cappa2) mapped to Chromosome 6 near Ptn. The {alpha}3 gene (Cappa3) also mapped to Chromosome 6, approximately 68 cM distal from Cappa2 near Kras2. One mouse mutation, de, maps in the vicinity of the {alpha}1 gene. No known mouse mutations map to regions near the {alpha}2 or {alpha}3 genes. 29 refs., 3 figs., 1 tab.

Infundibular neurons of the human hypothalamus simultaneously reactive with antisera against endorphins, ACTH, MSH and beta-LPH.

Science.gov (United States)

Bugnon, C; Bloch, B; Lenys, D; Fellmann, D

1979-06-27

In man, discrete neurons of the infundibular (arcuate) nucleus contain compounds that can be stained with anti-endorphin (alpha and beta), anti-ACTH, anti-MSH (alpha and beta) and anti-beta-LPH immune sera (I.S.). In the fetus, certain neurons stain with anti-beta-endorphin or anti((17--39)ACTH starting from the 11th week of fetal life. At the ultrastructural level, these neurons contain elementary granules that are immunoreactive with anti-beta-endorphin. In the adult, neurons immunoreactive with anti-beta-endorphin are found in the infundibular nucleus. Their axonal fibers terminate around blood vessels in the neurovascular zone and in the pituitary stalk, or establish contacts with non-immunoreactive perikarya of the infundibular nucleus. These neurons can be stained with anti(17--39)ACTH and anti-beta-endorphin I.S. The most reactive are also stained moderately with anti-alpha-MSH, anti-beta-MSH, anti-beta-LPH, anti-alpha-endorphin, or anti(1--24)ACTH I.S. These results indicate that, in man, compound(s) identical with or immunologically related to endorphins, beta-LPH, ACTH and MSH are secreted by certain hypothalamic neurons. These agents probably originate from a common precursor molecula similar to the so-called pro-opiocortin.

Estrogen modulates potassium currents and expression of the Kv4.2 subunit in GT1-7 cells.

Science.gov (United States)

Farkas, Imre; Varju, Patricia; Liposits, Zsolt

2007-03-01

The proper maintenance of reproduction requires the pulsatile secretion of gonadotropin-releasing hormone (GnRH), which is ensured by synchronized periodic firing of multiple GnRH neurons. Both hormone secretion and electrophysiological properties of GnRH cells are influenced by estrogen. The impact of 17beta-estradiol treatment on the function of voltage gated A- and K-type potassium channels, known modulators of firing rate, was therefore examined in our experiments using immortalized GnRH-producing GT1-7 neurons. Whole cell patch clamp recordings showed the absence of the A-type current in GT1-7 cells cultured in estrogen-free medium and after 8h 17beta-estradiol treatment. Exposure of the cells to 17beta-estradiol for 24 and 48 h, respectively, resulted in the appearance of the A-type current. The induction of the A-type current by 17beta-estradiol was dose-related (50 pM to 15 nM range). In contrast, the K-type potassium current was apparent in the estrogen-free environment and 17beta-estradiol administration significantly decreased its amplitude. Co-administration of 17beta-estradiol and estrogen receptor blocker, Faslodex (ICI 182,780; 1 microM) abolished the occurrence of the A-type current. Real-time PCR data demonstrated that expression of the Kv4.2 subunit of the A-type channel was low at 0, 0.5, 2 and 8h, peaked at 24h and diminished at 48 h 17beta-estradiol treatment (15 nM). These data indicate that potassium channels of GT1-7 neurons are regulated by estrogen a mechanism that might contribute to modulation of firing rate and hormone secretion in GnRH neurons.

Direct interaction of beta-amyloid with Na,K-ATPase as a putative regulator of the enzyme function

Science.gov (United States)

Petrushanko, Irina Yu.; Mitkevich, Vladimir A.; Anashkina, Anastasia A.; Adzhubei, Alexei A.; Burnysheva, Ksenia M.; Lakunina, Valentina A.; Kamanina, Yulia V.; Dergousova, Elena A.; Lopina, Olga D.; Ogunshola, Omolara O.; Bogdanova, Anna Yu.; Makarov, Alexander A.

2016-06-01

By maintaining the Na+ and K+ transmembrane gradient mammalian Na,K-ATPase acts as a key regulator of neuronal electrotonic properties. Na,K-ATPase has an important role in synaptic transmission and memory formation. Accumulation of beta-amyloid (Aβ) at the early stages of Alzheimer’s disease is accompanied by reduction of Na,K-ATPase functional activity. The molecular mechanism behind this phenomenon is not known. Here we show that the monomeric Aβ(1-42) forms a tight (Kd of 3 μM), enthalpy-driven equimolar complex with α1β1 Na,K-ATPase. The complex formation results in dose-dependent inhibition of the enzyme hydrolytic activity. The binding site of Aβ(1-42) is localized in the “gap” between the alpha- and beta-subunits of Na,K-ATPase, disrupting the enzyme functionality by preventing the subunits from shifting towards each other. Interaction of Na,K-ATPase with exogenous Aβ(1-42) leads to a pronounced decrease of the enzyme transport and hydrolytic activity and Src-kinase activation in neuroblastoma cells SH-SY5Y. This interaction allows regulation of Na,K-ATPase activity by short-term increase of the Aβ(1-42) level. However prolonged increase of Aβ(1-42) level under pathological conditions could lead to chronical inhibition of Na,K-ATPase and disruption of neuronal function. Taken together, our data suggest the role of beta-amyloid as a novel physiological regulator of Na,K-ATPase.

Relation between the 2{nu}{beta}{beta} and 0{nu}{beta}{beta} nuclear matrix elements

Energy Technology Data Exchange (ETDEWEB)

Vogel, Petr [Kellogg Radiation Laboratory, Caltech, Pasadena, CA 91125 (United States); Simkovic, Fedor [Department of Nuclear Physics and Biophysics, Comenius University, Mlynska dolina F1, SK-84248 Bratislava (Slovakia)

2011-12-16

A formal relation between the GT part of the nuclear matrix elements M{sub GT}{sup 0{nu}} of 0{nu}{beta}{beta} decay and the closure matrix elements M{sub cl}{sup 2{nu}} of 2{nu}{beta}{beta} decay is established. This relation is based on the integral representation of these quantities in terms of their dependence on the distance r between the two nucleons undergoing transformation. We also discuss the difficulties in determining the correct values of the closure 2{nu}{beta}{beta} decay matrix elements.

Ionospheric earthquake precursors

International Nuclear Information System (INIS)

Bulachenko, A.L.; Oraevskij, V.N.; Pokhotelov, O.A.; Sorokin, V.N.; Strakhov, V.N.; Chmyrev, V.M.

1996-01-01

Results of experimental study on ionospheric earthquake precursors, program development on processes in the earthquake focus and physical mechanisms of formation of various type precursors are considered. Composition of experimental cosmic system for earthquake precursors monitoring is determined. 36 refs., 5 figs

Measurement of the absolute activity of alpha or beta emitters by measuring product nuclei (daughter) activity increase or by studing its radioactive decay

International Nuclear Information System (INIS)

Campos, L.C. de.

1981-01-01

A new method for determining absolute activity of alpha or beta emitters by measuring daughter product radioactive decay is presented. The separation method of UX from hexahydrated uranyl nitrate UO 2 (NO 3 ) 2 6H 2 O based on its dissolution in ethyl ether is described and the accuracy of this method is shown. The factors which accuate on total efficiency of a Geiger Mueller detector for beta particles are determined. The possibility to determine the mass of precursor element by daughter nuclei activity is shown. The results are compared with the one obtained by direct measurement of the mass (or number of atoms) of precursor radioactive substance and with theoretical values calculated for isotopes in secular equilibrium. (Author) [pt

NSs protein of rift valley fever virus promotes posttranslational downregulation of the TFIIH subunit p62.

Science.gov (United States)

Kalveram, Birte; Lihoradova, Olga; Ikegami, Tetsuro

2011-07-01

Rift Valley fever virus (RVFV; family Bunyaviridae, genus Phlebovirus) is an important emerging pathogen of humans and ruminants. Its NSs protein has previously been identified as a major virulence factor that suppresses host defense through three distinct mechanisms: it directly inhibits beta interferon (IFN-β) promoter activity, it promotes the degradation of double-stranded RNA-dependent protein kinase (PKR), and it suppresses host transcription by disrupting the assembly of the basal transcription factor TFIIH through sequestration of its p44 subunit. Here, we report that in addition to PKR, NSs also promotes the degradation of the TFIIH subunit p62. Infection of cells with the RVFV MP-12 vaccine strain reduced p62 protein levels to below the detection limit early in the course of infection. This NSs-mediated downregulation of p62 was posttranslational, as it was unaffected by pharmacological inhibition of transcription or translation and MP-12 infection had no effect on p62 mRNA levels. Treatment of cells with proteasome inhibitors but not inhibition of lysosomal acidification or nuclear export resulted in a stabilization of p62 in the presence of NSs. Furthermore, p62 could be coprecipitated with NSs from lysates of infected cells. These data suggest that the RVFV NSs protein is able to interact with the TFIIH subunit p62 inside infected cells and promotes its degradation, which can occur directly in the nucleus.

Triploidia fetal associada à diminuição da subunidade beta e do estriol não-conjugado no soro materno Fetal triploidy associated with low levels of unconjugated estriol and beta-subunit in maternal serum

Directory of Open Access Journals (Sweden)

Eduardo Vieira Neto

1999-05-01

Full Text Available Relatamos um caso de triploidia fetal não-molar detectada na 20ª semana gestacional por cordocentese realizada em razão de estudo ultra-sonográfico que revelou retardo do crescimento intra-uterino e grave oligoidrâmnio. Na 19ª semana foram verificados acentuada diminuição da subunidade beta livre da gonadotrofina coriônica humana e do estriol não-conjugado e níveis de alfa-fetoproteína normais, apontando para um risco aumentado de síndrome de Edwards. Houve morte fetal um dia após a cordocentese e a resolução do caso foi por parto vaginal induzido com misoprostol e ocitocina, sob analgesia peridural. Estudo cromossômico das células sangüíneas fetais revelou o cariótipo 69,XXX. O grave retardo do crescimento intra-uterino, a macrocefalia, constatada no estudo anatomopatológico do feto, e os níveis muito baixos de hCG e de estriol não-conjugado sugerem um caso de triploidia por diginia, fertilização de um óvulo diplóide por um espermatozóide haplóide.We report a case of nonmolar fetal triploidy detected by fetal blood sampling at 20 weeks of gestation, performed as an investigation of intrauterine growth retardation and severe oligohydramnios found by ultrasound scan. At 19 weeks of gestation very low levels of maternal free serum beta-subunit of human chorionic gonadotropin and unconjugated estriol, and normal levels of alpha-fetoprotein were found, which were interpreted as a high risk of fetal Edwards syndrome. Fetal death supervened the day after fetal blood sampling, and the pregnancy was terminated by vaginal delivery induced by misoprostol and oxytocin, under epidural anesthesia. Chromosome study of the fetal blood cells showed a 69,XXX karyotype. The severe intrauterine growth retardation and macrocephaly noted on pathological review plus the very low levels of hCG and unconjugated estriol suggest a fetal gynoid triploidy case, caused by the fertilization of a diploid egg by a haploid sperm.

Interactions Between the Canonical WNT/Beta-Catenin Pathway and PPAR Gamma on Neuroinflammation, Demyelination, and Remyelination in Multiple Sclerosis.

Science.gov (United States)

Vallée, Alexandre; Vallée, Jean-Noël; Guillevin, Rémy; Lecarpentier, Yves

2018-05-01

Multiple sclerosis (MS) is marked by neuroinflammation and demyelination with loss of oligodendrocytes in the central nervous system. The immune response is regulated by WNT/beta-catenin pathway in MS. Activated NF-kappaB, a major effector of neuroinflammation, and upregulated canonical WNT/beta-catenin pathway positively regulate each other. Demyelinating events present an upregulation of WNT/beta-catenin pathway, whereas proper myelinating phases show a downregulation of WNT/beta-catenin pathway essential for the promotion of oligodendrocytes precursors cells proliferation and differentiation. The activation of WNT/beta-catenin pathway results in differentiation failure and impairment in remyelination. However, PI3K/Akt pathway and TCF7L2, two downstream targets of WNT/beta-catenin pathway, are upregulated and promote proper remyelination. The interactions of these signaling pathways remain unclear. PPAR gamma activation can inhibit NF-kappaB, and can also downregulate the WNT/beta-catenin pathway. PPAR gamma and canonical WNT/beta-catenin pathway act in an opposite manner. PPAR gamma agonists appear as a promising treatment for the inhibition of demyelination and the promotion of proper remyelination through the control of both NF-kappaB activity and canonical WNT/beta-catenin pathway.

Role of regulatory subunits and protein kinase inhibitor (PKI) in determining nuclear localization and activity of the catalytic subunit of protein kinase A.

Science.gov (United States)

Wiley, J C; Wailes, L A; Idzerda, R L; McKnight, G S

1999-03-05

Regulation of protein kinase A by subcellular localization may be critical to target catalytic subunits to specific substrates. We employed epitope-tagged catalytic subunit to correlate subcellular localization and gene-inducing activity in the presence of regulatory subunit or protein kinase inhibitor (PKI). Transiently expressed catalytic subunit distributed throughout the cell and induced gene expression. Co-expression of regulatory subunit or PKI blocked gene induction and prevented nuclear accumulation. A mutant PKI lacking the nuclear export signal blocked gene induction but not nuclear accumulation, demonstrating that nuclear export is not essential to inhibit gene induction. When the catalytic subunit was targeted to the nucleus with a nuclear localization signal, it was not sequestered in the cytoplasm by regulatory subunit, although its activity was completely inhibited. PKI redistributed the nuclear catalytic subunit to the cytoplasm and blocked gene induction, demonstrating that the nuclear export signal of PKI can override a strong nuclear localization signal. With increasing PKI, the export process appeared to saturate, resulting in the return of catalytic subunit to the nucleus. These results demonstrate that both the regulatory subunit and PKI are able to completely inhibit the gene-inducing activity of the catalytic subunit even when the catalytic subunit is forced to concentrate in the nuclear compartment.

Epoxidation of limonene over Ti MCM 41 and Ti BETA

International Nuclear Information System (INIS)

Cubillos Lobo, Jairo Antonio; Gonzalez Rodriguez, Lina Maria; Montes de Correa, Consuelo

2002-01-01

Ti MCM 41 were synthesized and evaluated in the epoxidation of limonene, using peroxide of hydrogen (H 2 O) as agent oxidizer. The characteristic hexagonal phase of Ti MCM 41 was obtained by heating the precursor gel during three days at 100 centigrade degrees. Further heating up to ten days leads to a decrease of this phase. The increase (Ti) in the synthesis gel also decreases that phase. The increase of Ti in the synthesis gel also decreases that phase UV VIS and FTIR spectroscopy indicates that Ti was incorporated in the lattice of Ti MCM 41 as well as, in Ti BETA. SEM micrographs of Ti MCM 41 show that the morphology changes with the Ti loading. Ti MCM 41 was most active than Ti BETA for limonene epoxidation even though both show high selectivity to epoxides

Thermoluminescent dosimetry of beta radiations of {sup 90} Sr/ {sup 90} Y using amorphous ZrO{sub 2}; Dosimetria termoluminiscente de radiaciones beta de {sup 90} Sr/ {sup 90} Y usando ZrO{sub 2} amorfo

Energy Technology Data Exchange (ETDEWEB)

Rivera M, T. [CICATA-Legaria, IPN, Legaria Num. 694, 11500 Mexico D.F. (Mexico); Olvera T, L.; Azorin N, J.; Barrera R, M.; Soto E, A.M. [UAM-I, 09340 Mexico D.F. (Mexico)

2005-07-01

In this work the results of studying the thermoluminescent properties (Tl) of the zirconium oxide in its amorphous state (ZrO{sub 2}-a) before beta radiations of {sup 90} Sr/ {sup 90} Y are presented. The amorphous powders of the zirconium oxide were synthesized by means of the sol-gel technique. The sol-gel process using alkoxides like precursors, is an efficient method to prepare a matrix of zirconium oxide by hydrolysis - condensation of the precursor to form chains of Zr-H{sub 3} and Zr-O{sub 2}. One of the advantages of this technique is the obtention of gels at low temperatures with very high purity and homogeneity. The powders were characterized by means of thermal analysis and by X-ray diffraction. The powders of ZrO{sub 2}-a, previously irradiated with beta particles of {sup 90} Sr/{sup 90} Y, presented a thermoluminescent curve with two peaks at 150 and 257 C. The dissipation of the information of the one ZrO{sub 2}-a was of 40% the first 2 hours remaining constant the information for the following 30 days. The reproducibility of the information was of {+-} 2.5% in standard deviation. The studied characteristics allow to propose to the amorphous zirconium oxide as thermoluminescent dosemeter for the detection of beta radiation. (Author)

Acetylcholine Receptor: Complex of Homologous Subunits

Science.gov (United States)

Raftery, Michael A.; Hunkapiller, Michael W.; Strader, Catherine D.; Hood, Leroy E.

1980-06-01

The acetylcholine receptor from the electric ray Torpedo californica is composed of five subunits; two are identical and the other three are structurally related to them. Microsequence analysis of the four polypeptides demonstrates amino acid homology among the subunits. Further sequence analysis of both membrane-bound and Triton-solubilized, chromatographically purified receptor gave the stoichiometry of the four subunits (40,000:50,000:60,000:65,000 daltons) as 2:1:1:1, indicating that this protein is a pentameric complex with a molecular weight of 255,000 daltons. Genealogical analysis suggests that divergence from a common ancestral gene occurred early in the evolution of the receptor. This shared ancestry argues that each of the four subunits plays a functional role in the receptor's physiological action.

Targeting dysfunctional beta-cell signaling for the potential treatment of type 1 diabetes mellitus.

Science.gov (United States)

Fenske, Rachel J; Kimple, Michelle E

2018-03-01

Since its discovery and purification by Frederick Banting in 1921, exogenous insulin has remained almost the sole therapy for type 1 diabetes mellitus. While insulin alleviates the primary dysfunction of the disease, many other aspects of the pathophysiology of type 1 diabetes mellitus are unaffected. Research aimed towards the discovery of novel type 1 diabetes mellitus therapeutics targeting different cell signaling pathways is gaining momentum. The focus of these efforts has been almost entirely on the impact of immunomodulatory drugs, particularly those that have already received FDA-approval for other autoimmune diseases. However, these drugs can often have severe side effects, while also putting already immunocompromised individuals at an increased risk for other infections. Potential therapeutic targets in the insulin-producing beta-cell have been largely ignored by the type 1 diabetes mellitus field, save the glucagon-like peptide 1 receptor. While there is preliminary evidence to support the clinical exploration of glucagon-like peptide 1 receptor-based drugs as type 1 diabetes mellitus adjuvant therapeutics, there is a vast space for other putative therapeutic targets to be explored. The alpha subunit of the heterotrimeric G z protein (Gα z ) has been shown to promote beta-cell inflammation, dysfunction, death, and failure to replicate in the context of diabetes in a number of mouse models. Genetic loss of Gα z or inhibition of the Gα z signaling pathway through dietary interventions is protective against the development of insulitis and hyperglycemia. The multifaceted effects of Gα z in regards to beta-cell health in the context of diabetes make it an ideal therapeutic target for further study. It is our belief that a low-risk, effective therapy for type 1 diabetes mellitus will involve a multidimensional approach targeting a number of regulatory systems, not the least of which is the insulin-producing beta-cell. Impact statement The expanding

Reversible oxidative modification: a key mechanism of Na+-K+ pump regulation.

Science.gov (United States)

Figtree, Gemma A; Liu, Chia-Chi; Bibert, Stephanie; Hamilton, Elisha J; Garcia, Alvaro; White, Caroline N; Chia, Karin K M; Cornelius, Flemming; Geering, Kaethi; Rasmussen, Helge H

2009-07-17

Angiotensin II (Ang II) inhibits the cardiac sarcolemmal Na(+)-K(+) pump via protein kinase (PK)C-dependent activation of NADPH oxidase. We examined whether this is mediated by oxidative modification of the pump subunits. We detected glutathionylation of beta(1), but not alpha(1), subunits in rabbit ventricular myocytes at baseline. beta(1) Subunit glutathionylation was increased by peroxynitrite (ONOO(-)), paraquat, or activation of NADPH oxidase by Ang II. Increased glutathionylation was associated with decreased alpha(1)/beta(1) subunit coimmunoprecipitation. Glutathionylation was reversed after addition of superoxide dismutase. Glutaredoxin 1, which catalyzes deglutathionylation, coimmunoprecipitated with beta(1) subunit and, when included in patch pipette solutions, abolished paraquat-induced inhibition of myocyte Na(+)-K(+) pump current (I(p)). Cysteine (Cys46) of the beta(1) subunit was the likely candidate for glutathionylation. We expressed Na(+)-K(+) pump alpha(1) subunits with wild-type or Cys46-mutated beta(1) subunits in Xenopus oocytes. ONOO(-) induced glutathionylation of beta(1) subunit and a decrease in Na(+)-K(+) pump turnover number. This was eliminated by mutation of Cys46. ONOO(-) also induced glutathionylation of the Na(+)-K(+) ATPase beta(1) subunit from pig kidney. This was associated with a approximately 2-fold decrease in the rate-limiting E(2)-->E(1) conformational change of the pump, as determined by RH421 fluorescence. We propose that kinase-dependent regulation of the Na(+)-K(+) pump occurs via glutathionylation of its beta(1) subunit at Cys46. These findings have implications for pathophysiological conditions characterized by neurohormonal dysregulation, myocardial oxidative stress and raised myocyte Na(+) levels.

Subunits of highly Fluorescent Protein R-Phycoerythrin as Probes for Cell Imaging and Single-Molecule Detection

Energy Technology Data Exchange (ETDEWEB)

Isailovic, Dragan [Iowa State Univ., Ames, IA (United States)

2005-01-01

The purposes of our research were: (1) To characterize subunits of highly fluorescent protein R-Phycoerythrin (R-PE) and check their suitability for single-molecule detection (SMD) and cell imaging, (2) To extend the use of R-PE subunits through design of similar proteins that will be used as probes for microscopy and spectral imaging in a single cell, and (3) To demonstrate a high-throughput spectral imaging method that will rival spectral flow cytometry in the analysis of individual cells. We first demonstrated that R-PE subunits have spectroscopic and structural characteristics that make them suitable for SMD. Subunits were isolated from R-PE by high-performance liquid chromatography (HPLC) and detected as single molecules by total internal reflection fluorescence microscopy (TIRFM). In addition, R-PE subunits and their enzymatic digests were characterized by several separation and detection methods including HPLC, capillary electrophoresis, sodium dodecyl sulfate-polyacrilamide gel electrophoresis (SDS-PAGE) and HPLC-electrospray ionization mass spectrometry (ESI-MS). Favorable absorption and fluorescence of the R-PE subunits and digest peptides originate from phycoerythrobilin (PEB) and phycourobilin (PUB) chromophores that are covalently attached to cysteine residues. High absorption coefficients and strong fluorescence (even under denaturing conditions), broad excitation and emission fluorescence spectra in the visible region of electromagnetic spectrum, and relatively low molecular weights make these molecules suitable for use as fluorescence labels of biomolecules and cells. We further designed fluorescent proteins both in vitro and in vivo (in Escherichia coli) based on the highly specific attachment of PEB chromophore to genetically expressed apo-subunits of R-PE. In one example, apo-alpha and apo-beta R-PE subunits were cloned from red algae Polisiphonia boldii (P. boldii), and expressed in E. coli. Although expressed apo-subunits formed inclusion

SH2 domains of the p85 alpha subunit of phosphatidylinositol 3-kinase regulate binding to growth factor receptors.

Science.gov (United States)

McGlade, C J; Ellis, C; Reedijk, M; Anderson, D; Mbamalu, G; Reith, A D; Panayotou, G; End, P; Bernstein, A; Kazlauskas, A

1992-01-01

The binding of cytoplasmic signaling proteins such as phospholipase C-gamma 1 and Ras GTPase-activating protein to autophosphorylated growth factor receptors is directed by their noncatalytic Src homology region 2 (SH2) domains. The p85 alpha regulatory subunit of phosphatidylinositol (PI) 3-kinase, which associates with several receptor protein-tyrosine kinases, also contains two SH2 domains. Both p85 alpha SH2 domains, when expressed individually as fusion proteins in bacteria, bound stably to the activated beta receptor for platelet-derived growth factor (PDGF). Complex formation required PDGF stimulation and was dependent on receptor tyrosine kinase activity. The bacterial p85 alpha SH2 domains recognized activated beta PDGF receptor which had been immobilized on a filter, indicating that SH2 domains contact autophosphorylated receptors directly. Several receptor tyrosine kinases within the PDGF receptor subfamily, including the colony-stimulating factor 1 receptor and the Steel factor receptor (Kit), also associate with PI 3-kinase in vivo. Bacterially expressed SH2 domains derived from the p85 alpha subunit of PI 3-kinase bound in vitro to the activated colony-stimulating factor 1 receptor and to Kit. We infer that the SH2 domains of p85 alpha bind to high-affinity sites on these receptors, whose creation is dependent on receptor autophosphorylation. The SH2 domains of p85 are therefore primarily responsible for the binding of PI 3-kinase to activated growth factor receptors. Images PMID:1372092

Structural characterization by NMR of the natively unfolded extracellular domain of beta-dystroglycan: toward the identification of the binding epitope for alpha-dystroglycan.

Science.gov (United States)

Bozzi, Manuela; Bianchi, Marzia; Sciandra, Francesca; Paci, Maurizio; Giardina, Bruno; Brancaccio, Andrea; Cicero, Daniel O

2003-11-25

Dystroglycan (DG) is an adhesion molecule playing a crucial role for tissue stability during both early embriogenesis and adulthood and is composed by two tightly interacting subunits: alpha-DG, membrane-associated and highly glycosylated, and the transmembrane beta-DG. Recently, by solid-phase binding assays and NMR experiments, we have shown that the C-terminal domain of alpha-DG interacts with a recombinant extracellular fragment of beta-DG (positions 654-750) independently from glycosylation and that the linear binding epitope is located between residues 550 and 565 of alpha-DG. In order to elucidate which moieties of beta-DG are specifically involved in the complex with alpha-DG, the ectodomain has been recombinantly expressed and purified in a labeled ((13)C,(15)N) form and studied by multidimensional NMR. Although it represents a natively unfolded protein domain, we obtained an almost complete backbone assignment. Chemical shift index, (1)H-(15)N heteronuclear single-quantum coherence and nuclear Overhauser effect (HSQC-NOESY) spectra and (3)J(HN,H)(alpha) coupling constant values confirm that this protein is highly disordered, but (1)H-(15)N steady-state NOE experiments indicate that the protein presents two regions of different mobility. The first one, between residues 659 and 722, is characterized by a limited degree of mobility, whereas the C-terminal portion, containing about 30 amino acids, is highly flexible. The binding of beta-DG(654-750) to the C-terminal region of the alpha subunit, alpha-DG(485-620), has been investigated, showing that the region of beta-DG(654-750) between residues 691 and 719 is involved in the interaction.

Functions of phenylalanine residues within the beta-barrel stem of the anthrax toxin pore.

Directory of Open Access Journals (Sweden)

Jie Wang

2009-07-01

Full Text Available A key step of anthrax toxin action involves the formation of a protein-translocating pore within the endosomal membrane by the Protective Antigen (PA moiety. Formation of this transmembrane pore by PA involves interaction of the seven 2beta2-2beta3 loops of the heptameric precursor to generate a 14-strand transmembrane beta barrel.We examined the effects on pore formation, protein translocation, and cytotoxicity, of mutating two phenylalanines, F313 and F314, that lie at the tip the beta barrel, and a third one, F324, that lies part way up the barrel.Our results show that the function of these phenylalanine residues is to mediate membrane insertion and formation of stable transmembrane channels. Unlike F427, a key luminal residue in the cap of the pore, F313, F314, and F324 do not directly affect protein translocation through the pore. Our findings add to our knowledge of structure-function relationships of a key virulence factor of the anthrax bacillus.

Beta-amyloid precursor protein transgenic mice that harbor diffuse A beta deposits but do not form plaques show increased ischemic vulnerability: role of inflammation

Czech Academy of Sciences Publication Activity Database

Koistinaho, M.; Kettunen, M. I.; Goldsteins, G.; Keinänen, R.; Salminen, A.; Ort, Michael; Bureš, Jan; Liu, D.; Kauppinen, R. A.; Higgins, L. S.; Koistinaho, J.

2002-01-01

Roč. 99, č. 3 (2002), s. 1610-1615 ISSN 0027-8424 R&D Projects: GA ČR GA309/00/1656 Institutional research plan: CEZ:AV0Z5011922 Keywords : Beta-amyloid * Alzheimer disease * brain ischemia Subject RIV: FH - Neurology Impact factor: 10.701, year: 2002

Subunit stoichiometry of the chloroplast photosystem I complex

International Nuclear Information System (INIS)

Bruce, B.D.; Malkin, R.

1988-01-01

A native photosystem I (PS I) complex and a PS I core complex depleted of antenna subunits has been isolated from the uniformly 14 C-labeled aquatic higher plant, Lemna. These complexes have been analyzed for their subunit stoichiometry by quantitative sodium dodecyl sulfate-polyacrylamide gel electrophoresis methods. The results for both preparations indicate that one copy of each high molecular mass subunit is present per PS I complex and that a single copy of most low molecular mass subunits is also present. These results suggest that iron-sulfur center X, an early PS I electron acceptor proposed to bind to the high molecular mass subunits, contains a single [4Fe-4S] cluster which is bound to a dimeric structure of high molecular mass subunits, each providing 2 cysteine residues to coordinate this cluster

Blood-brain barrier dysfunction and amyloid precursor protein accumulation in microvascular compartment following ischemia-reperfusion brain injury with 1-year survival.

Science.gov (United States)

Pluta, R

2003-01-01

This study examined the late microvascular consequences of brain ischemia due to cardiac arrest in rats. In reacted vibratome sections scattered foci of extravasated horseradish peroxidase were noted throughout the brain and did not appear to be restricted to any specific area of brain. Ultrastructural investigation of leaky sites frequently presented platelets adhering to the endothelium of venules and capillaries. Endothelial cells demonstrated pathological changes with evidence of perivascular astrocytic swelling. At the same time, we noted C-terminal of amyloid precursor protein/beta-amyloid peptide (CAPP/betaA) deposits in cerebral blood vessels, with a halo of CAPP/betaA immunoreactivity in the surrounding parenchyma suggested diffusion of CAPP/betaA out of the vascular compartment. Changes predominated in the hippocampus, cerebral and entorhinal cortex, corpus callosum, thalamus, basal ganglia and around the lateral ventricles. These data implicate delayed abnormal endothelial function of vessels following ischemia-reperfusion brain injury as a primary event in the pathogenesis of the recurrent cerebral infarction.

Antimicrobial actions of the human epididymis 2 (HE2 protein isoforms, HE2alpha, HE2beta1 and HE2beta2

Directory of Open Access Journals (Sweden)

French Frank S

2004-08-01

Full Text Available Abstract Background The HE2 gene encodes a group of isoforms with similarities to the antimicrobial beta-defensins. We demonstrated earlier that the antimicrobial activity of HE2 proteins and peptides is salt resistant and structure dependent and involves permeabilization of bacterial membranes. In this study, we further characterize the antimicrobial properties of HE2 peptides in terms of the structural changes induced in E. coli and the inhibition of macromolecular synthesis. Methods E. coli treated with 50 micro g/ml of HE2alpha, HE2beta1 or HE2beta2 peptides for 30 and 60 min were visualized using transmission and scanning electron microscopy to investigate the impact of these peptides on bacterial internal and external structure. The effects of HE2alpha, HE2beta1 and HE2beta2 on E. coli macromolecular synthesis was assayed by incubating the bacteria with 2, 10 and 25 micro g/ml of the individual peptides for 0–60 min and measuring the incorporation of the radioactive precursors [methyl-3H]thymidine, [5-3H]uridine and L-[4,5-3H(N]leucine into DNA, RNA and protein. Statistical analyses using Student's t-test were performed using Sigma Plot software. Values shown are Mean ± S.D. Results E. coli treated with HE2alpha, HE2beta1 and HE2beta2 peptides as visualized by transmission electron microscopy showed extensive damage characterized by membrane blebbing, thickening of the membrane, highly granulated cytoplasm and appearance of vacuoles in contrast to the smooth and continuous membrane structure of the untreated bacteria. Similarly, bacteria observed by scanning electron microscopy after treating with HE2alpha, HE2beta1 or HE2beta2 peptides exhibited membrane blebbing and wrinkling, leakage of cellular contents, especially at the dividing septa, and external accumulation of fibrous materials. In addition, HE2alpha, HE2beta1 and HE2beta2 peptides inhibited E. coli DNA, RNA and protein synthesis. Conclusions The morphological changes observed

RNA helicase DDX3 is a regulatory subunit of casein kinase 1 in Wnt-beta-catenin signaling

NARCIS (Netherlands)

Cruciat, C.M.; Dolde, C.; de Groot, R.E.; Ohkawara, B.; Reinhard, C.; Korswagen, H.C.; Niehrs, C.

2013-01-01

Casein kinase 1 (CK1) members play key roles in numerous biological processes. They are considered "rogue" kinases, because their enzymatic activity appears unregulated. Contrary to this notion, we have identified the DEAD-box RNA helicase DDX3 as a regulator of the Wnt-beta-catenin network, where

Soybean glycinin subunits: Characterization of physicochemical and adhesion properties.

Science.gov (United States)

Mo, Xiaoqun; Zhong, Zhikai; Wang, Donghai; Sun, Xiuzhi

2006-10-04

Soybean proteins have shown great potential for applications as renewable and environmentally friendly adhesives. The objective of this work was to study physicochemical and adhesion properties of soy glycinin subunits. Soybean glycinin was extracted from soybean flour and then fractionated into acidic and basic subunits with an estimated purity of 90 and 85%, respectively. Amino acid composition of glycinin subunits was determined. The high hydrophobic amino acid content is a major contributor to the solubility behavior and water resistance of the basic subunits. Acidic subunits and glycinin had similar solubility profiles, showing more than 80% solubility at pH 2.0-4.0 or 6.5-12.0, whereas basic subunits had considerably lower solubility with the minimum at pH 4.5-8.0. Thermal analysis using a differential scanning calorimeter suggested that basic subunits form new oligomeric structures with higher thermal stability than glycinin but no highly ordered structures present in isolated acidic subunits. The wet strength of basic subunits was 160% more than that of acidic subunits prepared at their respective isoelectric points (pI) and cured at 130 degrees C. Both pH and the curing temperature significantly affected adhesive performance. High-adhesion water resistance was usually observed for adhesives from protein prepared at their pI values and cured at elevated temperatures. Basic subunits are responsible for the water resistance of glycinin and are a good starting material for the development of water-resistant adhesives.

Mutations in sodium channel {beta}-subunit SCN3B are associated with early-onset lone atrial fibrillation

DEFF Research Database (Denmark)

Olesen, Morten Salling; Jespersen, Thomas; Nielsen, Jonas Bille

2011-01-01

AIMS: Atrial fibrillation (AF) is the most frequent arrhythmia. Screening of SCN5A-the gene encoding the a-subunit of the cardiac sodium channel-has indicated that disturbances of the sodium current may play a central role in the mechanism of lone AF. We tested the hypothesis that lone AF in young...... across species. Electrophysiological studies on the SCN3B mutation were carried out and all three SCN3B mutations caused a functionally reduced sodium channel current. One synonymous variant was found in SCN4B. CONCLUSION: In 192 young lone AF patients, we found three patients with suspected disease...

One precursor, three apolipoproteins: the relationship between two crustacean lipoproteins, the large discoidal lipoprotein and the high density lipoprotein/β-glucan binding protein.

Science.gov (United States)

Stieb, Stefanie; Roth, Ziv; Dal Magro, Christina; Fischer, Sabine; Butz, Eric; Sagi, Amir; Khalaila, Isam; Lieb, Bernhard; Schenk, Sven; Hoeger, Ulrich

2014-12-01

The novel discoidal lipoprotein (dLp) recently detected in the crayfish, differs from other crustacean lipoproteins in its large size, apoprotein composition and high lipid binding capacity, We identified the dLp sequence by transcriptome analyses of the hepatopancreas and mass spectrometry. Further de novo assembly of the NGS data followed by BLAST searches using the sequence of the high density lipoprotein/1-glucan binding protein (HDL-BGBP) of Astacus leptodactylus as query revealed a putative precursor molecule with an open reading frame of 14.7 kb and a deduced primary structure of 4889 amino acids. The presence of an N-terminal lipid bind- ing domain and a DUF 1943 domain suggests the relationship with the large lipid transfer proteins. Two-putative dibasic furin cleavage sites were identified bordering the sequence of the HDL-BGBP. When subjected to mass spectroscopic analyses, tryptic peptides of the large apoprotein of dLp matched the N-terminal part of the precursor, while the peptides obtained for its small apoprotein matched the C-terminal part. Repeating the analysis in the prawn Macrobrachium rosenbergii revealed a similar protein with identical domain architecture suggesting that our findings do not represent an isolated instance. Our results indicate that the above three apolipoproteins (i.e HDL-BGBP and both the large and the small subunit of dLp) are translated as a large precursor. Cleavage at the furin type sites releases two subunits forming a heterodimeric dLP particle, while the remaining part forms an HDL-BGBP whose relationship with other lipoproteins as well as specific functions are yet to be elucidated.

Mesenchymal precursor cells maintain the differentiation and proliferation potentials of breast epithelial cells

Science.gov (United States)

2014-01-01

Introduction Stromal-epithelial interactions play a fundamental role in tissue homeostasis, controlling cell proliferation and differentiation. Not surprisingly, aberrant stromal-epithelial interactions contribute to malignancies. Studies of the cellular and molecular mechanisms underlying these interactions require ex vivo experimental model systems that recapitulate the complexity of human tissue without compromising the differentiation and proliferation potentials of human primary cells. Methods We isolated and characterized human breast epithelial and mesenchymal precursors from reduction mammoplasty tissue and tagged them with lentiviral vectors. We assembled heterotypic co-cultures and compared mesenchymal and epithelial cells to cells in corresponding monocultures by analyzing growth, differentiation potentials, and gene expression profiles. Results We show that heterotypic culture of non-immortalized human primary breast epithelial and mesenchymal precursors maintains their proliferation and differentiation potentials and constrains their growth. We further describe the gene expression profiles of stromal and epithelial cells in co-cultures and monocultures and show increased expression of the tumor growth factor beta (TGFβ) family member inhibin beta A (INHBA) in mesenchymal cells grown as co-cultures compared with monocultures. Notably, overexpression of INHBA in mesenchymal cells increases colony formation potential of epithelial cells, suggesting that it contributes to the dynamic reciprocity between breast mesenchymal and epithelial cells. Conclusions The described heterotypic co-culture system will prove useful for further characterization of the molecular mechanisms mediating interactions between human normal or neoplastic breast epithelial cells and the stroma, and will provide a framework to test the relevance of the ever-increasing number of oncogenomic alterations identified in human breast cancer. PMID:24916766

BETA digital beta radiometer

International Nuclear Information System (INIS)

Borovikov, N.V.; Kosinov, G.A.; Fedorov, Yu.N.

1989-01-01

Portable transportable digital beta radiometer providing for measuring beta-decay radionuclide specific activity in the range from 5x10 -9 up to 10 -6 Cu/kg (Cu/l) with error of ±25% is designed and introduced into commercial production for determination of volume and specific water and food radioactivity. The device specifications are given. Experience in the BETA radiometer application under conditions of the Chernobyl' NPP 30-km zone has shown that it is convenient for measuring specific activity of the order of 10 -8 Cu/kg, and application of a set of different beta detectors gives an opportunity to use it for surface contamination measurement in wide range of the measured value

LHCb: $2\\beta_s$ measurement at LHCb

CERN Multimedia

Conti, G

2009-01-01

A measurement of $2\\beta_s$, the phase of the $B_s-\\bar{B_s}$ oscillation amplitude with respect to that of the ${\\rm b} \\rightarrow {\\rm c^{+}}{\\rm W^{-}}$ tree decay amplitude, is one of the key goals of the LHCb experiment with first data. In the Standard Model (SM), $2\\beta_s$ is predicted to be $0.0360^{+0.0020}_{-0.0016} \\rm rad$. The current constraints from the Tevatron are: $2\\beta_{s}\\in[0.32 ; 2.82]$ at 68$\\%$CL from the CDF experiment and $2\\beta_{s}=0.57^{+0.24}_{-0.30}$ from the D$\\oslash$ experiment. Although the statistical uncertainties are large, these results hint at the possible contribution of New Physics in the $B_s-\\bar{B_s}$ box diagram. After one year of data taking at LHCb at an average luminosity of $\\mathcal{L}\\sim2\\cdot10^{32}\\rm cm^{-2} \\rm s^{-1}$ (integrated luminosity $\\mathcal{L}_{\\rm int}\\sim 2 \\rm fb^{-1}$), the expected statistical uncertainty on the measurement is $\\sigma(2\\beta_s)\\simeq 0.03$. This uncertainty is similar to the $2\\beta_s$ value predicted by the SM.

Bleogens: Cactus-Derived Anti-Candida Cysteine-Rich Peptides with Three Different Precursor Arrangements

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Shining Loo

2017-12-01

Full Text Available Cysteine-rich peptides (CRPs play important host-defense roles in plants. However, information concerning CRPs in the Cactaceae (cactus family is limited, with only a single cactus-derived CRP described to date. Here, we report the identification of 15 novel CRPs with three different precursor architectures, bleogens pB1-15 from Pereskia bleo of the Cactaceae family. By combining proteomic and transcriptomic methods, we showed that the prototype, bleogen pB1, contained 36 amino acid residues, a six-cysteine motif typical of the six-cysteine-hevein-like peptide (6C-HLP family, and a type I two-domain precursor consisting of an endoplasmic reticulum (ER and a mature domain. In contrast, the precursors of the other 14 bleogens contained a type II three-domain architecture with a propeptide domain inserted between the ER and the mature bleogen domain. Four of these 14 bleogens display a third type of architecture with a tandemly repeating bleogen domain. A search of the Onekp database revealed that <1% plant species possess three different precursor architectures for the biosynthesis of 6C-HLPs, including Lophophora williamsii, Pereskia aculeate, Portulaca cryptopetala, Portulaca oleracea, Portulaca suffruticosa, and Talinum sp. NMR analysis confirmed that bleogen pB1 has cystine-knot disulfide connectivity as well as a two-beta-sheet and a four-loop structural fold that is similar to other 6C-HLPs. Sequence analysis, structural studies, and in silico modeling revealed that bleogen pB1 has a cation-polar-cation motif, a signature heparin-binding motif that was confirmed by heparin affinity chromatography. Cell-based assays showed that bleogen pB1 is non-toxic to mammalian cells but functions as an anti-Candida peptide. Taken together, our findings provide insight into the occurrence, functions and precursor architectures of CRPs in the cactus family.

Synthesis of beta zeolite with mesopores from a milk containing precursor and its performance in naphthalene isopropylation

Czech Academy of Sciences Publication Activity Database

Tokarová, V.; Šťávová, G.; Nováková, J.; Stiborová, S.; Kašpárek, A.; Zukal, Arnošt

2017-01-01

Roč. 222, č. 1 (2017), s. 343-356 ISSN 1878-5190 Institutional support: RVO:61388955 Keywords : Beta zeolite * Mesopores * Naphthalene isopropylation Subject RIV: CF - Physical ; Theoretical Chemistry OBOR OECD: Physical chemistry Impact factor: 1.264, year: 2016

Increased Expression of Laminin Subunit Alpha 1 Chain by dCas9-VP160

Directory of Open Access Journals (Sweden)

Arnaud Perrin

2017-03-01

Full Text Available Laminin-111 protein complex links the extracellular matrix to integrin α7β1 in sarcolemma, thus replacing in dystrophic muscles links normally insured by the dystrophin complex. Laminin-111 injection in mdx mouse stabilized sarcolemma, restored serum creatine kinase to wild-type levels, and protected muscles from exercised-induced damages. These results suggested that increased laminin-111 is a potential therapy for DMD. Laminin subunit beta 1 and laminin subunit gamma 1 are expressed in adult human muscle, but laminin subunit alpha 1 (LAMA1 gene is expressed only during embryogenesis. We thus developed an alternative method to laminin-111 protein repeated administration by inducing expression of the endogenous mouse Lama1 gene. This was done with the CRSPR/Cas9 system, i.e., by targeting the Lama1 promoter with one or several gRNAs and a dCas9 coupled with the VP160 transcription activation domain. Lama1 mRNA (qRT-PCR and proteins (immunohistochemistry and western blot were not detected in the control C2C12 myoblasts and in control muscles. However, significant expression was observed in cells transfected and in mouse muscles electroporated with plasmids coding for dCas9-VP160 and a gRNA. Larger synergic increases were observed by using two or three gRNAs. The increased Lama1 expression did not modify the expression of the α7 and β1 integrins. Increased expression of Lama1 by the CRISPR/Cas9 system will have to be further investigated by systemic delivery of the CRISPR/Cas9 components to verify whether this could be a treatment for several myopathies.

The morphological and chemical characteristics of striatal neurons immunoreactive for the alpha1-subunit of the GABA(A) receptor in the rat.

Science.gov (United States)

Waldvogel, H J; Kubota, Y; Trevallyan, S C; Kawaguchi, Y; Fritschy, J M; Mohler, H; Faull, R L

1997-10-01

The distribution, morphology and chemical characteristics of neurons immunoreactive for the alpha1-subunit of the GABA(A) receptor in the striatum of the basal ganglia in the rat brain were investigated at the light, confocal and electron microscope levels using single, double and triple immunohistochemical labelling techniques. The results showed that alpha1-subunit immunoreactive neurons were sparsely distributed throughout the rat striatum. Double and triple labelling results showed that all the alpha1-subunit-immunoreactive neurons were positive for glutamate decarboxylase and immunoreactive for the beta2,3 and gamma2 subunits of the GABA(A) receptor. Three types of alpha1-subunit-immunoreactive neurons were identified in the striatum on the basis of cellular morphology and chemical characteristics. The most numerous alpha1-subunit-immunoreactive neurons were medium-sized, aspiny neurons with a widely branching dendritic tree. They were parvalbumin-negative and were located mainly in the dorsolateral regions of the striatum. Electron microscopy showed that these neurons had an indented nuclear membrane, typical of striatal interneurons, and were surrounded by small numbers of axon terminals which established alpha1-subunit-immunoreactive synaptic contacts with the soma and dendrites. These cells were classified as type 1 alpha1-subunit-immunoreactive neurons and comprised 75% of the total population of alpha1-subunit-immunoreactive neurons in the striatum. The remaining alpha1-subunit-immunoreactive neurons comprised of a heterogeneous population of large-sized neurons localized in the ventral and medial regions of the striatum. The most numerous large-sized cells were parvalbumin-negative, had two to three relatively short branching dendrites and were designated type 2 alpha1-subunit-immunoreactive neurons. Electron microscopy showed that the type 2 neurons were characterized by a highly convoluted nuclear membrane and were sparsely covered with small axon

The first transmembrane domain (TM1) of β2-subunit binds to the transmembrane domain S1 of α-subunit in BK potassium channels

Science.gov (United States)

Morera, Francisco J.; Alioua, Abderrahmane; Kundu, Pallob; Salazar, Marcelo; Gonzalez, Carlos; Martinez, Agustin D.; Stefani, Enrico; Toro, Ligia; Latorre, Ramon

2012-01-01

The BK channel is one of the most broadly expressed ion channels in mammals. In many tissues, the BK channel pore-forming α-subunit is associated to an auxiliary β-subunit that modulates the voltage- and Ca2+-dependent activation of the channel. Structural components present in β-subunits that are important for the physical association with the α-subunit are yet unknown. Here, we show through co-immunoprecipitation that the intracellular C-terminus, the second transmembrane domain (TM2) and the extracellular loop of the β2-subunit are dispensable for association with the α-subunit pointing transmembrane domain 1 (TM1) as responsible for the interaction. Indeed, the TOXCAT assay for transmembrane protein–protein interactions demonstrated for the first time that TM1 of the β2-subunit physically binds to the transmembrane S1 domain of the α-subunit. PMID:22710124

Effect of gamma-irradiation on the levels of total and cis/trans isomers of beta-carotene in dehydrated parsley

Energy Technology Data Exchange (ETDEWEB)

Sebastiao, K.I. E-mail: kisebas@net.ipen.br; Almeida-Muradian, L.B. E-mail: ligiabi@usp.br; Romanelli, Maria Fernanda; Koseki, Paula Massae; Villavicencio, A.L.C.H. E-mail: villavic@net.ipen.br

2002-03-01

Ionizing radiation is a method for preservation of foods that use the high energy of gamma rays or accelerated electrons, thereby ionizing molecules. The most important precursor of vitamin A is {beta}-carotene, a carotenoid with pro-vitamin A activity. The highly unsaturated chain confers the instability of {beta}-carotene, and some reactions, as isomerisation, can reduce the characteristics of pro-vitamin A. The present study investigated whether increasing doses of 0, 10 and 20 kGy lower the total {beta}-carotene level and if an enhancement of cis-isomers occurred on samples of dehydrated parsley. No differences were observed of either fractions analyzed at doses applied in this study, nor did it contribute to the decrease of vitamin A.

Nature and regulation of the insulin receptor: structure and function

International Nuclear Information System (INIS)

Czech, M.P.

1985-01-01

Native, cell-surface insulin receptor consists of two glycoprotein subunit types with apparent masses of about 125,000 daltons (alpha subunit) and 90,000 daltons (beta subunit). The alpha and beta insulin-receptor subunits seem to have distinct functions such that alpha appears to bind hormone whereas beta appears to possess intrinsic tyrosine kinase activity. In detergent extracts, insulin activates receptor autophosphorylation of tyrosine residues on its beta subunit, whereas in the presence of reductant, the alpha subunit is also phosphorylated. In intact cells, insulin activates serine/threonine phosphorylation of insulin receptor beta subunit as well as tyrosine phosphorylation. The biological role of the receptor-associated tyrosine kinase is not known. The insulin receptor kinase is regulated by beta-adrenergic agonists and other agents that elevate cAMP in adipocytes, presumably via the cAMP-dependent protein kinase. Such agents decrease receptor affinity for insulin and partially uncouple receptor tyrosine kinase activity from activation by insulin. These effects appear to contribute to the biological antagonism between insulin and beta-agonists. These data suggest the hypothesis that a complex network of tyrosine and serine/threonine phosphorylations on the insulin receptor modulate its binding and kinase activities in an antagonistic manner

The interaction of CK2alpha and CK2beta, the subunits of protein kinase CK2, requires CK2beta in a preformed conformation and is enthalpically driven

DEFF Research Database (Denmark)

Raaf, Jennifer; Brunstein, Elena; Issinger, Olaf-Georg

2008-01-01

. In contrast to the cyclins in the case of the cyclin-dependent kinases CK2beta is no on-switch of CK2alpha; rather the formation of the CK2 holoenzyme is accompanied with an overall change of the enzyme's profile including a modulation of the substrate specificity, an increase of the thermostability......, and an allocation of docking sites for membranes and other proteins. In this study we used C-terminal deletion variants of human CK2alpha and CK2beta that were enzymologically fully competent and in particular able to form a heterotetrameric holoenzyme. With differential scanning calorimetry (DSC) we confirmed...

28 CFR 51.6 - Political subunits.

Science.gov (United States)

2010-07-01

... 28 Judicial Administration 2 2010-07-01 2010-07-01 false Political subunits. 51.6 Section 51.6 Judicial Administration DEPARTMENT OF JUSTICE (CONTINUED) PROCEDURES FOR THE ADMINISTRATION OF SECTION 5 OF THE VOTING RIGHTS ACT OF 1965, AS AMENDED General Provisions § 51.6 Political subunits. All political...

Interactions between two beta-sheets. Energetics of beta/beta packing in proteins.

Science.gov (United States)

Chou, K C; Némethy, G; Rumsey, S; Tuttle, R W; Scheraga, H A

1986-04-20

The analysis of the interactions between regularly folded segments of the polypeptide chain contributes to an understanding of the energetics of protein folding. Conformational energy-minimization calculations have been carried out to determine the favorable ways of packing two right-twisted beta-sheets. The packing of two five-stranded beta-sheets was investigated, with the strands having the composition CH3CO-(L-Ile)6-NHCH3 in one beta-sheet and CH3CO-(L-Val)6-NHCH3 in the other. Two distinct classes of low-energy packing arrangements were found. In the class with lowest energies, the strands of the two beta-sheets are aligned nearly parallel (or antiparallel) with each other, with a preference for a negative orientation angle, because this arrangement corresponds to the best complementary packing of the two twisted saddle-shaped beta-sheets. In the second class, with higher interaction energies, the strands of the two beta-sheets are oriented nearly perpendicular to each other. While the surfaces of the two beta-sheets are not complementary in this arrangement, there is good packing between the corner of one beta-sheet and the interior part of the surface of the other, resulting in a favorable energy of packing. Both classes correspond to frequently observed orientations of beta-sheets in proteins. In proteins, the second class of packing is usually observed when the two beta-sheets are covalently linked, i.e. when a polypeptide strand passes from one beta-sheet to the other, but we have shown here that a large contribution to the stabilization of this packing arrangement arises from noncovalent interactions.

NSs Protein of Rift Valley Fever Virus Promotes Posttranslational Downregulation of the TFIIH Subunit p62▿

Science.gov (United States)

Kalveram, Birte; Lihoradova, Olga; Ikegami, Tetsuro

2011-01-01

Rift Valley fever virus (RVFV; family Bunyaviridae, genus Phlebovirus) is an important emerging pathogen of humans and ruminants. Its NSs protein has previously been identified as a major virulence factor that suppresses host defense through three distinct mechanisms: it directly inhibits beta interferon (IFN-β) promoter activity, it promotes the degradation of double-stranded RNA-dependent protein kinase (PKR), and it suppresses host transcription by disrupting the assembly of the basal transcription factor TFIIH through sequestration of its p44 subunit. Here, we report that in addition to PKR, NSs also promotes the degradation of the TFIIH subunit p62. Infection of cells with the RVFV MP-12 vaccine strain reduced p62 protein levels to below the detection limit early in the course of infection. This NSs-mediated downregulation of p62 was posttranslational, as it was unaffected by pharmacological inhibition of transcription or translation and MP-12 infection had no effect on p62 mRNA levels. Treatment of cells with proteasome inhibitors but not inhibition of lysosomal acidification or nuclear export resulted in a stabilization of p62 in the presence of NSs. Furthermore, p62 could be coprecipitated with NSs from lysates of infected cells. These data suggest that the RVFV NSs protein is able to interact with the TFIIH subunit p62 inside infected cells and promotes its degradation, which can occur directly in the nucleus. PMID:21543505

Interaction with beta-arrestin determines the difference in internalization behavor between beta1- and beta2-adrenergic receptors.

Science.gov (United States)

Shiina, T; Kawasaki, A; Nagao, T; Kurose, H

2000-09-15

The beta(1)-adrenergic receptor (beta(1)AR) shows the resistance to agonist-induced internalization. As beta-arrestin is important for internalization, we examine the interaction of beta-arrestin with beta(1)AR with three different methods: intracellular trafficking of beta-arrestin, binding of in vitro translated beta-arrestin to intracellular domains of beta(1)- and beta(2)ARs, and inhibition of betaAR-stimulated adenylyl cyclase activities by beta-arrestin. The green fluorescent protein-tagged beta-arrestin 2 translocates to and stays at the plasma membrane by beta(2)AR stimulation. Although green fluorescent protein-tagged beta-arrestin 2 also translocates to the plasma membrane, it returns to the cytoplasm 10-30 min after beta(1)AR stimulation. The binding of in vitro translated beta-arrestin 1 and beta-arrestin 2 to the third intracellular loop and the carboxyl tail of beta(1)AR is lower than that of beta(2)AR. The fusion protein of beta-arrestin 1 with glutathione S-transferase inhibits the beta(1)- and beta(2)AR-stimulated adenylyl cyclase activities, although inhibition of the beta(1)AR-stimulated activity requires a higher concentration of the fusion protein than that of the beta(2)AR-stimulated activity. These results suggest that weak interaction of beta(1)AR with beta-arrestins explains the resistance to agonist-induced internalization. This is further supported by the finding that beta-arrestin can induce internalization of beta(1)AR when beta-arrestin 1 does not dissociate from beta(1)AR by fusing to the carboxyl tail of beta(1)AR.

Complete deficiency of mitochondrial trifunctional protein due to a novel mutation within the beta-subunit of the mitochondrial trifunctional protein gene leads to failure of long-chain fatty acid beta-oxidation with fatal outcome

NARCIS (Netherlands)

Schwab, Karl Otfried; Ensenauer, Regina; Matern, Dietrich; Uyanik, Gökhan; Schnieders, Birgit; Wanders, Ronald A.; Lehnert, Willy

2003-01-01

The mitochondrial trifunctional protein (MTP) is a multienzyme complex which catalyses three of the four chain-shortening reactions in the beta-oxidation of long-chain fatty acids. Clinically, failure of long-chain fatty acid beta-oxidation leads to hypoketotic hypoglycaemia associated with coma,

Precursors and preinvasive lesions of the breast: the role of molecular prognostic markers in the diagnostic and therapeutic dilemma

Directory of Open Access Journals (Sweden)

Zografos George C

2007-05-01

Full Text Available Abstract Precursors and preinvasive lesions of the breast include atypical ductal hyperplasia (ADH, ductal carcinoma in situ (DCIS, and lobular neoplasia (LN. There is a significant debate regarding the classification, diagnosis, prognosis and management of these lesions. This review article describes the current theories regarding the pathogenesis and molecular evolution of these lesions. It reviews the implication of a variety of molecules in the continuum of breast lesions: estrogen receptors (ER-alpha and ER-beta, c-erb-B2 (Her2/neu, p53, Ki-67, bcl-2, E-cadherin, transforming growth factor-beta (TGF-beta, p27 (Kip1, p16 (INK4a, p21 (Waf1, vascular endothelial growth factor (VEGF. With respect to the aforementioned molecules, this article reviews their pathophysiological importance, and puts the stress on whether they confer additional risk for invasive breast cancer or not. This knowledge has the potential to be of importance in the therapeutic decisions presenting in the common clinical practice.

Involvement of proteasomal subunits zeta and iota in RNA degradation.

Science.gov (United States)

Petit, F; Jarrousse, A S; Dahlmann, B; Sobek, A; Hendil, K B; Buri, J; Briand, Y; Schmid, H P

1997-01-01

We have identified two distinct subunits of 20 S proteasomes that are associated with RNase activity. Proteasome subunits zeta and iota, eluted from two-dimensional Western blots, hydrolysed tobacco mosaic virus RNA, whereas none of the other subunits degraded this substrate under the same conditions. Additionally, proteasomes were dissociated by 6 M urea, and subunit zeta, containing the highest RNase activity, was isolated by anion-exchange chromatography and gel filtration. Purified subunit zeta migrated as a single spot on two-dimensional PAGE with a molecular mass of approx. 28 kDa. Addition of anti-(subunit zeta) antibodies led to the co-precipitation of this proteasome subunit and nuclease activity. This is the first evidence that proteasomal alpha-type subunits are associated with an enzymic activity, and our results provide further evidence that proteasomes may be involved in cellular RNA metabolism. PMID:9337855

Subunit Stoichiometry of Human Muscle Chloride Channels

OpenAIRE

Fahlke, Christoph; Knittle, Timothy; Gurnett, Christina A.; Campbell, Kevin P.; George, Alfred L.

1997-01-01

Voltage-gated Cl? channels belonging to the ClC family appear to function as homomultimers, but the number of subunits needed to form a functional channel is controversial. To determine subunit stoichiometry, we constructed dimeric human skeletal muscle Cl? channels in which one subunit was tagged by a mutation (D136G) that causes profound changes in voltage-dependent gating. Sucrose-density gradient centrifugation experiments indicate that both monomeric and dimeric hClC-1 channels in their ...

Caspase activation increases beta-amyloid generation independently of caspase cleavage of the beta-amyloid precursor protein (APP).

Science.gov (United States)

Tesco, Giuseppina; Koh, Young Ho; Tanzi, Rudolph E

2003-11-14

The amyloid precursor protein (APP) undergoes "alternative" proteolysis mediated by caspases. Three major caspase recognition sites have been identified in the APP, i.e. one at the C terminus (Asp720) and two at the N terminus (Asp197 and Asp219). Caspase cleavage at Asp720 has been suggested as leading to increased production of Abeta. Thus, we set out to determine which putative caspase sites in APP, if any, are cleaved in Chinese hamster ovary cell lines concurrently with the increased Abeta production that occurs during apoptosis. We found that cleavage at Asp720 occurred concurrently with caspase 3 activation and the increased production of total secreted Abeta and Abeta1-42 in association with staurosporine- and etoposide-induced apoptosis. To investigate the contribution of caspase cleavage of APP to Abeta generation, we expressed an APP mutant truncated at Asp720 that mimics APP caspase cleavage at the C-terminal site. This did not increase Abeta generation but, in contrast, dramatically decreased Abeta production in Chinese hamster ovary cells. Furthermore, the ablation of caspase-dependent cleavage at Asp720, Asp197, and Asp219 (by site-directed mutagenesis) did not prevent enhanced Abeta production following etoposide-induced apoptosis. These findings indicate that the enhanced Abeta generation associated with apoptosis does not require cleavage of APP at its C-terminal (Asp720) and/or N-terminal caspase sites.

EST Table: DC548736 [KAIKOcDNA[Archive

Lifescience Database Archive (English)

Full Text Available milar to Glucosylceramidase precursor (Beta-glucocerebrosidase) (Acid beta-glucosidase) (D-glucosyl-N-acylsp...ucosylceramidase precursor (Beta-glucocerebrosidase) (Acid beta-glucosidase) (D-g....1| PREDICTED: similar to Glucosylceramidase precursor (Beta-glucocerebrosidase) (Acid beta-glucosidase) (D-

Quantification of beta A4 protein deposition in the medial temporal lobe: a comparison of Alzheimer's disease and senile dementia of the Lewy body type.

Science.gov (United States)

Gentleman, S M; Williams, B; Royston, M C; Jagoe, R; Clinton, J; Perry, R H; Ince, P G; Allsop, D; Polak, J M; Roberts, G W

1992-08-03

The distribution of beta-amyloid protein (beta A4) was examined in the medial temporal lobes from cases of Alzheimer's disease (AD) (n = 13), senile dementia of Lewy body type (SDLT) (n = 12) and age matched controls (n = 9). Using a previously described image analysis technique the extent of beta A4 pathology was determined in ten distinct anatomical sites within the medial temporal lobe. AD and SDLT cases contained very similar amounts of beta A4 in the areas sampled and both contained significantly more beta A4 than the age matched controls, particularly in the dentate and parahippocampal gyri. The similarity of the beta A4 load in the two conditions is in contrast to reported differences in the number of neurofibrillary tangles which can be observed. It is suggested that AD and SDLT represent a spectrum of pathology which centres around the aberrant processing of the beta A4 precursor protein.

Effects of transforming growth factor-beta on long-term human cord blood monocyte cultures

International Nuclear Information System (INIS)

Orcel, P.; Bielakoff, J.; De Vernejoul, M.C.

1990-01-01

Transforming growth factor-beta (TGF-beta) modulates growth and differentiation in many cell types and is abundant in bone matrix. We recently showed that human cord blood monocytes cultured in the presence of 1,25(OH)2D3 acquire some features of osteoclast precursors. Since TGF-beta has been shown to influence bone resorption in organ culture, we have studied the effect of TGF-beta (1-1,000 pg/ml) on cord blood monocyte cultures. These cells were cultured on plastic substrate during 3 weeks in the presence of 20% horse serum and 10(-9) M 1,25(OH)2D3. TGF-beta, from a concentration of 10 pg/ml in the culture medium, decreased in a dose dependent manner the formation of multinucleated cells. At a concentration of TGF-beta of 1 ng/ml, the multinucleated cells were reduced to 2.1% +/- 0.3%, compared to 19.3% +/- 1.5% in control cultures. TGF-beta inhibited in a dose-dependent manner the proliferation of cord blood monocytes as assessed by 3H-thymidine incorporation at 7 and 14 days of culture. The fusion index was also decreased by 3 weeks of treatment with TGF-beta. Indomethacin did not reverse the inhibitory effects of TGF-beta. The expression of the osteoclastic phenotype was assessed using two different antibodies: 23C6, a monoclonal antibody directed against the vitronectin receptor, which is highly expressed by osteoclasts but not by adult monocytes, and an antibody to HLA-DR, which is not present on osteoclast. TGF-beta decreased the expression of HLA-DR and increased in a dose-dependent manner the proportion of 23C6-labeled cells; these results suggest that TGF-beta could modulate a differentiation effect to the osteoclastic phenotype. However, when cord blood monocytes were cultured on devitalized rat calvariae prelabeled with 45Ca, TGF-beta did not induce any 45Ca release from bone cultured with monocytes

Hydrothermal synthesis of porous triphasic hydroxyapatite/(alpha and beta) tricalcium phosphate.

Science.gov (United States)

Vani, R; Girija, E K; Elayaraja, K; Prakash Parthiban, S; Kesavamoorthy, R; Narayana Kalkura, S

2009-12-01

A novel, porous triphasic calcium phosphate composed of nonresorbable hydroxyapatite (HAp) and resorbable tricalcium phosphate (alpha- and beta-TCP) has been synthesized hydrothermally at a relatively low temperature. The calcium phosphate precursor for hydrothermal treatment was prepared by gel method in the presence of ascorbic acid. XRD, FT-IR, Raman analyses confirmed the presence of HAp/TCP. The surface area and average pore size of the samples were found to be 28 m2/g and 20 nm, respectively. The samples were found to be bioactive in simulated body fluid (SBF).

Characterization of fimbrial subunits from Bordetella species

NARCIS (Netherlands)

Mooi, F.R.; Heide, H.G.J. van der; Avest, A.R. ter; Welinder, K.G.; Livey, I.; Zeijst, B.A.M. van der; Gaastra, W.

Using antisera raised against serotype 2 and 3 fimbrial subunits from Bordetella pertussis, serologically related polypeptides were detected in Bordetella bronchiseptica, Bordetella parapertussis and Bordetella avium strains. The two B. pertussis fimbrial subunits, and three of the serologically

Precursors prior to type IIn supernova explosions are common: Precursor rates, properties, and correlations

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Ofek, Eran O.; Steinbok, Aviram; Arcavi, Iair; Gal-Yam, Avishay; Tal, David; Ben-Ami, Sagi; Yaron, Ofer [Benoziyo Center for Astrophysics, Weizmann Institute of Science, 76100 Rehovot (Israel); Sullivan, Mark [School of Physics and Astronomy, University of Southampton, Southampton SO17 1BJ (United Kingdom); Shaviv, Nir J. [Racah Institute of Physics, The Hebrew University, 91904 Jerusalem (Israel); Kulkarni, Shrinivas R. [Cahill Center for Astronomy and Astrophysics, California Institute of Technology, Pasadena, CA 91125 (United States); Nugent, Peter E. [Lawrence Berkeley National Laboratory, 1 Cyclotron Road, Berkeley, CA 94720 (United States); Kasliwal, Mansi M. [Observatories of the Carnegie Institution for Science, 813 Santa Barbara Street, Pasadena, CA 91101 (United States); Cenko, S. Bradley [Astrophysics Science Division, NASA/Goddard Space Flight Center, Mail Code 661, Greenbelt, MD 20771 (United States); Laher, Russ; Surace, Jason [Spitzer Science Center, California Institute of Technology, M/S 314-6, Pasadena, CA 91125 (United States); Bloom, Joshua S.; Filippenko, Alexei V. [Department of Astronomy, University of California, Berkeley, CA 94720-3411 (United States); Silverman, Jeffrey M. [Department of Astronomy, University of Texas, Austin, TX 78712 (United States)

2014-07-10

There is a growing number of Type IIn supernovae (SNe) which present an outburst prior to their presumably final explosion. These precursors may affect the SN display, and are likely related to poorly charted phenomena in the final stages of stellar evolution. By coadding Palomar Transient Factory (PTF) images taken prior to the explosion, here we present a search for precursors in a sample of 16 Type IIn SNe. We find five SNe IIn that likely have at least one possible precursor event (PTF 10bjb, SN 2010mc, PTF 10weh, SN 2011ht, and PTF 12cxj), three of which are reported here for the first time. For each SN we calculate the control time. We find that precursor events among SNe IIn are common: at the one-sided 99% confidence level, >50% of SNe IIn have at least one pre-explosion outburst that is brighter than 3 × 10{sup 7} L{sub ☉} taking place up to 1/3 yr prior to the SN explosion. The average rate of such precursor events during the year prior to the SN explosion is likely ≳ 1 yr{sup –1}, and fainter precursors are possibly even more common. Ignoring the two weakest precursors in our sample, the precursors rate we find is still on the order of one per year. We also find possible correlations between the integrated luminosity of the precursor and the SN total radiated energy, peak luminosity, and rise time. These correlations are expected if the precursors are mass-ejection events, and the early-time light curve of these SNe is powered by interaction of the SN shock and ejecta with optically thick circumstellar material.

Food choice in hyperthyroidism: potential influence of the autonomic nervous system and brain serotonin precursor availability.

Science.gov (United States)

Pijl, H; de Meijer, P H; Langius, J; Coenegracht, C I; van den Berk, A H; Chandie Shaw, P K; Boom, H; Schoemaker, R C; Cohen, A F; Burggraaf, J; Meinders, A E

2001-12-01

We explored energy and macronutrient intake in patients with Graves' hyperthyroidism. We specifically hypothesized that hyperthyroidism is associated with increased appetite for carbohydrates, because of enhanced sympathetic tone and diminished serotonin mediated neurotransmission in the brain. To test this hypothesis, we measured food intake by dietary history and food selected for lunch in the laboratory in 14 patients with Graves' hyperthyroidism. Twenty-four-hour catecholamine excretion was used as a measure of activity of the sympathetic nervous system (SNS) and the plasma [Trp]/[NAA] ratio was measured to estimate (rate limiting) precursor availability for brain 5-hydroxytryptamine synthesis. All measurements were repeated after the subjects had been treated to establish euthyroidism. In addition, the effects of nonselective beta-adrenoceptor blockade upon these parameters were studied to evaluate the influence of beta-adrenergic hyperactivity on food intake. Hyperthyroidism was marked by increased SNS activity and reduced plasma [Trp]/[NAA] ratio. Accordingly, energy intake was considerably and significantly increased in hyper vs. euthyroidism, which was fully attributable to enhanced carbohydrate consumption, as protein and fat intake were not affected. These results suggest that hyperthyroidism alters the neurophysiology of food intake regulation. Increased SNS activity and reduced Trp precursor availability for 5-hydroxytryptamine synthesis in the brain may drive the marked hyperphagia and craving for carbohydrates that appears to characterize hyperthyroid patients. Because propranolol did not affect food intake in hyperthyroidism, the potential effect of catecholamines on food intake might be mediated by alpha-adrenoceptors.

The ectopic expression of Pax4 in the mouse pancreas converts progenitor cells into alpha and subsequently beta cells.

Science.gov (United States)

Collombat, Patrick; Xu, Xiaobo; Ravassard, Philippe; Sosa-Pineda, Beatriz; Dussaud, Sébastien; Billestrup, Nils; Madsen, Ole D; Serup, Palle; Heimberg, Harry; Mansouri, Ahmed

2009-08-07

We have previously reported that the loss of Arx and/or Pax4 gene activity leads to a shift in the fate of the different endocrine cell subtypes in the mouse pancreas, without affecting the total endocrine cell numbers. Here, we conditionally and ectopically express Pax4 using different cell-specific promoters and demonstrate that Pax4 forces endocrine precursor cells, as well as mature alpha cells, to adopt a beta cell destiny. This results in a glucagon deficiency that provokes a compensatory and continuous glucagon+ cell neogenesis requiring the re-expression of the proendocrine gene Ngn3. However, the newly formed alpha cells fail to correct the hypoglucagonemia since they subsequently acquire a beta cell phenotype upon Pax4 ectopic expression. Notably, this cycle of neogenesis and redifferentiation caused by ectopic expression of Pax4 in alpha cells is capable of restoring a functional beta cell mass and curing diabetes in animals that have been chemically depleted of beta cells.

Increased Expression of Laminin Subunit Alpha 1 Chain by dCas9-VP160.

Science.gov (United States)

Perrin, Arnaud; Rousseau, Joël; Tremblay, Jacques P

2017-03-17

Laminin-111 protein complex links the extracellular matrix to integrin α7β1 in sarcolemma, thus replacing in dystrophic muscles links normally insured by the dystrophin complex. Laminin-111 injection in mdx mouse stabilized sarcolemma, restored serum creatine kinase to wild-type levels, and protected muscles from exercised-induced damages. These results suggested that increased laminin-111 is a potential therapy for DMD. Laminin subunit beta 1 and laminin subunit gamma 1 are expressed in adult human muscle, but laminin subunit alpha 1 (LAMA1) gene is expressed only during embryogenesis. We thus developed an alternative method to laminin-111 protein repeated administration by inducing expression of the endogenous mouse Lama1 gene. This was done with the CRSPR/Cas9 system, i.e., by targeting the Lama1 promoter with one or several gRNAs and a dCas9 coupled with the VP160 transcription activation domain. Lama1 mRNA (qRT-PCR) and proteins (immunohistochemistry and western blot) were not detected in the control C2C12 myoblasts and in control muscles. However, significant expression was observed in cells transfected and in mouse muscles electroporated with plasmids coding for dCas9-VP160 and a gRNA. Larger synergic increases were observed by using two or three gRNAs. The increased Lama1 expression did not modify the expression of the α7 and β1 integrins. Increased expression of Lama1 by the CRISPR/Cas9 system will have to be further investigated by systemic delivery of the CRISPR/Cas9 components to verify whether this could be a treatment for several myopathies. Copyright © 2016 The Authors. Published by Elsevier Inc. All rights reserved.

Spontaneous quaternary and tertiary T-R transitions of human hemoglobin in molecular dynamics simulation.

Directory of Open Access Journals (Sweden)

Jochen S Hub

2010-05-01

Full Text Available We present molecular dynamics simulations of unliganded human hemoglobin (Hb A under physiological conditions, starting from the R, R2, and T state. The simulations were carried out with protonated and deprotonated HC3 histidines His(beta146, and they sum up to a total length of 5.6 micros. We observe spontaneous and reproducible T-->R quaternary transitions of the Hb tetramer and tertiary transitions of the alpha and beta subunits, as detected from principal component projections, from an RMSD measure, and from rigid body rotation analysis. The simulations reveal a marked asymmetry between the alpha and beta subunits. Using the mutual information as correlation measure, we find that the beta subunits are substantially more strongly linked to the quaternary transition than the alpha subunits. In addition, the tertiary populations of the alpha and beta subunits differ substantially, with the beta subunits showing a tendency towards R, and the alpha subunits showing a tendency towards T. Based on the simulation results, we present a transition pathway for coupled quaternary and tertiary transitions between the R and T conformations of Hb.

Proteomic analysis of osteogenic differentiation of dental follicle precursor cells

DEFF Research Database (Denmark)

Morsczeck, Christian; Petersen, Jørgen; Völlner, Florian

2009-01-01

of differentiation. In the present study we applied 2-DE combined with capillary-LC-MS/MS analysis to profile differentially regulated proteins upon differentiation of dental follicle precursor cells (DFPCs). Out of 115 differentially regulated proteins, glutamine synthetase, lysosomal proteinase cathepsin B....... The bioinformatic analyses suggest that proteins associated with cell cycle progression and protein metabolism were down-regulated and proteins involved in catabolism, cell motility and biological quality were up-regulated. These results display the general physiological state of DFPCs before and after osteogenic...... proteins, plastin 3 T-isoform, beta-actin, superoxide dismutases, and transgelin were found to be highly up-regulated, whereas cofilin-1, pro-alpha 1 collagen, destrin, prolyl 4-hydrolase and dihydrolipoamide dehydrogenase were found to be highly down-regulated. The group of up-regulated proteins...

Molecular determinants of desensitization and assembly of the chimeric GABA(A) receptor subunits (alpha1/gamma2) and (gamma2/alpha1) in combinations with beta2 and gamma2

DEFF Research Database (Denmark)

Elster, L; Kristiansen, U; Pickering, D S

2001-01-01

Two gamma-aminobutyric acid(A) (GABA(A)) receptor chimeras were designed in order to elucidate the structural requirements for GABA(A) receptor desensitization and assembly. The (alpha1/gamma2) and (gamma2/alpha1) chimeric subunits representing the extracellular N-terminal domain of alpha1 or gamma......, as opposed to the staining of the (gamma2/alpha1)-containing receptors, which was only slightly higher than background. To explain this, the (alpha1/gamma2) and (gamma2/alpha1) chimeras may act like alpha1 and gamma2 subunits, respectively, indicating that the extracellular N-terminal segment is important...... for assembly. However, the (alpha1/gamma2) chimeric subunit had characteristics different from the alpha1 subunit, since the (alpha1/gamma2) chimera gave rise to no desensitization after GABA stimulation in whole-cell patch-clamp recordings, which was independent of whether the chimera was expressed...

Assembly of the epithelial Na+ channel evaluated using sucrose gradient sedimentation analysis.

Science.gov (United States)

Cheng, C; Prince, L S; Snyder, P M; Welsh, M J

1998-08-28

Three subunits, alpha, beta, and gamma, contribute to the formation of the epithelial Na+ channel. To investigate the oligomeric assembly of the channel complex, we used sucrose gradient sedimentation analysis to determine the sedimentation properties of individual subunits and heteromultimers comprised of multiple subunits. When the alpha subunit was expressed alone, it first formed an oligomeric complex with a sedimentation coefficient of 11 S, and then generated a higher order multimer of 25 S. In contrast, individual beta and gamma subunits predominately assembled into 11 S complexes. We obtained similar results with expression in cells and in vitro. When we co-expressed beta with alpha or with alpha plus gamma, the beta subunit assembled into a 25 S complex. Glycosylation of the alpha subunit was not required for assembly into a 25 S complex. We found that the alpha subunit formed intra-chain disulfide bonds. Although such bonds were not required to generate an oligomeric complex, under nonreducing conditions the alpha subunit formed a complex that migrated more homogeneously at 25 S. This suggests that intra-chain disulfide bonds may stabilize the complex. These data suggest that the epithelial Na+ channel subunits form high order oligomeric complexes and that the alpha subunit contains the information that facilitates such formation. Interestingly, the ability of the alpha, but not the beta or gamma, subunit to assemble into a 25 S homomeric complex correlates with the ability of these subunits to generate functional channels when expressed alone.

TFIIH subunit alterations causing xeroderma pigmentosum and trichothiodystrophy specifically disturb several steps during transcription.

Science.gov (United States)

Singh, Amita; Compe, Emanuel; Le May, Nicolas; Egly, Jean-Marc

2015-02-05

Mutations in genes encoding the ERCC3 (XPB), ERCC2 (XPD), and GTF2H5 (p8 or TTD-A) subunits of the transcription and DNA-repair factor TFIIH lead to three autosomal-recessive disorders: xeroderma pigmentosum (XP), XP associated with Cockayne syndrome (XP/CS), and trichothiodystrophy (TTD). Although these diseases were originally associated with defects in DNA repair, transcription deficiencies might be also implicated. By using retinoic acid receptor beta isoform 2 (RARB2) as a model in several cells bearing mutations in genes encoding TFIIH subunits, we observed that (1) the recruitment of the TFIIH complex was altered at the activated RARB2 promoter, (2) TFIIH participated in the recruitment of nucleotide excision repair (NER) factors during transcription in a manner different from that observed during NER, and (3) the different TFIIH variants disturbed transcription by having distinct consequences on post-translational modifications of histones, DNA-break induction, DNA demethylation, and gene-loop formation. The transition from heterochromatin to euchromatin was disrupted depending on the variant, illustrating the fact that TFIIH, by contributing to NER factor recruitment, orchestrates chromatin remodeling. The subtle transcriptional differences found between various TFIIH variants thus participate in the phenotypic variability observed among XP, XP/CS, and TTD individuals. Copyright © 2015 The American Society of Human Genetics. Published by Elsevier Inc. All rights reserved.

Sequence swapping does not result in conformation swapping for the beta4/beta5 and beta8/beta9 beta-hairpin turns in human acidic fibroblast growth factor.

Science.gov (United States)

Kim, Jaewon; Lee, Jihun; Brych, Stephen R; Logan, Timothy M; Blaber, Michael

2005-02-01

The beta-turn is the most common type of nonrepetitive structure in globular proteins, comprising ~25% of all residues; however, a detailed understanding of effects of specific residues upon beta-turn stability and conformation is lacking. Human acidic fibroblast growth factor (FGF-1) is a member of the beta-trefoil superfold and contains a total of five beta-hairpin structures (antiparallel beta-sheets connected by a reverse turn). beta-Turns related by the characteristic threefold structural symmetry of this superfold exhibit different primary structures, and in some cases, different secondary structures. As such, they represent a useful system with which to study the role that turn sequences play in determining structure, stability, and folding of the protein. Two turns related by the threefold structural symmetry, the beta4/beta5 and beta8/beta9 turns, were subjected to both sequence-swapping and poly-glycine substitution mutations, and the effects upon stability, folding, and structure were investigated. In the wild-type protein these turns are of identical length, but exhibit different conformations. These conformations were observed to be retained during sequence-swapping and glycine substitution mutagenesis. The results indicate that the beta-turn structure at these positions is not determined by the turn sequence. Structural analysis suggests that residues flanking the turn are a primary structural determinant of the conformation within the turn.

Metabolic engineering of Saccharomyces cerevisiae for production of germacrene A, a precursor of beta-elemene

DEFF Research Database (Denmark)

Hu, Yating; Zhou, Yongjin J.; Bao, Jichen

2017-01-01

inefficient and suffers from limited natural resources. Here, we engineered a yeast cell factory for the sustainable production of germacrene A, which can be transformed to beta-elemene by a one-step chemical reaction in vitro. Two heterologous germacrene A synthases (GASs) converting farnesyl pyrophosphate...... (FPP) to germacrene A were evaluated in yeast for their ability to produce germacrene A. Thereafter, several metabolic engineering strategies were used to improve the production level. Overexpression of truncated 3-hydroxyl-3-methylglutaryl-CoA reductase and fusion of FPP synthase with GAS, led...

The Role of Oxidative Stress and Hypoxia in Pancreatic Beta-Cell Dysfunction in Diabetes Mellitus.

Science.gov (United States)

Gerber, Philipp A; Rutter, Guy A

2017-04-01

Metabolic syndrome is a frequent precursor of type 2 diabetes mellitus (T2D), a disease that currently affects ∼8% of the adult population worldwide. Pancreatic beta-cell dysfunction and loss are central to the disease process, although understanding of the underlying molecular mechanisms is still fragmentary. Recent Advances: Oversupply of nutrients, including glucose and fatty acids, and the subsequent overstimulation of beta cells, are believed to be an important contributor to insulin secretory failure in T2D. Hypoxia has also recently been implicated in beta-cell damage. Accumulating evidence points to a role for oxidative stress in both processes. Although the production of reactive oxygen species (ROS) results from enhanced mitochondrial respiration during stimulation with glucose and other fuels, the expression of antioxidant defense genes is unusually low (or disallowed) in beta cells. Not all subjects with metabolic syndrome and hyperglycemia go on to develop full-blown diabetes, implying an important role in disease risk for gene-environment interactions. Possession of common risk alleles at the SLC30A8 locus, encoding the beta-cell granule zinc transporter ZnT8, may affect cytosolic Zn 2+ concentrations and thus susceptibility to hypoxia and oxidative stress. Loss of normal beta-cell function, rather than total mass, is increasingly considered to be the major driver for impaired insulin secretion in diabetes. Better understanding of the role of oxidative changes, its modulation by genes involved in disease risk, and effects on beta-cell identity may facilitate the development of new therapeutic strategies to this disease. Antioxid. Redox Signal. 26, 501-518.

Myristoylated α subunits of guanine nucleotide-binding regulatory proteins

International Nuclear Information System (INIS)

Buss, J.E.; Mumby, S.M.; Casey, P.J.; Gilman, A.G.; Sefton, B.M.

1987-01-01

Antisera directed against specific subunits of guanine nucleotide-binding regulatory proteins (G proteins) were used to immunoprecipitate these polypeptides from metabolically labeled cells. This technique detects, in extracts of a human astrocytoma cell line, the α subunits of G/sub s/ (stimulatory) (α 45 and α 52 ), a 41-kDa subunit of G/sub i/ (inhibitory) (α 41 ), a 40-kDa protein (α 40 ), and the 36-kDa β subunit. No protein that comigrated with the α subunit of G 0 (unknown function) (α 39 ) was detected. In cells grown in the presence of [ 3 H]myristic acid, α 41 and α 40 contained 3 H label, while the β subunit did not. Chemical analysis of lipids attached covalently to purified α 41 and α 39 from bovine brain also revealed myristic acid. Similar analysis of brain G protein β and γ subunits and of G/sub t/ (Transducin) subunits (α, β, and γ) failed to reveal fatty acids. The fatty acid associated with α 41 , α 40 , and α 39 was stable to treatment with base, suggesting that the lipid is linked to the polypeptide via an amide bond. These GTP binding proteins are thus identified as members of a select group of proteins that contains myristic acid covalently attached to the peptide backbone. Myristate may play an important role in stabilizing interactions of G proteins with phospholipid or with membrane-bound proteins

The Subunit Principle in Scar Face Revision.

Science.gov (United States)

Elshahat, Ahmed; Lashin, Riham

2017-06-01

Facial scaring is considered one of the most difficult cosmetic problems for any plastic surgeon to solve. The condition is more difficult if the direction of the scar is not parallel to relaxed skin tension lines. Attempts to manage this difficult situation included revisions using geometric designs, Z plasties or W plasties to camouflage the straight line visible scaring. The use of long-lasting resorbable sutures was tried too. Recently, the use of botulinum toxin during revision improved the results. Fractional CO2 lasers, microfat grafts, and platelet-rich plasma were added to the armamentarium. The scar is least visible if placed in the junction between the facial subunits. The aim of this study is to investigate the use of the subunit principle to improve the results of scar revision. Four patients were included in this study. Tissue expansion of the intact part of the subunit allowed shifting the scar to the junction between the affected subunit and the adjacent one. Tissue expansion, delivery of the expanders, and advancement of the flaps were successful in all patients. The fact that this is a 2-stage procedure and sacrifices some of the intact skin from the affected facial subunit, makes this technique reserved to patients with ugly facial scars who are ambitious to improve their appearance.

High non-specific binding of the {beta}{sub 1}-selective radioligand 2-{sup 125}I-ICI-H

Energy Technology Data Exchange (ETDEWEB)

Riemann, B. [Muenster Univ. (Germany). Department of Nuclear Medicine; Law, M.P. [Muenster Univ. (Germany). Department of Nuclear Medicine; Hammersmith Hospital, London (United Kingdom). MRC Clinical Sciences Centre; Kopka, K. [Muenster Univ. (DE). Department of Nuclear Medicine] [and others

2003-08-01

Aim: As results of cardiac biopsies suggest, myocardial {beta}{sub 1}-adrenoceptor density is reduced in patients with chronic heart failure. However, changes in cardiac {beta}{sub 2}-adrenoceptors vary. With suitable radiopharmaceuticals single photon emission computed tomography (SPECT) and positron emission tomography (PET) offer the opportunity to assess {beta}-adrenoceptors non-invasively. Among the novel racemic analogues of the established {beta}{sub 1}-selective adrenoceptor antagonist ICI 89.406 the iodinated 2-I-ICI-H showed high affinity and selectivity to {beta}{sub 1}-adrenoceptors in murine ventricular membranes. The aim of this study was its evaluation as a putative subtype selective {beta}{sub 1}-adrenergic radioligand in cardiac imaging. Methods: Competition studies in vitro and in vivo were used to investigate the kinetics of 2-I-ICI-H binding to cardiac {beta}-adrenoceptors in mice and rats. In addition, the radiosynthesis of 2-{sup 125}I-ICI-H from the silylated precursor 2-SiMe{sub 3}-ICI-H was established. The specific activity was 80 GBq/{mu}mol, the radiochemical yield ranged from 70 to 80%. Results: The unlabelled compound 2-I-ICI-H showed high {beta}{sub 1}-selectivity and -affinity in the in vitro competition studies. In vivo biodistribution studies apparently showed low affinity to cardiac {beta}-adrenoceptors. The radiolabelled counterpart 2-{sup 125}I-ICI-H showed a high degree of non-specific binding in vitro and no specific binding to cardiac {beta}{sub 1}-adrenoceptors in vivo. Conclusion: Because of its high non-specific binding 2-{sup 125}I-ICI-H is no suitable radiotracer for imaging in vivo. (orig.)

Semen quality in men with disseminated testicular cancer : relation with human chorionic gonadotropin beta-subunit and pituitary gonadal hormones

NARCIS (Netherlands)

de Bruin, Daphne; de Jong, Igle J.; Arts, Eugene G. J. M.; Nuver, Janine; Dullaart, Robin P. F.; Sluiter, Willem J.; Hoekstra, Harald J.; Sleijfer, Dirk T.; Gietema, Jourik A.

Objective: To compare the semen quality and hormonal status between patients with testicular cancer and normal versus increased serum levels of beta-hCG. Design: Retrospective study. Setting: Academic research environment. Patient(s): All 203 patients with testicular cancer who required chemotherapy

Expression of transforming growth factor beta (TGF beta) receptors and expression of TGF beta 1, TGF beta 2 and TGF beta 3 in human small cell lung cancer cell lines

DEFF Research Database (Denmark)

Damstrup, L; Rygaard, K; Spang-Thomsen, M

1993-01-01

A panel of 21 small cell lung cancer cell (SCLC) lines were examined for the presence of Transforming growth factor beta receptors (TGF beta-r) and the expression of TGF beta mRNAs. By the radioreceptor assay we found high affinity receptors to be expressed in six cell lines. scatchard analysis......(r) = 65,000 and 90,000 and the betaglycan (type III) with M(r) = 280,000. Northern blotting showed expression of TGF beta 1 mRNA in ten, TGF beta 2 mRNA in two and TGF beta 3 mRNA in seven cell lines. Our results provide, for the first time, evidence that a large proportion of a broad panel of SCLC cell...... lines express TGF beta-receptors and also produce TGF beta mRNAs....

Intestinal epithelial restitution. Involvement of specific laminin isoforms and integrin laminin receptors in wound closure of a transformed model epithelium

DEFF Research Database (Denmark)

Lotz, M M; Nusrat, A; Madara, J L

1997-01-01

laminins 5, 6, and 7 as indicated by immunostaining using laminin subunit-specific monoclonal antibodies (MAbs). A MAb (BM2) specific for the laminin alpha 3 subunit, a component of laminins 5, 6, and 7, completely inhibited the closure of mechanical wounds in T84 monolayers. Confocal microscopy using MAbs...... BM2 (laminin alpha 3 subunit) and 6F12 (laminin beta 3 subunit) revealed that laminin-5 is deposited in a basal matrix that extends into the wound. The MAbs 4E10 (laminin beta 1 subunit) and C4 (laminin beta 2 subunit) stained the lateral membranes between T84 cells. This staining was enhanced...

Efficient expression of functional (α6β22β3 AChRs in Xenopus oocytes from free subunits using slightly modified α6 subunits.

Directory of Open Access Journals (Sweden)

Carson Kai-Kwong Ley

Full Text Available Human (α6β2(α4β2β3 nicotinic acetylcholine receptors (AChRs are essential for addiction to nicotine and a target for drug development for smoking cessation. Expressing this complex AChR is difficult, but has been achieved using subunit concatamers. In order to determine what limits expression of α6* AChRs and to efficiently express α6* AChRs using free subunits, we investigated expression of the simpler (α6β22β3 AChR. The concatameric form of this AChR assembles well, but is transported to the cell surface inefficiently. Various chimeras of α6 with the closely related α3 subunit increased expression efficiency with free subunits and produced pharmacologically equivalent functional AChRs. A chimera in which the large cytoplasmic domain of α6 was replaced with that of α3 increased assembly with β2 subunits and transport of AChRs to the oocyte surface. Another chimera replacing the unique methionine 211 of α6 with leucine found at this position in transmembrane domain 1 of α3 and other α subunits increased assembly of mature subunits containing β3 subunits within oocytes. Combining both α3 sequences in an α6 chimera increased expression of functional (α6β22β3 AChRs to 12-fold more than with concatamers. This is pragmatically useful, and provides insights on features of α6 subunit structure that limit its expression in transfected cells.

Effects of light and the regulatory Beta subunit composition of protein phosphatase 2A on the susceptibility of Arabidopsis thaliana to aphid (Myzus persicae infestation

Directory of Open Access Journals (Sweden)

Brwa eRasool

2014-08-01

Full Text Available The interactions between biotic and abiotic stress signalling pathways are complex and poorly understood but protein kinase/phosphatase cascades are potentially important components. Aphid fecundity and susceptibility to Pseudomonas syringae infection were determined in the low light-grown Arabidopsis thaliana wild type and in mutant lines defective in either the protein phosphatase (PP2A regulatory subunit B’γ (gamma; pp2a-b’γ or B’ζ (zeta; pp2a-b’ζ1-1 and pp2a-b’ζ1-2 and in gamma zeta double mutants (pp2a-b’γζ lacking both subunits. All the mutants except for pp2a-b’ζ1-1 had significantly lower leaf areas than the wild type. Susceptibility to P. syringae was similar in all genotypes. In contrast, aphid fecundity was significantly decreased in the pp2a-b’γ mutant relative to the wild type but not in the pp2a-b’γζ double mutant. A high light pre-treatment, which led to a significant increase in rosette growth in all mutant lines but not in the wild type, led to a significant decrease in aphid fecundity in all genotypes. The high light pre-treatment abolished the differences in aphid resistance observed in the pp2a-b’γ mutant relative to the wild type. The light and CO2 response curves for photosynthesis were changed in response to the high light pre-treatment, but the high light effects were similar in all genotypes. These data demonstrate that a pre-exposure to high light and the composition of subunits on the trimeric PP2A holoenzymes are important in regulating plant resistance to aphids. The functional specificity for the individual regulatory B-subunits may therefore limit aphid colonisation, depending on the prevailing abiotic stress environment.

Beta-energy averaging and beta spectra

International Nuclear Information System (INIS)

Stamatelatos, M.G.; England, T.R.

1976-07-01

A simple yet highly accurate method for approximately calculating spectrum-averaged beta energies and beta spectra for radioactive nuclei is presented. This method should prove useful for users who wish to obtain accurate answers without complicated calculations of Fermi functions, complex gamma functions, and time-consuming numerical integrations as required by the more exact theoretical expressions. Therefore, this method should be a good time-saving alternative for investigators who need to make calculations involving large numbers of nuclei (e.g., fission products) as well as for occasional users interested in restricted number of nuclides. The average beta-energy values calculated by this method differ from those calculated by ''exact'' methods by no more than 1 percent for nuclides with atomic numbers in the 20 to 100 range and which emit betas of energies up to approximately 8 MeV. These include all fission products and the actinides. The beta-energy spectra calculated by the present method are also of the same quality

The functional performance of microencapsulated human pancreatic islet-derived precursor cells.

Science.gov (United States)

Montanucci, Pia; Pennoni, Ilaria; Pescara, Teresa; Blasi, Paolo; Bistoni, Giovanni; Basta, Giuseppe; Calafiore, Riccardo

2011-12-01

We have examined long-term cultured, human islet-derived stem/precursor cells (hIPC). Whole human islets (HI) were obtained by multi-enzymatic digestion of cadaveric donor pancreases, plated on tissue flasks, and allowed to adhere and expand for several in vitro passages, in order to obtain hIPC. We detected specific stem cell markers (Oct-4, Sox-2, Nanog, ABCG2, Klf-4, CD117) in both intact HI and hIPC. Moreover, hIPC while retaining the expression of Glut-2, Pdx-1, CK-19, and ICA-512, started re-expressing Ngn3, thereby indicating acquisition of a specific pancreatic islet beta cell-oriented phenotype identity. The intrinsic plasticity of hIPC was documented by their ability to differentiate into various germ layer-derived cell phenotypes (ie, osteocytic, adipocytic and neural), including endocrine cells associated with insulin secretory capacity. To render hIPC suitable for transplantation we have enveloped them within our highly purified, alginate-based microcapsules. Upon intraperitoneal graft in NOD/SCID mice we have observed that the microcapsules acted as three-dimensional niches favouring post-transplant hIPC differentiation and acquisition of beta cell-like functional competence. Copyright © 2011 Elsevier Ltd. All rights reserved.

Plasma-assisted co-evaporation of {beta}-indium sulfide thin films

Energy Technology Data Exchange (ETDEWEB)

Kosaraju, Sreenivas; Marino, Joseph A.; Harvey, John A.; Wolden, Colin A. [Department of Chemical Engineering, Colorado School of Mines, Golden, CO 80401 (United States)

2006-05-05

This paper describes the development of plasma-assisted co-evaporation (PACE) for the formation of {beta}-In{sub 2}S{sub 3} thin films. Indium was supplied by conventional thermal evaporation, while the chalcogen gas precursor (H{sub 2}S) was activated using an inductively coupled plasma (ICP) source. Using a combination of optical emission spectroscopy and mass spectrometry it was shown that the ICP effectively dissociated H{sub 2}S, producing atomic sulfur. Transport modeling was used to quantify the flux distributions of the co-evaporated metal and the plasma-generated species impinging the substrate. Model predictions were validated by measurements of deposition rate and film properties. Substantial improvements in both materials utilization and substrate temperature reduction were realized with respect to conventional co-evaporation. {beta}-In{sub 2}S{sub 3} was formed as low as 100{sup o}C and it was observed that quality was a strong function of S/In ratio. The grain size decreased and the optical band gap increased as the substrate temperature was reduced. (author)

Genetic analysis of the cytoplasmic dynein subunit families.

Science.gov (United States)

Pfister, K Kevin; Shah, Paresh R; Hummerich, Holger; Russ, Andreas; Cotton, James; Annuar, Azlina Ahmad; King, Stephen M; Fisher, Elizabeth M C

2006-01-01

Cytoplasmic dyneins, the principal microtubule minus-end-directed motor proteins of the cell, are involved in many essential cellular processes. The major form of this enzyme is a complex of at least six protein subunits, and in mammals all but one of the subunits are encoded by at least two genes. Here we review current knowledge concerning the subunits, their interactions, and their functional roles as derived from biochemical and genetic analyses. We also carried out extensive database searches to look for new genes and to clarify anomalies in the databases. Our analysis documents evolutionary relationships among the dynein subunits of mammals and other model organisms, and sheds new light on the role of this diverse group of proteins, highlighting the existence of two cytoplasmic dynein complexes with distinct cellular roles.

Genetic analysis of the cytoplasmic dynein subunit families.

Directory of Open Access Journals (Sweden)

K Kevin Pfister

2006-01-01

Full Text Available Cytoplasmic dyneins, the principal microtubule minus-end-directed motor proteins of the cell, are involved in many essential cellular processes. The major form of this enzyme is a complex of at least six protein subunits, and in mammals all but one of the subunits are encoded by at least two genes. Here we review current knowledge concerning the subunits, their interactions, and their functional roles as derived from biochemical and genetic analyses. We also carried out extensive database searches to look for new genes and to clarify anomalies in the databases. Our analysis documents evolutionary relationships among the dynein subunits of mammals and other model organisms, and sheds new light on the role of this diverse group of proteins, highlighting the existence of two cytoplasmic dynein complexes with distinct cellular roles.

Effect of SiO2/Al2O3 Ratio on Micro-Mesopore Formation for Pt/Beta-MCM-41 via NaOH Treatment and the Catalytic Performance in N-heptane Hydro isomerization

Science.gov (United States)

Gao, Li; Shi, Zhiyuan; Liu, Yingming; Zhao, Yuanshou; Liu, Qinghua; Xu, Chengguo; Bai, Peng; Yan, Zifeng

2018-01-01

Micro-mesoporous composite material Beta-MCM-41(BM) were hydrothermally synthesized by treating parent beta with molar SiO2/Al2O3 ratios of 12.5, 20 and 30 as precursors. The influence of SiO2/Al2O3 ratio of zeolite beta on effective micro-mesoporous composite formation was studied by investigating the crystallinity, morphology, chemical composition, acidity and textural property of Beta-MCM-41 through XRD, nitrogen adsorption, SEM, TEM, NH3-TPD, FTIR and Pyridine-FTIR. The catalytic performance was evaluated in terms of n-heptane hydro isomerization. The results demonstrated that Beta-MCM-41 supported Pt catalysts showed higher selectivity to isoheptanes than Pt/Beta. It was attributed to the superiorities of the pore structure and mesoporous accelerated the diffusion of larger molecules of isoheptanes.

Protein kinase CK2 structure-function relationship

DEFF Research Database (Denmark)

Boldyreff, B; Meggio, F; Pinna, L A

1994-01-01

Protein kinase CK2 subunits alpha and beta were expressed either separately or together in a bacterial expression system (pT7-7/BL21(DE3)) and purified to homogeneity. After mixing the subunits, a CK2 holoenzyme (alpha 2 beta 2) was spontaneously reconstituted, which displays identical features...... subunit have been prepared and assayed for their ability to assemble with the catalytic alpha subunit to give a fully competent CK2 holoenzyme. The beta subunit contains an acidic stretch (amino acid 55-64), which is obviously responsible for a negative control of enzyme activity since mutations...

Speculative Betas

OpenAIRE

Harrison Hong; David Sraer

2012-01-01

We provide a model for why high beta assets are more prone to speculative overpricing than low beta ones. When investors disagree about the common factor of cash-flows, high beta assets are more sensitive to this macro-disagreement and experience a greater divergence-of-opinion about their payoffs. Short-sales constraints for some investors such as retail mutual funds result in high beta assets being over-priced. When aggregate disagreement is low, expected return increases with beta due to r...

Characterization of cDNAs encoding human pyruvate dehydrogenase α subunit

International Nuclear Information System (INIS)

Ho, Lap; Wexler, I.D.; Liu, Techung; Thekkumkara, T.J.; Patel, M.S.

1989-01-01

A cDNA clone (1,423 base pairs) comprising the entire coding region of the precursor form of the α subunit of pyruvate dehydrogenase (E 1 α) has been isolated from a human liver cDNA library in phage λgt11. The first 29 amino acids deduced from the open reading frame correspond to a typical mitochondrial targeting leader sequence. The remaining 361 amino acids, starting at the N terminus with phenylalanine, represent the mature mitochondrial E 1 α peptide. The cDNA has 43 base pairs in the 5' untranslated region and 210 base pairs in the 3' untranslated region, including a polyadenylylation signal and a short poly(A) tract. The nucleotide sequence of human liver E 1 α cDNA was confirmed by the nucleotide sequences of three overlapping fragments generated from human liver and fibroblast RNA by reverse transcription and DNA amplification by the polymerase chain reaction. This consensus nucleotide sequence of human liver E 1 α cDNA resolves existing discrepancies among three previously reported human E 1 α cDNAs and provides the unambiguous reference sequence needed for the characterization of genetic mutations in pyruvate dehydrogenase-deficient patients

Roles of the β subunit hinge domain in ATP synthase F1 sector: Hydrophobic network formed by introduced βPhe174 inhibits subunit rotation

International Nuclear Information System (INIS)

Nakanishi-Matsui, Mayumi; Kashiwagi, Sachiko; Kojima, Masaki; Nonaka, Takamasa; Futai, Masamitsu

2010-01-01

The ATP synthase β subunit hinge domain (βPhe148 ∼ βGly186, P-loop/α-helixB/loop/β-sheet4, Escherichia coli residue numbering) dramatically changes in conformation upon nucleotide binding. We previously reported that F 1 with the βSer174 to Phe mutation in the domain lowered the γ subunit rotation speed, and thus decreased the ATPase activity [M. Nakanishi-Matsui, S. Kashiwagi, T. Ubukata, A. Iwamoto-Kihara, Y. Wada, M. Futai, Rotational catalysis of Escherichia coli ATP synthase F 1 sector. Stochastic fluctuation and a key domain of the β subunit, J. Biol. Chem. 282 (2007) 20698-20704.]. Homology modeling indicates that the amino acid replacement induces a hydrophobic network, in which the βMet159, βIle163, and βAla167 residues of the β subunit are involved together with the mutant βPhe174. The network is expected to stabilize the conformation of β DP (nucleotide-bound form of the β subunit), resulting in increased activation energy for transition to β E (empty β subunit). The modeling further predicts that replacement of βMet159 with Ala or Ile weakens the hydrophobic network. As expected, these two mutations experimentally suppressed the ATPase activities as well as subunit rotation of βS174F. Furthermore, the rotation rate decreased with the increase of the strength in the hydrophobic network. These results indicate that the smooth conformational change of the β subunit hinge domain is pertinent for the rotational catalysis.

Studies on the subunits of human glycoprotein hormones in relation to reproduction

International Nuclear Information System (INIS)

Hagen, C.

1977-01-01

In this review summarising present knowledge of the biological and immunological activity of the subunits of human glycoprotein hormones, the specificity of the α-subunit and β-subunit radioimmunoassays are discussed. The crossreaction studies performed with the α-subunit radioimmunoassays are aummarised in one table while those with the β-subunit radioimmunoassays are presented in a second table. (JIW)

Role of Tim50 in the transfer of precursor proteins from the outer to the inner membrane of mitochondria.

Science.gov (United States)

Mokranjac, Dejana; Sichting, Martin; Popov-Celeketić, Dusan; Mapa, Koyeli; Gevorkyan-Airapetov, Lada; Zohary, Keren; Hell, Kai; Azem, Abdussalam; Neupert, Walter

2009-03-01

Transport of essentially all matrix and a number of inner membrane proteins is governed, entirely or in part, by N-terminal presequences and requires a coordinated action of the translocases of outer and inner mitochondrial membranes (TOM and TIM23 complexes). Here, we have analyzed Tim50, a subunit of the TIM23 complex that is implicated in transfer of precursors from TOM to TIM23. Tim50 is recruited to the TIM23 complex via Tim23 in an interaction that is essentially independent of the rest of the translocase. We find Tim50 in close proximity to the intermembrane space side of the TOM complex where it recognizes both types of TIM23 substrates, those that are to be transported into the matrix and those destined to the inner membrane, suggesting that Tim50 recognizes presequences. This function of Tim50 depends on its association with TIM23. We conclude that the efficient transfer of precursors between TOM and TIM23 complexes requires the concerted action of Tim50 with Tim23.

Evaluation of the chemical reactivity in lignin precursors using the Fukui function.

Science.gov (United States)

Martinez, Carmen; Rivera, José L; Herrera, Rafael; Rico, José L; Flores, Nelly; Rutiaga, José G; López, Pablo

2008-02-01

The hydroxycinnamyl alcohols: p-coumarol, coniferol and sinapol are considered the basic units and precursors of lignins models. In this work, the specific reactivity of these molecules was studied. We investigate their intrinsic chemical reactivity in terms of the Fukui function, applying the principle of hard and soft acids and bases (HSAB) in the framework of the density functional theory (DFT). Comparisons of their nucleophilic, electrophilic and free radical reactivity show their most probably sites to form linkages among them. It is found that the most reactive sites, for reactions involving free radicals, are the carbons at the beta-position in the p-coumarol and sinapol molecules, whilst the regions around the carbon-oxygen bond of the phenoxyl group are the most reactive in coniferol.

Regulation of KV channel voltage-dependent activation by transmembrane β subunits

Directory of Open Access Journals (Sweden)

Xiaohui eSun

2012-04-01

Full Text Available Voltage-activated K+ (KV channels are important for shaping action potentials and maintaining resting membrane potential in excitable cells. KV channels contain a central pore-gate domain (PGD surrounded by four voltage-sensing domains (VSD. The VSDs will change conformation in response to alterations of the membrane potential thereby inducing the opening of the PGD. Many KV channels are heteromeric protein complexes containing auxiliary β subunits. These β subunits modulate channel expression and activity to increase functional diversity and render tissue specific phenotypes. This review focuses on the KV β subunits that contain transmembrane (TM segments including the KCNE family and the β subunits of large conductance, Ca2+- and voltage-activated K+ (BK channels. These TM β subunits affect the voltage-dependent activation of KV α subunits. Experimental and computational studies have described the structural location of these β subunits in the channel complexes and the biophysical effects on VSD activation, PGD opening and VSD-PGD coupling. These results reveal some common characteristics and mechanistic insights into KV channel modulation by TM β subunits.

ORF Alignment: NC_005810 [GENIUS II[Archive

Lifescience Database Archive (English)

Full Text Available NC_005810 gi|45441027 >1p71A 1 90 1 91 3e-24 ... ref|YP_069949.1| integration host fa...ctor beta-subunit [Yersinia pseudotuberculosis ... IP 32953] ref|NP_670080.1| integration host factor ... ... (IHF), beta subunit [Yersinia pestis KIM] ... gb|AAS61443.1| integration host factor beta-su...bunit ... [Yersinia pestis biovar Medievalis str. 91001] ... ref|NP_992566.1| integration...ration host factor (IHF), beta ... subunit [Yersinia pestis KIM] ref|NP_404986.1| ... integratio

Labelling of. beta. -endorphin (. beta. -END) and. beta. -lipotropin (. beta. -LPH) by /sup 125/I

Energy Technology Data Exchange (ETDEWEB)

Deby-Dupont, G.; Joris, J.; Franchimont, P. (Universite de Liege (Belgique)); Reuter, A.M.; Vrindts-Gevaert, Y. (Institut des Radioelements, Fleurus (Belgique))

1983-01-01

5 ..mu..g of human ..beta..-endorphin were labelled with 2 mCi /sup 125/I by the chloramine T technique. After two gel filtrations on Sephadex G-15 and on Sephadex G-50 in phosphate buffer with EDTA, Trasylol and mercapto-ethanol, a pure tracer was obtained with a specific activity about 150 ..mu..Ci/..mu..g.Kept at + 4/sup 0/C, the tracer remained utilizable for 30 days without loss of immunoreactivity. The labelling with lactoperoxydase and the use of another gel filtration method (filtration on Aca 202) gave a /sup 125/I ..beta..-END tracer with the same immunoreactivity. The binding of this tracer to the antibody of an anti-..beta..-END antiserum diluted at 1/8000 was 32% with a non specific binding of 2%. 5 ..mu..g of human ..beta..-lipotropin were labelled with 0.5 mCi /sup 125/I by the lactoperoxydase method. After two gel filtrations on Sephadex G-25 and on Sephadex G-75 in phosphate buffer with EDTA, Trasylol and mercapto-ethanol, a pure tracer with a specific activity of 140 ..mu..Ci/..mu..g was obtained. It remained utilizable for 30 days when kept at + 4/sup 0/C. Gel filtration on Aca 202 did not give good purification, while gel filtration on Aca 54 was good but slower than on Sephadex G-75. The binding to antibody in absence of unlabelled ..beta..-LPH was 32% for an anti-..beta..-LPH antiserum diluted at 1/4000. The non specific binding was 2.5%.

A remarkably stable TipE gene cluster: evolution of insect Para sodium channel auxiliary subunits

Directory of Open Access Journals (Sweden)

Li Jia

2011-11-01

Full Text Available Abstract Background First identified in fruit flies with temperature-sensitive paralysis phenotypes, the Drosophila melanogaster TipE locus encodes four voltage-gated sodium (NaV channel auxiliary subunits. This cluster of TipE-like genes on chromosome 3L, and a fifth family member on chromosome 3R, are important for the optional expression and functionality of the Para NaV channel but appear quite distinct from auxiliary subunits in vertebrates. Here, we exploited available arthropod genomic resources to trace the origin of TipE-like genes by mapping their evolutionary histories and examining their genomic architectures. Results We identified a remarkably conserved synteny block of TipE-like orthologues with well-maintained local gene arrangements from 21 insect species. Homologues in the water flea, Daphnia pulex, suggest an ancestral pancrustacean repertoire of four TipE-like genes; a subsequent gene duplication may have generated functional redundancy allowing gene losses in the silk moth and mosquitoes. Intronic nesting of the insect TipE gene cluster probably occurred following the divergence from crustaceans, but in the flour beetle and silk moth genomes the clusters apparently escaped from nesting. Across Pancrustacea, TipE gene family members have experienced intronic nesting, escape from nesting, retrotransposition, translocation, and gene loss events while generally maintaining their local gene neighbourhoods. D. melanogaster TipE-like genes exhibit coordinated spatial and temporal regulation of expression distinct from their host gene but well-correlated with their regulatory target, the Para NaV channel, suggesting that functional constraints may preserve the TipE gene cluster. We identified homology between TipE-like NaV channel regulators and vertebrate Slo-beta auxiliary subunits of big-conductance calcium-activated potassium (BKCa channels, which suggests that ion channel regulatory partners have evolved distinct lineage

Development of a Subunit Vaccine for Contagious Bovine ...

International Development Research Centre (IDRC) Digital Library (Canada)

Their work has set the stage for commercial development of a sub-unit vaccine. ... The sub-unit vaccine will be cost-effective, easy to produce, and safe. How it will make a ... IDRC invites applications for the IDRC Doctoral Research Awards.

Interaction of the p85 subunit of PI 3-kinase and its N-terminal SH2 domain with a PDGF receptor phosphorylation site: structural features and analysis of conformational changes.

Science.gov (United States)

Panayotou, G; Bax, B; Gout, I; Federwisch, M; Wroblowski, B; Dhand, R; Fry, M J; Blundell, T L; Wollmer, A; Waterfield, M D

1992-01-01

Circular dichroism and fluorescence spectroscopy were used to investigate the structure of the p85 alpha subunit of the PI 3-kinase, a closely related p85 beta protein, and a recombinant SH2 domain-containing fragment of p85 alpha. Significant spectral changes, indicative of a conformational change, were observed on formation of a complex with a 17 residue peptide containing a phosphorylated tyrosine residue. The sequence of this peptide is identical to the sequence surrounding Tyr751 in the kinase-insert region of the platelet-derived growth factor beta-receptor (beta PDGFR). The rotational correlation times measured by fluorescence anisotropy decay indicated that phosphopeptide binding changed the shape of the SH2 domain-containing fragment. The CD and fluorescence spectroscopy data support the secondary structure prediction based on sequence analysis and provide evidence for flexible linker regions between the various domains of the p85 proteins. The significance of these results for SH2 domain-containing proteins is discussed. Images PMID:1330535

Liposome-Based Adjuvants for Subunit Vaccines: Formulation Strategies for Subunit Antigens and Immunostimulators

Directory of Open Access Journals (Sweden)

Signe Tandrup Schmidt

2016-03-01

Full Text Available The development of subunit vaccines has become very attractive in recent years due to their superior safety profiles as compared to traditional vaccines based on live attenuated or whole inactivated pathogens, and there is an unmet medical need for improved vaccines and vaccines against pathogens for which no effective vaccines exist. The subunit vaccine technology exploits pathogen subunits as antigens, e.g., recombinant proteins or synthetic peptides, allowing for highly specific immune responses against the pathogens. However, such antigens are usually not sufficiently immunogenic to induce protective immunity, and they are often combined with adjuvants to ensure robust immune responses. Adjuvants are capable of enhancing and/or modulating immune responses by exposing antigens to antigen-presenting cells (APCs concomitantly with conferring immune activation signals. Few adjuvant systems have been licensed for use in human vaccines, and they mainly stimulate humoral immunity. Thus, there is an unmet demand for the development of safe and efficient adjuvant systems that can also stimulate cell-mediated immunity (CMI. Adjuvants constitute a heterogeneous group of compounds, which can broadly be classified into delivery systems or immunostimulators. Liposomes are versatile delivery systems for antigens, and they can carefully be customized towards desired immune profiles by combining them with immunostimulators and optimizing their composition, physicochemical properties and antigen-loading mode. Immunostimulators represent highly diverse classes of molecules, e.g., lipids, nucleic acids, proteins and peptides, and they are ligands for pattern-recognition receptors (PRRs, which are differentially expressed on APC subsets. Different formulation strategies might thus be required for incorporation of immunostimulators and antigens, respectively, into liposomes, and the choice of immunostimulator should ideally be based on knowledge regarding the

Expression of class III beta tubulin in cervical cancer patients administered preoperative radiochemotherapy: correlation with response to treatment and clinical outcome.

Science.gov (United States)

Ferrandina, Gabriella; Martinelli, Enrica; Zannoni, Gian Franco; Distefano, Mariagrazia; Paglia, Amelia; Ferlini, Cristiano; Scambia, Giovanni

2007-02-01

Alterations of the beta subunit of tubulin have been reported to be predictive of resistance to radiation and antitubulin agents in several solid tumors. The aim of the study was to investigate the clinical role of beta III tubulin expression as prognostic factor for survival and as a predictive parameter of response to preoperative radiochemotherapy in a single institutional series of locally advanced cervical cancer (LACC) patients. The study included 98 LACC patients admitted to the Gynecologic Oncology Unit, Catholic University of Rome and Campobasso between January 1998 and January 2005. Immunohistochemistry was performed by using the polyclonal rabbit anti-beta III tubulin antibody (Covance, Princeton, NJ, USA). The value of 10% immunostained tumor cells was arbitrarily chosen as cut-off value to distinguish cases with high versus low beta III tubulin content. In the whole series, beta III tubulin immunoreaction was detectable in 66/98 cases (67.3%), and the percentage of positively stained cells ranged from 0 to 100% (median=10%). The percentages of cases with high beta III tubulin expression were shown not to be differently distributed according to clinico-pathological characteristics. There was no statistically significant difference in the distribution of cases with high beta III tubulin expression according to clinical and pathological response to treatment. During the follow-up period, recurrence and death of disease occurred in 15 and 13 cases, respectively. There was no difference in disease-free and overall survival in cases with high versus low beta III tubulin expression. The assessment of class III beta tubulin status seems of little usefulness in order to identify LACC patients with poor chance of response to concomitant radiochemotherapy and unfavorable prognosis.

Positive selection within the Schizophrenia-associated GABA(A receptor beta(2 gene.

Directory of Open Access Journals (Sweden)

Wing-Sze Lo

Full Text Available The gamma-aminobutyric acid type-A (GABA(A receptor plays a major role in inhibitory neurotransmissions. Intronic SNPs and haplotypes in GABRB2, the gene for GABA(A receptor beta(2 subunit, are associated with schizophrenia and correlated with the expression of two alternatively spliced beta(2 isoforms. In the present study, using chimpanzee as an ancestral reference, high frequencies were observed for the derived (D alleles of the four SNPs rs6556547, rs187269, rs1816071 and rs1816072 in GABRB2, suggesting the occurrence of positive selection for these derived alleles. Coalescence-based simulation showed that the population frequency spectra and the frequencies of H56, the haplotype having all four D alleles, significantly deviated from neutral-evolution expectation in various demographic models. Haplotypes containing the derived allele of rs1816072 displayed significantly less diversity compared to haplotypes containing its ancestral allele, further supporting positive selection. The variations in DD-genotype frequencies in five human populations provided a snapshot of the evolutionary history, which suggested that the positive selections of the D alleles are recent and likely ongoing. The divergence between the DD-genotype profiles of schizophrenic and control samples pointed to the schizophrenia-relevance of positive selections, with the schizophrenic samples showing weakened selections compared to the controls. These DD-genotypes were previously found to increase the expression of beta(2, especially its long isoform. Electrophysiological analysis showed that this long beta(2 isoform favored by the positive selections is more sensitive than the short isoform to the inhibition of GABA(A receptor function by energy depletion. These findings represent the first demonstration of positive selection in a schizophrenia-associated gene.

The ectopic expression of Pax4 in the mouse pancreas converts progenitor cells into alpha and subsequently beta cells

DEFF Research Database (Denmark)

Collombat, Patrick; Xu, Xiaobo; Ravassard, Philippe

2009-01-01

We have previously reported that the loss of Arx and/or Pax4 gene activity leads to a shift in the fate of the different endocrine cell subtypes in the mouse pancreas, without affecting the total endocrine cell numbers. Here, we conditionally and ectopically express Pax4 using different cell......-specific promoters and demonstrate that Pax4 forces endocrine precursor cells, as well as mature alpha cells, to adopt a beta cell destiny. This results in a glucagon deficiency that provokes a compensatory and continuous glucagon+ cell neogenesis requiring the re-expression of the proendocrine gene Ngn3. However......, the newly formed alpha cells fail to correct the hypoglucagonemia since they subsequently acquire a beta cell phenotype upon Pax4 ectopic expression. Notably, this cycle of neogenesis and redifferentiation caused by ectopic expression of Pax4 in alpha cells is capable of restoring a functional beta cell...

Liposome-Based Adjuvants for Subunit Vaccines: Formulation Strategies for Subunit Antigens and Immunostimulators

DEFF Research Database (Denmark)

Schmidt, Signe Tandrup; Foged, Camilla; Korsholm, Karen Smith

2016-01-01

be classified into delivery systems or immunostimulators. Liposomes are versatile delivery systems for antigens, and they can carefully be customized towards desired immune profiles by combining them with immunostimulators and optimizing their composition, physicochemical properties and antigen-loading mode......The development of subunit vaccines has become very attractive in recent years due to their superior safety profiles as compared to traditional vaccines based on live attenuated or whole inactivated pathogens, and there is an unmet medical need for improved vaccines and vaccines against pathogens...... of immunostimulators and antigens, respectively, into liposomes, and the choice of immunostimulator should ideally be based on knowledge regarding the specific PRR expression profile of the target APCs. Here, we review state-of-the-art formulation approaches employed for the inclusion of immunostimulators and subunit...

Summary Report of 1st Research Coordination Meeting on Development of Reference Database for Beta-delayed Neutron Emission

International Nuclear Information System (INIS)

Dillmann, Iris; Dimitriou, Paraskevi; Singh, Balraj

2014-03-01

A summary is given of the 1st Research Coordination Meeting of the new IAEA Coordinated Research Project (CRP) on Development of a Reference Database for Beta-delayed neutron emission data. Participants presented their work, reviewed the current status of the field with regards to individual precursors and aggregate data, and discussed the scope of the work to be undertaken. A list of priorities and task assignments was produced. (author)

Catalytic Subunit 1 of Protein Phosphatase 2A Is a Subunit of the STRIPAK Complex and Governs Fungal Sexual Development.

Science.gov (United States)

Beier, Anna; Teichert, Ines; Krisp, Christoph; Wolters, Dirk A; Kück, Ulrich

2016-06-21

The generation of complex three-dimensional structures is a key developmental step for most eukaryotic organisms. The details of the molecular machinery controlling this step remain to be determined. An excellent model system to study this general process is the generation of three-dimensional fruiting bodies in filamentous fungi like Sordaria macrospora Fruiting body development is controlled by subunits of the highly conserved striatin-interacting phosphatase and kinase (STRIPAK) complex, which has been described in organisms ranging from yeasts to humans. The highly conserved heterotrimeric protein phosphatase PP2A is a subunit of STRIPAK. Here, catalytic subunit 1 of PP2A was functionally characterized. The Δpp2Ac1 strain is sterile, unable to undergo hyphal fusion, and devoid of ascogonial septation. Further, PP2Ac1, together with STRIPAK subunit PRO22, governs vegetative and stress-related growth. We revealed in vitro catalytic activity of wild-type PP2Ac1, and our in vivo analysis showed that inactive PP2Ac1 blocks the complementation of the sterile deletion strain. Tandem affinity purification, followed by mass spectrometry and yeast two-hybrid analysis, verified that PP2Ac1 is a subunit of STRIPAK. Further, these data indicate links between the STRIPAK complex and other developmental signaling pathways, implying the presence of a large interconnected signaling network that controls eukaryotic developmental processes. The insights gained in our study can be transferred to higher eukaryotes and will be important for understanding eukaryotic cellular development in general. The striatin-interacting phosphatase and kinase (STRIPAK) complex is highly conserved from yeasts to humans and is an important regulator of numerous eukaryotic developmental processes, such as cellular signaling and cell development. Although functional insights into the STRIPAK complex are accumulating, the detailed molecular mechanisms of single subunits are only partially understood

Nuclear beta decay induced by intense electromagnetic fields: Forbidden transition examples

International Nuclear Information System (INIS)

Reiss, H.R.

1983-01-01

A formalism developed earlier for the effect on nuclear beta decay of an intense plane-wave electromagnetic field is applied to three examples of forbidden beta transitions. The examples represent cases where the nuclear ''fragment'' contains one, two, and three nucleons; where the nuclear fragment is defined to be that smallest sub-unit of the nucleus containing the nucleon which undergoes beta decay plus any other nucleons directly angular-momentum coupled to it in initial or final states. The single-nucleon-fragment example is 113 Cd, which has a fourth-forbidden transition. The two-nucleon-fragment example is 90 Sr, which is first-forbidden. The three-nucleon-fragment example is 87 Rb, which is third-forbidden. An algebraic closed-form transition probability is found in each case. At low external-field intensity, the transition probability is proportional to z/sup L/, where z is the field intensity parameter and L is the degree of forbiddenness. At intermediate intensities, the transition probability behaves as z/sup L/-(1/2). At higher intensities, the transition probability contains the z/sup L/-(1/2) factor, a declining exponential factor, and an alternating polynomial in z. This high-intensity transition probability possesses a maximum value, which is found for each of the examples. A general rule, z = q 2 (2L-1), where q is the number of particles in the fragment, is found for giving an upper limit on the intensity at which the maximum transition probability occurs. Field-induced beta decay half-lives for all the examples are dramatically reduced from natural half-lives when evaluated at the optimum field intensity. Relative half-life reduction is greater the higher the degree of forbiddenness

Beta-hydroxy-beta-methylbutyrate (HMB) stimulates myogenic cell proliferation, differentiation and survival via the MAPK/ERK and PI3K/Akt pathways.

Science.gov (United States)

Kornasio, Reut; Riederer, Ingo; Butler-Browne, Gillian; Mouly, Vincent; Uni, Zehava; Halevy, Orna

2009-05-01

Beta-hydroxy-beta-methylbutyrate (HMB), a leucine catabolite, has been shown to prevent exercise-induced protein degradation and muscle damage. We hypothesized that HMB would directly regulate muscle-cell proliferation and differentiation and would attenuate apoptosis, the latter presumably underlying satellite-cell depletion during muscle degradation or atrophy. Adding various concentrations of HMB to serum-starved myoblasts induced cell proliferation and MyoD expression as well as the phosphorylation of MAPK/ERK. HMB induced differentiation-specific markers, increased IGF-I mRNA levels and accelerated cell fusion. Its inhibition of serum-starvation- or staurosporine-induced apoptosis was reflected by less apoptotic cells, reduced BAX expression and increased levels of Bcl-2 and Bcl-X. Annexin V staining and flow cytometry analysis showed reduced staurosporine-induced apoptosis in human myoblasts in response to HMB. HMB enhanced the association of the p85 subunit of PI3K with tyrosine-phosphorylated proteins. HMB elevated Akt phosphorylation on Thr308 and Ser473 and this was inhibited by Wortmannin, suggesting that HMB acts via Class I PI3K. Blocking of the PI3K/Akt pathway with specific inhibitors revealed its requirement in mediating the promotive effects of HMB on muscle cell differentiation and fusion. These direct effects of HMB on myoblast differentiation and survival resembling those of IGF-I, at least in culture, suggest its positive influence in preventing muscle wasting.

INTRINSIC REGULATION OF HEMOGLOBIN EXPRESSION BY VARIABLE SUBUNIT INTERFACE STRENGTHS

Science.gov (United States)

Manning, James M.; Popowicz, Anthony M.; Padovan, Julio C.; Chait, Brian T.; Manning, Lois R.

2012-01-01

SUMMARY The expression of the six types of human hemoglobin subunits over time is currently considered to be regulated mainly by transcription factors that bind to upstream control regions of the gene (the “extrinsic” component of regulation). Here we describe how subunit pairing and further assembly to tetramers in the liganded state is influenced by the affinity of subunits for one another (the “intrinsic” component of regulation). The adult hemoglobin dimers have the strongest subunit interfaces and the embryonic hemoglobins are the weakest with fetal hemoglobins of intermediate strength, corresponding to the temporal order of their expression. These variable subunit binding strengths and the attenuating effects of acetylation contribute to the differences with which these hemoglobin types form functional O2-binding tetramers consistent with gene switching. PMID:22129306

Immunochemical aspects of crotoxim and its subunits

International Nuclear Information System (INIS)

Nakazone, A.K.

1979-01-01

Crotamine and crotoxin with the subunits - phospholipase A and crotapotin - were obtained by purification from Crotalus durissus terrificus venom. Interaction studies of the subunits using crotalic antiserum, indicated that: crotoxin is formed of crotapotin and phospholipase A with the molar ratio of 1 to 1; using crotapotin 125 I the presence of a soluble complex was shown with the same antiserum. Immunological precipitation reactions demonstrated that crotapotin is antigenic: crotapotin and phospholipase A presented similar antigenic determinants; crotoxin antiserum reacted with each one of the submits; when the subunits are mixed to form synthetic crotoxin some antigenic determinants are masked in the process of interaction. Crotamine, interacted with crotapotin 1:1, without hidden antigenic determinants crotapotin antigenic site seems to be formed by, at least, one lysine. Enzimatical activity of phospholipase A apreared to be dependent on some reaction conditions when its arginine residues are blocked. Tyrosines of phospholipase A are more susceptible to labelling with 131 I than crotapotin. Gama irradiation of aqueous solutions of the subunits produced modifications in the ultraviolet spectra. A decrease of the enzymatic activity occured as a function of radiation dosis. Immunological activities of crotapotin and phospholipase A were not altered [pt

Structure of the Mr 140,000 growth hormone-dependent insulin-like growth factor binding protein complex: Determination by reconstitution and affinity-labeling

International Nuclear Information System (INIS)

Baxter, R.C.; Martin, J.L.

1989-01-01

To determine the structure of the high molecular weight, growth hormone-dependent complex between the insulin-like growth factors (IGF-I and IGF-II) and their binding proteins in human serum, we have reconstituted the complex from its purified component proteins and analyzed it by gel electrophoresis and autoradiography after covalent cross-linking. The proteins tested in reconstitution mixtures were an acid-labile Mr 84,000-86,000 glycoprotein doublet (alpha subunit), an acid-stable Mr 47,000-53,000 glycoprotein doublet with IGF-binding activity (BP-53 or beta subunit), and IGF-I or IGF-II (gamma subunit). In incubations containing any one of the three subunits 125I-labeled and the other two unlabeled, identical 125I-labeled alpha-beta-gamma complexes of Mr 140,000 were formed. Minor bands of Mr 120,000 and 90,000 were also seen, thought to represent a partially deglycosylated form of the alpha-beta-gamma complex, and an alpha-gamma complex arising as a cross-linking artifact. When serum samples from subjects of various growth hormone status were affinity-labeled with IGF-II tracer, a growth hormone-dependent Mr 140,000 band was seen, corresponding to the reconstituted alpha-beta-gamma complex. Other growth hormone-dependent labeled bands, of Mr 90,000 (corresponding to alpha-gamma), Mr 55,000-60,000 (corresponding to labeled beta-subunit doublet), and smaller bands of Mr 38,000, 28,000, and 23,000-25,000 (corresponding to labeled beta-subunit degradation products), were also seen in the affinity-labeled serum samples and in the complex reconstituted from pure proteins. All were immunoprecipitable with an anti-BP-53 antiserum. We conclude that the growth hormone-dependent Mr 140,000 IGF-binding protein complex in human serum has three components: the alpha (acid-labile) subunit, the beta (binding) subunit, and the gamma (growth factor) subunit

An X11alpha/FSBP complex represses transcription of the GSK3beta gene promoter.

LENUS (Irish Health Repository)

Lau, Kwok-Fai

2010-08-04

X11alpha is a neuronal adaptor protein that interacts with the amyloid precursor protein (APP) through a centrally located phosphotyrosine binding domain to inhibit the production of Abeta peptide that is deposited in Alzheimer\\'s disease brains. X11alpha also contains two C-terminal postsynaptic density-95, large discs, zona occludens 1 (PDZ) domains, and we show here that through its PDZ domains, X11alpha interacts with a novel transcription factor, fibrinogen silencer binding protein. Moreover, we show that an X11alpha\\/fibrinogen silencer binding protein complex signals to the nucleus to repress glycogen synthase kinase-3beta promoter activity. Glycogen synthase kinase-3beta is a favoured candidate kinase for phosphorylating tau in Alzheimer\\'s disease. Our findings show a new function for X11alpha that may impact on Alzheimer\\'s disease pathogenesis.

Wide range of interacting partners of pea Gβ subunit of G-proteins suggests its multiple functions in cell signalling.

Science.gov (United States)

Bhardwaj, Deepak; Lakhanpaul, Suman; Tuteja, Narendra

2012-09-01

Climate change is a major concern especially in view of the increasing global population and food security. Plant scientists need to look for genetic tools whose appropriate usage can contribute to sustainable food availability. G-proteins have been identified as some of the potential genetic tools that could be useful for protecting plants from various stresses. Heterotrimeric G-proteins consisting of three subunits Gα, Gβ and Gγ are important components of a number of signalling pathways. Their structure and functions are already well studied in animals but their potential in plants is now gaining attention for their role in stress tolerance. Earlier we have reported that over expressing pea Gβ conferred heat tolerance in tobacco plants. Here we report the interacting partners (proteins) of Gβ subunit of Pisum sativum and their putative role in stress and development. Out of 90 transformants isolated from the yeast-two-hybrid (Y2H) screening, seven were chosen for further investigation due to their recurrence in multiple experiments. These interacting partners were confirmed using β-galactosidase colony filter lift and ONPG (O-nitrophenyl-β-D-galactopyranoside) assays. These partners include thioredoxin H, histidine-containing phosphotransfer protein 5-like, pathogenesis-related protein, glucan endo-beta-1, 3-glucosidase (acidic isoform), glycine rich RNA binding protein, cold and drought-regulated protein (corA gene) and soluble inorganic pyrophosphatase 1. This study suggests the role of pea Gβ subunit in stress signal transduction and development pathways owing to its capability to interact with a wide range of proteins of multiple functions. Copyright © 2012 Elsevier Masson SAS. All rights reserved.

Dynamic fluorescence studies of beta-glycosidase mutants from Sulfolobus solfataricus: effects of single mutations on protein thermostability.

Science.gov (United States)

Bismuto, Ettore; Febbraio, Ferdinando; Limongelli, Simona; Briante, Raffaella; Nucci, Roberto

2003-04-01

Multiple sequence alignment on 73 proteins belonging to glycosyl hydrolase family 1 reveals the occurrence of a segment (83-124) in the enzyme sequences from hyperthermophilic archaea bacteria, which is absent in all the mesophilic members of the family. The alignment of the known three-dimensional structures of hyperthermophilic glycosidases with the known ones from mesophilic organisms shows a similar spatial organizations of beta-glycosidases except for this sequence segment whose structure is located on the external surface of each of four identical subunits, where it overlaps two alpha-helices. Site-directed mutagenesis substituting N97 or S101 with a cysteine residue in the sequence of beta-glycosidase from hyperthermophilic archaeon Sulfolobus solfataricus caused some changes in the structural and dynamic properties as observed by circular dichroism in far- and near-UV light, as well as by frequency domain fluorometry, with a simultaneous loss of thermostability. The results led us to hypothesize an important role of the sequence segment present only in hyperthermophilic beta-glycosidases, in the thermal adaptation of archaea beta-glycosidases. The thermostabilization mechanism could occur as a consequence of numerous favorable ionic interactions of the 83-124 sequence with the other part of protein matrix that becomes more rigid and less accessible to the insult of thermal-activated solvent molecules. Copyright 2003 Wiley-Liss, Inc.

Assessment of affinities of beta-CIT, beta-CIT-FE, and beta-CIT-FP for monoamine transporters permanently expressed in cell lines

International Nuclear Information System (INIS)

Okada, Tomoya; Fujita, Masahiro; Shimada, Shoichi; Sato, Kohji; Schloss, Patrick; Watanabe, Yoshiyuki; Itoh, Yasushi; Tohyama, Masaya; Nishimura, Tsunehiko

1998-01-01

We investigated the effects of three cocaine analogs, beta-CIT (2-beta-carbomethoxy-3-beta-(4-iodophenyl)-tropane), beta-CIT-FE (2-beta-carbomethoxy-3-beta-(4-iodophenyl)-N-(2-fluoroethyl)-nortropane), and beta-CIT-FP (2-beta-carbomethoxy-3-beta-(4-iodophenyl)-N-(3-fluoropropyl)-nortropane), on the uptake of [ 3 H]dopamine(DA), serotonin(5-HT), and 1-norepinephrine (NE) using cell lines permanently expressing DA, 5-HT, and NE transporters, respectively, to determine their affinities for these three transporters. We generated cell lines stably expressing DA, 5-HT, and NE transporters, respectively, by the Chen-Okayama method, and then tested the abilities of (-)cocaine, beta-CIT, beta-CIT-FE, beta-CIT-FP, and clomipramine to inhibit the uptake of [ 3 H]DA, 5-HT, and 1-NE. Ki values of beta-CIT, beta-CIT-FE, and beta-CIT-FP for [ 3 H]DA, 5-HT, 1-NE uptake were 6, 29, and 33 nM, 91, 133, and 130 nM, and 28, 113 and 70 nM, respectively, whereas those of cocaine and clomipramine were 316, 581, and 176 nM and > 10,000, 437, and 851 nM, respectively. Beta-CIT, beta-CIT-FE, and beta-CIT-FP were shown to be potent DA, 5-HT, and NE uptake inhibitors. Beta-CIT and beta-CIT-FP were highly potent and selective dopamine uptake inhibitors, and therefore might be useful for imaging of DA transporter with single photon emission computed tomography (SPECT) or positron emission tomography (PET)

Protein kinase CK2 and its role in cellular proliferation, development and pathology

DEFF Research Database (Denmark)

Guerra, B; Issinger, O G

1999-01-01

, signaling, proliferation and in various steps of development. The tetrameric holoenzyme (alpha2beta2) consists of two catalytic alpha-subunits and two regulatory beta-subunits. The structure of the catalytic subunit with the fixed positioning of the activation segment in the active conformation through its...

Mutations in the Atp1p and Atp3p subunits of yeast ATP synthase differentially affect respiration and fermentation in Saccharomyces cerevisiae.

Science.gov (United States)

Francis, Brian R; White, Karen H; Thorsness, Peter E

2007-04-01

ATP1-111, a suppressor of the slow-growth phenotype of yme1Delta lacking mitochondrial DNA is due to the substitution of phenylalanine for valine at position 111 of the alpha-subunit of mitochondrial ATP synthase (Atp1p in yeast). The suppressing activity of ATP1-111 requires intact beta (Atp2p) and gamma (Atp3p) subunits of mitochondrial ATP synthase, but not the stator stalk subunits b (Atp4p) and OSCP (Atp5p). ATP1-111 and other similarly suppressing mutations in ATP1 and ATP3 increase the growth rate of wild-type strains lacking mitochondrial DNA. These suppressing mutations decrease the growth rate of yeast containing an intact mitochondrial chromosome on media requiring oxidative phosphorylation, but not when grown on fermentable media. Measurement of chronological aging of yeast in culture reveals that ATP1 and ATP3 suppressor alleles in strains that contain mitochondrial DNA are longer lived than the isogenic wild-type strain. In contrast, the chronological life span of yeast cells lacking mitochondrial DNA and containing these mutations is shorter than that of the isogenic wild-type strain. Spore viability of strains bearing ATP1-111 is reduced compared to wild type, although ATP1-111 enhances the survival of spores that lacked mitochondrial DNA.

Analysis of the influence of two different milling processes in the properties of precursor powder and [Beta]-TCP cement

International Nuclear Information System (INIS)

Cardoso, H.A.I.; Pereira, C.H.R.; Zavaglia, C.A.C.; Motisuke, M.

2011-01-01

There are several characteristics that put calcium phosphate cements in evidence, like its bioactivity and in vivo resorption. The influence of two milling processes in the morphological properties of the [beta]-tricalcium phosphate powder, [beta]-TCP, and in the mechanical properties of the cement were analyzed. The powder was obtained by solid state reaction of CaCO_3 and CaHPO_4 at 1050 ° C. It showed high phase purity and absence of toxic elements. The powder was processed in ball mill (A) and high-energy vibratory mill (B), with posterior analyze by SEM and particle size distribution. The powders showed different average and distribution of grain size. Finally, the cement obtained by the process (B) showed values of axial tensile strength significantly greater than that obtained by the process (A). The milling process (B) is much more efficient than the process (A). (author)

Structural characterization of recombinant crustacyanin subunits from the lobster Homarus americanus

International Nuclear Information System (INIS)

Ferrari, Michele; Folli, Claudia; Pincolini, Elisa; McClintock, Timothy S.; Rössle, Manfred; Berni, Rodolfo; Cianci, Michele

2012-01-01

The two recombinant apo subunits H1 and H2 from H. americanus have been structurally characterized. Reconstitution studies with astaxanthin reproduced the bathochromic shift of 85–95 nm typical of the natural crustacyanin subunits. Crustacean crustacyanin proteins are linked to the production and modification of carapace colour, with direct implications for fitness and survival. Here, the structural and functional properties of the two recombinant crustacyanin subunits H 1 and H 2 from the American lobster Homarus americanus are reported. The two subunits are structurally highly similar to the corresponding natural apo crustacyanin CRTC and CRTA subunits from the European lobster H. gammarus. Reconstitution studies of the recombinant crustacyanin proteins H 1 and H 2 with astaxanthin reproduced the bathochromic shift of 85–95 nm typical of the natural crustacyanin subunits from H. gammarus in complex with astaxanthin. Moreover, correlations between the presence of crustacyanin genes in crustacean species and the resulting carapace colours with the spectral properties of the subunits in complex with astaxanthin confirmed this genotype–phenotype linkage

Probing the functional subunits of the tonoplast H+-ATPase

International Nuclear Information System (INIS)

Randall, S.K.; Lai, S.; Sze, H.

1986-01-01

The tonoplast ATPase of oat roots is composed of at least three polypeptides of 72, 60, and 16 kDa. The 16 kDA polypeptide covalently binds N,N'-dicyclohexylcarbodiimide and is postulated to be a component of the proton channel. Initial studies to identify other subunits indicate that both the 72 and 60 kDa subunits covalently bind 14 C]-7-chloro-4-nitrobenzo-2-oxa-1,3-diazole and [ 14 C]N-ethylamleimide, inhibitors of the tonoplast ATPase. ATP prevents binding of these inhibitors suggesting that both the 72 and 60 kDa subunits are involved in substrate binding. Polyclonal antibody has been made to the 72 kDa subunit. Western blot analysis of tonoplast vesicles reveals single reactive polypeptide (72 kDa). The antibody shows no cross-reactivity towards either the mitochondrial F 1 -ATPase or the plasma membrane ATPase. This antibody specifically inhibits ATP hydrolysis and ATP-dependent H + pumping in native tonoplast vesicles. The authors conclude that the 72 kDa subunit is intimately associated with the catalytic (or ATP-binding) site

Meat flavor precursors and factors influencing flavor precursors--A systematic review.

Science.gov (United States)

Khan, Muhammad Issa; Jo, Cheorun; Tariq, Muhammad Rizwan

2015-12-01

Flavor is the sensory impression sensed by taste and smell buds and is a leading factor determining the meat quality and purchasing decision of the consumer. Meat flavor is characteristic of volatiles produced as a result of reactions of non-volatile components that are induced thermally. The water soluble compounds having low molecular weight and meat lipids are important precursors of cooked meat flavor. The Maillard reaction, lipid oxidation, and vitamin degradation are leading reactions during cooking which develop meat flavor from uncooked meat with little aroma and bloody taste. The pre-slaughter and postmortem factors like animal breed, sex, age, feed, aging and cooking conditions contribute to flavor development of cooked meat. The objective of this review is to highlight the flavor chemistry, meat flavor precursors and factors affecting meat flavor precursors. Copyright © 2015 Elsevier Ltd. All rights reserved.

Loss of tumorigenic potential by human lung tumor cells in the presence of antisense RNA specific to the ectopically synthesized alpha subunit of human chorionic gonadotropin.

Science.gov (United States)

Rivera, R T; Pasion, S G; Wong, D T; Fei, Y B; Biswas, D K

1989-06-01

A clonal strain of human lung tumor cells in culture (ChaGo), derived from a bronchogenic carcinoma, synthesizes and secretes large amounts of alpha (alpha) and a comparatively lower level of beta (beta) subunit of the glycoprotein hormone, human chorionic gonadotropin (HCG). ChaGo cells lost their characteristic anchorage-independent growth phenotype in the presence of anti-alpha-HCG antibody. The effect of the antibody was partially reversed by addition of alpha-HCG to the culture medium. ChaGo cells were transfected with an expression vector (pRSV-anti-alpha-HCG), that directs synthesis of RNA complementary to alpha-HCG mRNA. The transfectants produced alpha-HCG antisense RNA which was associated with the reduced level of alpha-HCG. Transfectants also displayed several altered phenotypic properties, including altered morphology, less mitosis, reduced growth rate, loss of anchorage-independent growth, and loss of tumorigenicity in nude mice. Treatment of transfectants with 8,bromo-cAMP resulted in increased accumulation of alpha-HCG mRNA, no change in the level of alpha-HCG antisense RNA, release of the inhibition of [3H]thymidine incorporation, and restoration of anchorage-independent growth phenotype. The overexpression of c-myc, observed in ChaGo cells, was unaffected by the reduced level of alpha-HCG. These results suggest that ectopic synthesis of the alpha subunit of HCG plays a functional role in the transformation of these human lung cells.

Crystallization of the Nonameric Small Terminase Subunit of Bacteriophage P22

Energy Technology Data Exchange (ETDEWEB)

A Roy; A Bhardwaj; G Cingolani

2011-12-31

The packaging of viral genomes into preformed empty procapsids is powered by an ATP-dependent genome-translocating motor. This molecular machine is formed by a heterodimer consisting of large terminase (L-terminase) and small terminase (S-terminase) subunits, which is assembled into a complex of unknown stoichiometry, and a dodecameric portal protein. There is considerable confusion in the literature regarding the biologically relevant oligomeric state of terminases, which, like portal proteins, form ring-like structures. The number of subunits in a hollow oligomeric protein defines the internal diameter of the central channel and the ability to fit DNA inside. Thus, knowledge of the exact stoichiometry of terminases is critical to decipher the mechanisms of terminase-dependent DNA translocation. Here, the gene encoding bacteriophage P22 S-terminase in Escherichia coli has been overexpressed and the protein purified under native conditions. In the absence of detergents and/or denaturants that may cause disassembly of the native oligomer and formation of aberrant rings, it was found that P22 S-terminase assembles into a concentration-independent nonamer of {approx}168 kDa. Nonameric S-terminase was crystallized in two different crystal forms at neutral pH. Crystal form I belonged to space group P2{sub 1}2{sub 1}2, with unit-cell parameters a = 144.2, b = 144.2, c = 145.3 {angstrom}, and diffracted to 3.0 {angstrom} resolution. Crystal form II belonged to space group P2{sub 1}, with unit-cell parameters a = 76.48, b = 100.9, c = 89.95 {angstrom}, {beta} = 93.73{sup o}, and diffracted to 1.75 {angstrom} resolution. Preliminary crystallographic analysis of crystal form II confirms that the S-terminase crystals contain a nonamer in the asymmetric unit and are suitable for high-resolution structure determination.

Crystallization of the Nonameric Small Terminase Subunit of bacteriophage P22

Energy Technology Data Exchange (ETDEWEB)

A Roy; A Bhardwaj; G Cingoloni

2011-12-31

The packaging of viral genomes into preformed empty procapsids is powered by an ATP-dependent genome-translocating motor. This molecular machine is formed by a heterodimer consisting of large terminase (L-terminase) and small terminase (S-terminase) subunits, which is assembled into a complex of unknown stoichiometry, and a dodecameric portal protein. There is considerable confusion in the literature regarding the biologically relevant oligomeric state of terminases, which, like portal proteins, form ring-like structures. The number of subunits in a hollow oligomeric protein defines the internal diameter of the central channel and the ability to fit DNA inside. Thus, knowledge of the exact stoichiometry of terminases is critical to decipher the mechanisms of terminase-dependent DNA translocation. Here, the gene encoding bacteriophage P22 S-terminase in Escherichia coli has been overexpressed and the protein purified under native conditions. In the absence of detergents and/or denaturants that may cause disassembly of the native oligomer and formation of aberrant rings, it was found that P22 S-terminase assembles into a concentration-independent nonamer of {approx}168 kDa. Nonameric S-terminase was crystallized in two different crystal forms at neutral pH. Crystal form I belonged to space group P2{sub 1}2{sub 1}2, with unit-cell parameters a = 144.2, b = 144.2, c = 145.3 {angstrom}, and diffracted to 3.0 {angstrom} resolution. Crystal form II belonged to space group P2{sub 1}, with unit-cell parameters a = 76.48, b = 100.9, c = 89.95 {angstrom}, {beta} = 93.73{sup o}, and diffracted to 1.75 {angstrom} resolution. Preliminary crystallographic analysis of crystal form II confirms that the S-terminase crystals contain a nonamer in the asymmetric unit and are suitable for high-resolution structure determination.

Structure of Pfu Pop5, an archaeal RNase P protein.

Science.gov (United States)

Wilson, Ross C; Bohlen, Christopher J; Foster, Mark P; Bell, Charles E

2006-01-24

We have used NMR spectroscopy and x-ray crystallography to determine the three-dimensional structure of PF1378 (Pfu Pop5), one of four protein subunits of archaeal RNase P that shares a homolog in the eukaryotic enzyme. RNase P is an essential and ubiquitous ribonucleoprotein enzyme required for maturation of tRNA. In bacteria, the enzyme's RNA subunit is responsible for cleaving the single-stranded 5' leader sequence of precursor tRNA molecules (pre-tRNA), whereas the protein subunit assists in substrate binding. Although in bacteria the RNase P holoenzyme consists of one large catalytic RNA and one small protein subunit, in archaea and eukarya the enzyme contains several (> or =4) protein subunits, each of which lacks sequence similarity to the bacterial protein. The functional role of the proteins is poorly understood, as is the increased complexity in comparison to the bacterial enzyme. Pfu Pop5 has been directly implicated in catalysis by the observation that it pairs with PF1914 (Pfu Rpp30) to functionally reconstitute the catalytic domain of the RNA subunit. The protein adopts an alpha-beta sandwich fold highly homologous to the single-stranded RNA binding RRM domain. Furthermore, the three-dimensional arrangement of Pfu Pop5's structural elements is remarkably similar to that of the bacterial protein subunit. NMR spectra have been used to map the interaction of Pop5 with Pfu Rpp30. The data presented permit tantalizing hypotheses regarding the role of this protein subunit shared by archaeal and eukaryotic RNase P.

Cross-linking of hCG to luteal receptors

Energy Technology Data Exchange (ETDEWEB)

Ji, T.H.; Ji, I.

1985-01-01

Photoaffinity labeling of the lutropin/choriogonadotropin (LH/hCG) receptor system on porcine granulosa cells has demonstrated that both the ..cap alpha.. and ..beta.. subunits of hCG directly photoaffinity label the hormone receptor. Three new bands appear on SDS-PAGE as a consequence of photoaffinity labeling by each subunit: the molecular weights of the three bands (106K, 88K, and 83K) produced by the subunit are larger by approximately 10K than those of the three bands (96K, 76K, and 73K) labeled by the ..cap alpha.. subunit. Although it could be a coincidence that the molecular weight of the ..beta.. subunit is approximately 10K larger than that of the ..cap alpha.. subunit, the similarity in these differences suggests the possibility that both the ..cap alpha.. and ..beta.. subunits have labeled the same polypeptides.

Ubiquilin 1 modulates amyloid precursor protein trafficking and Abeta secretion.

Science.gov (United States)

Hiltunen, Mikko; Lu, Alice; Thomas, Anne V; Romano, Donna M; Kim, Minji; Jones, Phill B; Xie, Zhongcong; Kounnas, Maria Z; Wagner, Steven L; Berezovska, Oksana; Hyman, Bradley T; Tesco, Giuseppina; Bertram, Lars; Tanzi, Rudolph E

2006-10-27

Ubiquilin 1 (UBQLN1) is a ubiquitin-like protein, which has been shown to play a central role in regulating the proteasomal degradation of various proteins, including the presenilins. We recently reported that DNA variants in UBQLN1 increase the risk for Alzheimer disease, by influencing expression of this gene in brain. Here we present the first assessment of the effects of UBQLN1 on the metabolism of the amyloid precursor protein (APP). For this purpose, we employed RNA interference to down-regulate UBQLN1 in a variety of neuronal and non-neuronal cell lines. We demonstrate that down-regulation of UBQLN1 accelerates the maturation and intracellular trafficking of APP, while not interfering with alpha-, beta-, or gamma-secretase levels or activity. UBQLN1 knockdown increased the ratio of APP mature/immature, increased levels of full-length APP on the cell surface, and enhanced the secretion of sAPP (alpha- and beta-forms). Moreover, UBQLN1 knockdown increased levels of secreted Abeta40 and Abeta42. Finally, employing a fluorescence resonance energy transfer-based assay, we show that UBQLN1 and APP come into close proximity in intact cells, independently of the presence of the presenilins. Collectively, our findings suggest that UBQLN1 may normally serve as a cytoplasmic "gatekeeper" that may control APP trafficking from intracellular compartments to the cell surface. These findings suggest that changes in UBQLN1 steady-state levels affect APP trafficking and processing, thereby influencing the generation of Abeta.

Biosynthesis and release of beta-endorphin-, N-acetyl beta-endorphin-, beta-endorphin-(1-27)-, and N-acetyl beta-endorphin-(1-27)-like peptides by rat pituitary neurointermediate lobe: beta-endorphin is not further processed by anterior lobe

International Nuclear Information System (INIS)

Liotta, A.S.; Yamaguchi, H.; Krieger, D.T.

1981-01-01

Continuous labeling and pulse-chase techniques were employed to study the synthesis and secretion of multiple forms of immunoreactive beta-endorphin by cultured dispersed rat anterior lobe cells and intact neurointermediate pituitary lobe. Intact neurointermediate lobes incorporated radiolabeled amino acids into four to six forms of immunoreactive beta-endorphin. Four of these forms were physicochemically similar to authentic beta-endorphin, N-acetylated beta-endorphin, beta-endorphin-(1-27), and N-acetylated beta-endorphin-(1-27). Pulse-chase studies indicated that a beta-lipotropin-like molecule served as a metabolic intermediate for a beta-endorphin-like molecule. As beta-endorphin-like material accumulated in the cell, some of it was N-acetylated (approximately 18% at 2 hr chase and approximately 65% at 18 hr chase). At later chase times, beta-endorphin-(1-27)- and N-acetylated beta-endorphin-(1-27)-like peptides were the predominant molecular species detected. All endorphin forms were detected in unlabeled tissue maintained in culture or tissue continuously labeled for 72 hr and were released into the medium under basal, stimulatory (10(-8) M norepinephrine), or inhibitory (10(-7) M dopamine) incubation conditions. In all cases, beta-endorphin-(1-27)-like species were the predominant forms (more than 70% of total) present in the cells and released into the medium. In contrast, approximately 90% of radiolabeled immunoreactive beta-endorphin extracted from anterior lobe cells and medium similarly incubated appeared to represent the authentic beta-endorphin molecule. Continuous labeling (72 hr) revealed the beta-lipotropin/beta-endorphin molar ratio to be approximately 4. We conclude that, in anterior lobe, most of the beta-endorphin is not processed further and is released intact, while in neurointermediate lobe, it serves as a biosynthetic intermediate

The stream of precursors that colonizes the thymus proceeds selectively through the early T lineage precursor stage of T cell development

Science.gov (United States)

Benz, Claudia; Martins, Vera C.; Radtke, Freddy; Bleul, Conrad C.

2008-01-01

T cell development in the thymus depends on continuous colonization by hematopoietic precursors. Several distinct T cell precursors have been identified, but whether one or several independent precursor cell types maintain thymopoiesis is unclear. We have used thymus transplantation and an inducible lineage-tracing system to identify the intrathymic precursor cells among previously described thymus-homing progenitors that give rise to the T cell lineage in the thymus. Extrathymic precursors were not investigated in these studies. Both approaches show that the stream of T cell lineage precursor cells, when entering the thymus, selectively passes through the early T lineage precursor (ETP) stage. Immigrating precursor cells do not exhibit characteristics of double-negative (DN) 1c, DN1d, or DN1e stages, or of populations containing the common lymphoid precursor 2 (CLP-2) or the thymic equivalent of circulating T cell progenitors (CTPs). It remains possible that an unknown hematopoietic precursor cell or previously described extrathymic precursors with a CLP, CLP-2, or CTP phenotype feed into T cell development by circumventing known intrathymic T cell lineage progenitor cells. However, it is clear that of the known intrathymic precursors, only the ETP population contributes significant numbers of T lineage precursors to T cell development. PMID:18458114

Muscular subunits transplantation for facial reanimation

Directory of Open Access Journals (Sweden)

Hazan André Salo Buslik

2006-01-01

Full Text Available PURPOSE: To present an alternative technique for reconstruction of musculocutaneous damages in the face transferring innervated subsegments(subunits of the latissimus dorsi flap for replacement of various facial mimetic muscles. METHODS: One clinical case of trauma with skin and mimetic muscles damage is described as an example of the technique. The treatment was performed with microsurgical transfer of latissimus dorsi muscle subunits. Each subunit present shape and dimensions of the respective mimetic muscles replaced. The origin, insertions and force vectors for the mimicmuscle lost were considered. Each subsegment has its own arterial and venous supply with a motor nerve component for the muscular unit. RESULTS: Pre and one year postoperative photos registration of static and dynamic mimic aspects, as well as digital electromyography digital data of the patients were compared. The transplanted muscular units presented myoeletric activity, fulfilling both the functional and cosmetic aspect. CONCLUSION: This technique seems to be a promising way to deal with the complex musculocutaneous losses of the face as well as facial palsy.

Application of Monte Carlo method in study of the padronization for radionuclides with complex disintegration scheme in 4{pi}{beta}-{gamma} coincidence System; Aplicacao do metodo de Monte Carlo no estudo da padronizacao de radionuclideos com esquema de desintegracao complexos em sistema de coincidencias 4{pi}{beta}-{gamma}

Energy Technology Data Exchange (ETDEWEB)

Takeda, Mauro Noriaki

2006-07-01

The present work described a new methodology for modelling the behaviour of the activity in a 4{pi}{beta}-{gamma} coincidence system. The detection efficiency for electrons in the proportional counter and gamma radiation in the NaI(Tl) detector was calculated using the Monte Carlo program MCNP4C. Another Monte Carlo code was developed which follows the path in the disintegration scheme from the initial state of the precursor radionuclide, until the ground state of the daughter nucleus. Every step of the disintegration scheme is sorted by random numbers taking into account the probabilities of all {beta}{sup -} branches, electronic capture branches, transitions probabilities and internal conversion coefficients. Once the final state was reached beta, electronic capture events and gamma transitions are accounted for the three spectra: beta, gamma and coincidence variation in the beta efficiency was performed simulating energy cut off or use of absorbers (Collodion). The selected radionuclides for simulation were: {sup 134}Cs, {sup 72}Ga which disintegrate by {beta}{sup -} transition, {sup 133}Ba which disintegrates by electronic capture and {sup 35}S which is a beta pure emitter. For the latter, the Efficiency Tracing technique was simulated. The extrapolation curves obtained by Monte Carlo were filled by the Least Square Method with the experimental points and the results were compared to the Linear Extrapolation method. (author)

Pituitary glycoprotein hormone a-subunit secretion by cirrhotic patients

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Oliveira M.C.

1999-01-01

Full Text Available Secretion of the a-subunit of pituitary glycoprotein hormones usually follows the secretion of intact gonadotropins and is increased in gonadal failure and decreased in isolated gonadotropin deficiency. The aim of the present study was to determine the levels of the a-subunit in the serum of patients with cirrhosis of the liver and to compare the results obtained for eugonadal cirrhotic patients with those obtained for cirrhotic patients with hypogonadotropic hypogonadism. Forty-seven of 63 patients with cirrhosis (74.6% presented hypogonadism (which was central in 45 cases and primary in 2, 7 were eugonadal, and 9 women were in normal menopause. The serum a-subunit was measured by the fluorimetric method using monoclonal antibodies. Cross-reactivity with LH, TSH, FSH and hCG was 6.5, 1.2, 4.3 and 1.1%, respectively, with an intra-assay coefficient of variation (CV of less than 5% and an interassay CV of 5%, and sensitivity limit of 4 ng/l. The serum a-subunit concentration ranged from 36 to 6253 ng/l, with a median of 273 ng/l. The median was 251 ng/l for patients with central hypogonadism and 198 ng/l for eugonadal patients. The correlation between the a-subunit and basal LH levels was significant both in the total sample (r = 0.48, P<0.01 and in the cirrhotic patients with central hypogonadism (r = 0.33, P = 0.02. Among men with central hypogonadism there was a negative correlation between a-subunit levels and total testosterone levels (r = 0.54, P<0.01 as well as free testosterone levels (r = -0.53, P<0.01. In conclusion, although the a-subunit levels are correlated with LH levels, at present they cannot be used as markers for hypogonadism in patients with cirrhosis of the liver.

Polysaccharides of algae. Pt. 37. Characterization of hybrid structure of substituted agarose from Polysiphonia morrowii (Rhodophyta, Rhodomelaceae) using. beta. -agarase and /sup 13/C-NMR spectroscopy

Energy Technology Data Exchange (ETDEWEB)

Usov, A.I.; Ivanova, E.G.

1987-09-01

Structure of gel-forming galactan from Polysiphonia morrowii was analysed using bacterial ..beta..-agarase and /sup 13/C-nuclear magnetic resonance (/sup 13/C-NMR) spectroscopy. The polysaccharide was shown to contain: a) blocks composed of agarobiose residues, partly 6-O-methylated and 6-sulfated, which are sensitive to enzymolysis; b) extended blocks composed of agarobiose 6-sulfate residues, which are resistant to ..beta..-agarase action. The latter blocks contain also ..beta..-D-galactopyranosyl-(1->4)-..cap alpha..-L-galactopyranose 6.6'-disulfate residues (biogenetic precursors of agarobiose 6-sulfate), which are hardly detectable by /sup 13/C-NMR spectrum of the starting polysaccharide. Action of alkali on the enzyme-resistant fraction afforded a polysaccharide preparation having /sup 13/C-NMR spectrum of agarose 6-sulfate.

Ex Situ Formation of Metal Selenide Quantum Dots Using Bacterially Derived Selenide Precursors

Energy Technology Data Exchange (ETDEWEB)

Fellowes, Jonathan W.; Pattrick, Richard; Lloyd, Jon; Charnock, John M.; Coker, Victoria S.; Mosselmans, JFW; Weng, Tsu-Chien; Pearce, Carolyn I.

2013-04-12

Luminescent quantum dots were synthesized using bacterially derived selenide (SeII-) as the precursor. Biogenic SeII- was produced by the reduction of Se-IV by Veillonella atypica and compared directly against borohydride-reduced Se-IV for the production of glutathione-stabilized CdSe and beta-mercaptoethanol-stabilized ZnSe nanoparticles by aqueous synthesis. Biological SeII- formed smaller, narrower size distributed QDs under the same conditions. The growth kinetics of biologically sourced CdSe phases were slower. The proteins isolated from filter sterilized biogenic SeII- included a methylmalonyl-CoA decarboxylase previously characterized in the closely related Veillonella parvula. XAS analysis of the glutathione-capped CdSe at the S K-edge suggested that sulfur from the glutathione was structurally incorporated within the CdSe. A novel synchrotron based XAS technique was also developed to follow the nucleation of biological and inorganic selenide phases, and showed that biogenic SeII- is more stable and more resistant to beam-induced oxidative damage than its inorganic counterpart. The bacterial production of quantum dot precursors offers an alternative, 'green' synthesis technique that negates the requirement of expensive, toxic chemicals and suggests a possible link to the exploitation of selenium contaminated waste streams.

Monte Carlo simulation of {beta}-{gamma} coincidence system using plastic scintillators in 4{pi} geometry

Energy Technology Data Exchange (ETDEWEB)

Dias, M.S. [Instituto de Pesquisas Energeticas e Nucleares: IPEN-CNEN/SP, Av. Prof. Lineu Prestes 2242, 05508-000 Sao Paulo, SP (Brazil)], E-mail: msdias@ipen.br; Piuvezam-Filho, H. [Instituto de Pesquisas Energeticas e Nucleares: IPEN-CNEN/SP, Av. Prof. Lineu Prestes 2242, 05508-000 Sao Paulo, SP (Brazil); Baccarelli, A.M. [Departamento de Fisica-PUC/SP-Rua Marques de Paranagua 111, 01303-050 Sao Paulo, SP (Brazil); Takeda, M.N. [Universidade Santo Amaro, UNISA-Rua Prof. Eneas da Siqueira Neto 340, 04829-300 Sao Paulo, SP (Brazil); Koskinas, M.F. [Instituto de Pesquisas Energeticas e Nucleares: IPEN-CNEN/SP, Av. Prof. Lineu Prestes 2242, 05508-000 Sao Paulo, SP (Brazil)

2007-09-21

A modified version of a Monte Carlo code called Esquema, developed at the Nuclear Metrology Laboratory in IPEN, Sao Paulo, Brazil, has been applied for simulating a 4{pi}{beta}(PS)-{gamma} coincidence system designed for primary radionuclide standardisation. This system consists of a plastic scintillator in 4{pi} geometry, for alpha or electron detection, coupled to a NaI(Tl) counter for gamma-ray detection. The response curves for monoenergetic electrons and photons have been calculated previously by Penelope code and applied as input data to code Esquema. The latter code simulates all the disintegration processes, from the precursor nucleus to the ground state of the daughter radionuclide. As a result, the curve between the observed disintegration rate as a function of the beta efficiency parameter can be simulated. A least-squares fit between the experimental activity values and the Monte Carlo calculation provided the actual radioactive source activity, without need of conventional extrapolation procedures. Application of this methodology to {sup 60}Co and {sup 133}Ba radioactive sources is presented and showed results in good agreement with a conventional proportional counter 4{pi}{beta}(PC)-{gamma} coincidence system.

Computer modeling of siRNA knockdown effects indicates an essential role of the Ca2+ channel alpha2delta-1 subunit in cardiac excitation-contraction coupling.

Science.gov (United States)

Tuluc, Petronel; Kern, Georg; Obermair, Gerald J; Flucher, Bernhard E

2007-06-26

L-type Ca(2+) currents determine the shape of cardiac action potentials (AP) and the magnitude of the myoplasmic Ca(2+) signal, which regulates the contraction force. The auxiliary Ca(2+) channel subunits alpha(2)delta-1 and beta(2) are important regulators of membrane expression and current properties of the cardiac Ca(2+) channel (Ca(V)1.2). However, their role in cardiac excitation-contraction coupling is still elusive. Here we addressed this question by combining siRNA knockdown of the alpha(2)delta-1 subunit in a muscle expression system with simulation of APs and Ca(2+) transients by using a quantitative computer model of ventricular myocytes. Reconstitution of dysgenic muscle cells with Ca(V)1.2 (GFP-alpha(1C)) recapitulates key properties of cardiac excitation-contraction coupling. Concomitant depletion of the alpha(2)delta-1 subunit did not perturb membrane expression or targeting of the pore-forming GFP-alpha(1C) subunit into junctions between the outer membrane and the sarcoplasmic reticulum. However, alpha(2)delta-1 depletion shifted the voltage dependence of Ca(2+) current activation by 9 mV to more positive potentials, and it slowed down activation and inactivation kinetics approximately 2-fold. Computer modeling revealed that the altered voltage dependence and current kinetics exert opposing effects on the function of ventricular myocytes that in total cause a 60% prolongation of the AP and a 2-fold increase of the myoplasmic Ca(2+) concentration during each contraction. Thus, the Ca(2+) channel alpha(2)delta-1 subunit is not essential for normal Ca(2+) channel targeting in muscle but is a key determinant of normal excitation and contraction of cardiac muscle cells, and a reduction of alpha(2)delta-1 function is predicted to severely perturb normal heart function.

2-Azido-( sup 32 P)NAD+, a photoactivatable probe for G-protein structure: Evidence for holotransducin oligomers in which the ADP-ribosylated carboxyl terminus of alpha interacts with both alpha and gamma subunits

Energy Technology Data Exchange (ETDEWEB)

Vaillancourt, R.R.; Dhanasekaran, N.; Johnson, G.L.; Ruoho, A.E. (Univ. of Wisconsin Medical School, Madison (USA))

1990-05-01

A radioactive and photoactivatable derivative of NAD+, 2-azido-(adenylate-32P)NAD+, has been synthesized and used with pertussis toxin to ADP-ribosylate Cys347 of the alpha subunit (alpha T) of GT, the retinal guanine nucleotide-binding protein. ADP-ribosylation of alpha T followed by light activation of the azide moiety of 2-azido-(adenylate-32P)ADP-ribose produced four crosslinked species involving the alpha and gamma subunits of the GT heterotrimer: an alpha trimer (alpha-alpha-alpha), and alpha-alpha-gamma crosslink, an alpha dimer (alpha-alpha), and an alpha-gamma crosslink. The alpha trimer, alpha-alpha-gamma complex, alpha dimer, and alpha-gamma complexes were immunoreactive with alpha T antibodies. The alpha-alpha-gamma and the alpha-gamma complexes were immunoreactive with antisera recognizing gamma subunits. No evidence was found for crosslinking of alpha T to beta T subunits. Hydrolysis of the thioglycosidic bond between Cys347 and 2-azido-(adenylate-32P)ADP-ribose using mercuric acetate resulted in the transfer of radiolabel from Cys347 of alpha T in the crosslinked oligomers to alpha monomers, indicative of intermolecular photocrosslinking, and to gamma monomers, indicative of either intermolecular crosslinked complexes (between heterotrimers) or intramolecular crosslinked complexes (within the heterotrimer). These results demonstrate that GT exists as an oligomer and that ADP-ribosylated Cys347, which is four residues from the alpha T-carboxyl terminus, is oriented toward and in close proximity to the gamma subunit.

Mutation Analysis of 16 Mucolipidosis II and III Alpha/Beta Chinese Children Revealed Genotype-Phenotype Correlations.

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Shuang Liu

Full Text Available Mucolipidosis II and III alpha/beta are autosomal recessive diseases caused by mutations in the GNPTAB gene which encodes the α and β subunits of the N-acetylglucosamine-1-phosphotransferase. Clinically, mucolipidosis II (MLII is characterized by severe developmental delay, coarse facial features, skeletal deformities, and other systemic involvement. In contrast, MLIII alpha/beta is a much milder disorder, the symptoms of which include progressive joint stiffness, short stature, and scoliosis. To study the relationship between the genotypes and phenotypes of the MLII and MLIII alpha/beta patients, we analyzed the GNPTAB gene in 16 Chinese MLII and MLIII alpha/beta patients. We collected and analyzed the patients' available clinical data and all showed clinical features typical of MLII or MLIII alpha/beta. Moreover, the activity of several lysosomal enzymes was measured in the plasma and finally the GNPTAB gene was sequenced. We detected 30 mutant alleles out of 32 alleles in our patients. These include 10 new mutations (c.99delC, c.118-1G>A, c.523_524delAAinsG, c.1212C>G, c.2213C>A, c.2345C>T, c.2356C>T, c.2455G>T, c.2821dupA, and c.3136-2A>G and 5 previously reported mutations (c.1071G>A, c.1090C>T, c.2715+1G>A, c.2550_2554delGAAA, and c.3613C>T. The most frequent mutation was the splicing mutation c.2715+1G>A, which accounted for 28% of the mutations. The majority of the mutations reported in the Chinese patients (57% were located on exon 13 or in its intronic flanking regions.

Molecular characterization of the alpha subunit of complement component C8 (GcC8alpha) in the nurse shark (Ginglymostoma cirratum).

Science.gov (United States)

Aybar, Lydia; Shin, Dong-Ho; Smith, Sylvia L

2009-09-01

Target cell lysis by complement is achieved by the assembly and insertion of the membrane attack complex (MAC) composed of glycoproteins C5b through C9. The lytic activity of shark complement involves functional analogues of mammalian C8 and C9. Mammalian C8 is composed of alpha, beta, and gamma subunits. The subunit structure of shark C8 is not known. This report describes a 2341 nucleotide sequence that translates into a polypeptide of 589 amino acid residues, orthologue to mammalian C8alpha and has the same modular architecture with conserved cysteines forming the peptide bond backbone. The C8gamma-binding cysteine is conserved in the perforin-like domain. Hydrophobicity profile indicates the presence of hydrophobic residues essential for membrane insertion. It shares 41.1% and 47.4% identity with human and Xenopus C8alpha respectively. Southern blot analysis showed GcC8alpha exists as a single copy gene expressed in most tissues except the spleen with the liver being the main site of synthesis. Phylogenetic analysis places it in a clade with C8alpha orthologs and as a sister taxa to the Xenopus. 2009 Elsevier Ltd.

Purification of beta-acetylglucosaminase and beta-galactosidase from ram testis.

Science.gov (United States)

Caygill, J C; Roston, C P; Jevons, F R

1966-02-01

1. The presence of beta-galactosidase (EC 3.2.1.23) in an acetic acid extract of ram testis is reported. Some properties of the crude enzyme preparation were studied. 2. The purification of beta-acetylglucosaminase (EC 3.2.1.30) and of beta-galactosidase from the ram-testis extract by ammonium sulphate precipitation and chromatography on a CM-cellulose column is described. 3. The final purifications of the separated enzymes achieved were for the beta-acetylglucosaminase 35 times and for the beta-galactosidase 99 times. 4. The possibility of using DEAE-cellulose and Sephadex G-200 to purify the enzymes was investigated.

Catalytic Subunit 1 of Protein Phosphatase 2A Is a Subunit of the STRIPAK Complex and Governs Fungal Sexual Development

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Anna Beier

2016-06-01

Full Text Available The generation of complex three-dimensional structures is a key developmental step for most eukaryotic organisms. The details of the molecular machinery controlling this step remain to be determined. An excellent model system to study this general process is the generation of three-dimensional fruiting bodies in filamentous fungi like Sordaria macrospora. Fruiting body development is controlled by subunits of the highly conserved striatin-interacting phosphatase and kinase (STRIPAK complex, which has been described in organisms ranging from yeasts to humans. The highly conserved heterotrimeric protein phosphatase PP2A is a subunit of STRIPAK. Here, catalytic subunit 1 of PP2A was functionally characterized. The Δpp2Ac1 strain is sterile, unable to undergo hyphal fusion, and devoid of ascogonial septation. Further, PP2Ac1, together with STRIPAK subunit PRO22, governs vegetative and stress-related growth. We revealed in vitro catalytic activity of wild-type PP2Ac1, and our in vivo analysis showed that inactive PP2Ac1 blocks the complementation of the sterile deletion strain. Tandem affinity purification, followed by mass spectrometry and yeast two-hybrid analysis, verified that PP2Ac1 is a subunit of STRIPAK. Further, these data indicate links between the STRIPAK complex and other developmental signaling pathways, implying the presence of a large interconnected signaling network that controls eukaryotic developmental processes. The insights gained in our study can be transferred to higher eukaryotes and will be important for understanding eukaryotic cellular development in general.

Electrophysiology and Beyond: Multiple roles of Na+ channel β subunits in development and disease

Science.gov (United States)

Patino, Gustavo A.; Isom, Lori L.

2010-01-01

Voltage-gated Na+ channel (VGSC) β subunits are not “auxiliary.” These multifunctional molecules not only modulate Na+ current (INa), but also function as cell adhesion molecules (CAMs) – playing roles in aggregation, migration, invasion, neurite outgrowth, and axonal fasciculation. β subunits are integral members of VGSC signaling complexes at nodes of Ranvier, axon initial segments, and cardiac intercalated disks, regulating action potential propagation through critical intermolecular and cell-cell communication events. At least in vitro, many β subunit cell adhesive functions occur both in the presence and absence of pore-forming VGSC α subunits, and in vivo β subunits are expressed in excitable as well as non-excitable cells, thus β subunits may play important functional roles on their own, in the absence of α subunits. VGSC β1 subunits are essential for life and appear to be especially important during brain development. Mutations in β subunit genes result in a variety of human neurological and cardiovascular diseases. Moreover, some cancer cells exhibit alterations in β subunit expression during metastasis. In short, these proteins, originally thought of as merely accessory to α subunits, are critical players in their own right in human health and disease. Here we discuss the role of VGSC β subunits in the nervous system. PMID:20600605

Betting Against Beta

DEFF Research Database (Denmark)

Frazzini, Andrea; Heje Pedersen, Lasse

We present a model with leverage and margin constraints that vary across investors and time. We find evidence consistent with each of the model’s five central predictions: (1) Since constrained investors bid up high-beta assets, high beta is associated with low alpha, as we find empirically for U...... of the BAB factor is low; (4) Increased funding liquidity risk compresses betas toward one; (5) More constrained investors hold riskier assets........S. equities, 20 international equity markets, Treasury bonds, corporate bonds, and futures; (2) A betting-against-beta (BAB) factor, which is long leveraged low beta assets and short high-beta assets, produces significant positive risk-adjusted returns; (3) When funding constraints tighten, the return...

Conversion of beta-methylbutyric acid to beta-hydroxy-beta-methylbutyric acid by Galactomyces reessii.

OpenAIRE

Lee, I Y; Nissen, S L; Rosazza, J P

1997-01-01

beta-Hydroxy-beta-methylbutyric acid (HMB) has been shown to increase strength and lean mass gains in humans undergoing resistance-exercise training. HMB is currently marketed as a calcium salt of HMB, and thus, environmentally sound and inexpensive methods of manufacture are being sought. This study investigates the microbial conversion of beta-methylbutyric acid (MBA) to HMB by cultures of Galactomyces reessii. Optimal concentrations of MBA were in the range of 5 to 20 g/liter for HMB produ...

Spectral difference method applied in determination of beta-carotene, chlorophyll a and b from irradiated seeds of Calendula Officinalis

International Nuclear Information System (INIS)

Cucu, Delia-Irina; Dorobantu, Ioan

2002-01-01

Beta-carotene is part of the class of the carotenoid pigments that have a special function in the pigmentation of flowers and fruits. These pigments are essential in photosynthesis where they are retaining energy and are photoprotectors by forming the redox system. They are also precursors of vitamin A, pigments involved in the process of seeing. Beta-carotene is responsible for the growth and development of organisms, for the protection and recovery of epithelial tissue, etc. Next to chlorophyll, carotenoids are the major components of the complex pigment-protein from the thylacoide membrane, and some of them, such as beta-carotene, were for a long time considered to be protecting the membrane against destructive events caused by the over excitement of the chlorophyll. This paper presents spectral difference method for physico-chemical characterisation of three components (beta-carotene, chlorophyll a and b) from Calendula officinalis leaves and stalks, after irradiation of germinated seeds. The germinated seeds of Calendula officinalis has been irradiated at 8 different doses ( 1 k Rad, 3 k Rad, 5 k Rad, 10 k Rad, 20 k Rad, 50 k Rad, 100 k Rad, 500 krad), one set of seeds remaining unirradiated for comparison. The plants had been grown in the same conditions of light, temperature and humidity. The identification of beta-carotene was performed through thin layer chromatography. The chemical concentrations of beta-carotene, chlorophyll a and b were determined by spectral difference method. (authors)

Forward-Looking Betas

DEFF Research Database (Denmark)

Christoffersen, Peter; Jacobs, Kris; Vainberg, Gregory

Few issues are more important for finance practice than the computation of market betas. Existing approaches compute market betas using historical data. While these approaches differ in terms of statistical sophistication and the modeling of the time-variation in the betas, they are all backward......-looking. This paper introduces a radically different approach to estimating market betas. Using the tools in Bakshi and Madan (2000) and Bakshi, Kapadia and Madan (2003) we employ the information embedded in the prices of individual stock options and index options to compute our forward-looking market beta...

Arabidopsis CDS blastp result: AK063110 [KOME

Lifescience Database Archive (English)

Full Text Available AK063110 001-111-D06 At5g43430.1 electron transfer flavoprotein beta subunit family... protein contains Pfam profile: PF01012 electron transfer flavoprotein, beta subunit 1e-100 ...

Identification of the A2 adenosine receptor binding subunit by photoaffinity crosslinking

International Nuclear Information System (INIS)

Barrington, W.W.; Jacobson, K.A.; Hutchison, A.J.; Williams, M.; Stiles, G.L.

1989-01-01

A high-affinity iodinated agonist radioligand for the A2 adenosine receptor has been synthesized to facilitate studies of the A2 adenosine receptor binding subunit. The radioligand 125I-labeled PAPA-APEC (125I-labeled 2-[4-(2-[2-[(4- aminophenyl)methylcarbonylamino]ethylaminocarbonyl]- ethyl)phenyl]ethylamino-5'-N-ethylcarboxamidoadenosine) was synthesized and found to bind to the A2 adenosine receptor in bovine striatal membranes with high affinity (Kd = 1.5 nM) and A2 receptor selectivity. Competitive binding studies reveal the appropriate A2 receptor pharmacologic potency order with 5'-N-ethylcarboxamidoadenosine (NECA) greater than (-)-N6-[(R)-1-methyl- 2-phenylethyl]adenosine (R-PIA) greater than (+)-N6-[(S)-1-methyl-2- phenylethyl]adenosine (S-PIA). Adenylate cyclase assays, in human platelet membranes, demonstrate a dose-dependent stimulation of cAMP production. PAPA-APEC (1 microM) produces a 43% increase in cAMP production, which is essentially the same degree of increase produced by 5'-N- ethylcarboxamidoadenosine (the prototypic A2 receptor agonist). These findings combined with the observed guanine nucleotide-mediated decrease in binding suggest that PAPA-APEC is a full A2 agonist. The A2 receptor binding subunit was identified by photoaffinity-crosslinking studies using 125I-labeled PAPA-APEC and the heterobifunctional crosslinking agent N-succinimidyl 6-(4'-azido-2'-nitrophenylamino)hexanoate (SANPAH). After covalent incorporation, a single specifically radiolabeled protein with an apparent molecular mass of 45 kDa was observed on NaDodSO4/PAGE/autoradiography. Incorporation of 125I-labeled PAPA-APEC into this polypeptide is blocked by agonists and antagonists with the expected potency for A2 receptors and is decreased in the presence of 10(-4) M guanosine 5'-[beta, gamma-imido]triphosphate

Intracellular APP Domain Regulates Serine-Palmitoyl-CoA Transferase Expression and Is Affected in Alzheimer's Disease

Science.gov (United States)

Grimm, Marcus O. W.; Grösgen, Sven; Rothhaar, Tatjana L.; Burg, Verena K.; Hundsdörfer, Benjamin; Haupenthal, Viola J.; Friess, Petra; Müller, Ulrike; Fassbender, Klaus; Riemenschneider, Matthias; Grimm, Heike S.; Hartmann, Tobias

2011-01-01

Lipids play an important role as risk or protective factors in Alzheimer's disease (AD), a disease biochemically characterized by the accumulation of amyloid beta peptides (Aβ), released by proteolytic processing of the amyloid precursor protein (APP). Changes in sphingolipid metabolism have been associated to the development of AD. The key enzyme in sphingolipid de novo synthesis is serine-palmitoyl-CoA transferase (SPT). In the present study we identified a new physiological function of APP in sphingolipid synthesis. The APP intracellular domain (AICD) was found to decrease the expression of the SPT subunit SPTLC2, the catalytic subunit of the SPT heterodimer, resulting in that decreased SPT activity. AICD function was dependent on Fe65 and SPTLC2 levels are increased in APP knock-in mice missing a functional AICD domain. SPTLC2 levels are also increased in familial and sporadic AD postmortem brains, suggesting that SPT is involved in AD pathology. PMID:21660213

Mac-2 binding protein is a cell-adhesive protein of the extracellular matrix which self-assembles into ring-like structures and binds beta1 integrins, collagens and fibronectin

DEFF Research Database (Denmark)

Sasaki, T; Brakebusch, C; Engel, J

1998-01-01

Human Mac-2 binding protein (M2BP) was prepared in recombinant form from the culture medium of 293 kidney cells and consisted of a 92 kDa subunit. The protein was obtained in a native state as indicated by CD spectroscopy, demonstrating alpha-helical and beta-type structure, and by protease resis...... in the extracellular matrix of several mouse tissues....... in solid-phase assays to collagens IV, V and VI, fibronectin and nidogen, but not to fibrillar collagens I and III or other basement membrane proteins. The protein also mediated adhesion of cell lines at comparable strength with laminin. Adhesion to M2BP was inhibited by antibodies to integrin beta1...

Enantioselective synthesis of alpha,beta-disubstituted-beta-amino acids.

Science.gov (United States)

Sibi, Mukund P; Prabagaran, Narayanasamy; Ghorpade, Sandeep G; Jasperse, Craig P

2003-10-01

Highly diastereoselective and enantioselective addition of N-benzylhydroxylamine to imides 17 and 20-30 produces alpha,beta-trans-disubstituted N-benzylisoxazolidinones 19 and 31-41. These reactions proceed in 60-96% ee with 93-99% de's using 5 mol % of Mg(NTf2)2 and ligand 18. The product isoxazolidinones can be hydrogenolyzed directly to provide alpha,beta-disubstituted-beta-amino acids.

Subunit association as the stabilizing determinant for archaeal methionine adenosyltransferases.

Science.gov (United States)

Garrido, Francisco; Alfonso, Carlos; Taylor, John C; Markham, George D; Pajares, María A

2009-07-01

Archaea contain a class of methionine adenosyltransferases (MATs) that exhibit substantially higher stability than their mesophilic counterparts. Their sequences are highly divergent, but preserve the essential active site motifs of the family. We have investigated the origin of this increased stability using chemical denaturation experiments on Methanococcus jannaschii MAT (Mj-MAT) and mutants containing single tryptophans in place of tyrosine residues. The results from fluorescence, circular dichroism, hydrodynamic, and enzyme activity measurements showed that the higher stability of Mj-MAT derives largely from a tighter association of its subunits in the dimer. Local fluorescence changes, interpreted using secondary structure predictions, further identify the least stable structural elements as the C-terminal ends of beta-strands E2 and E6, and the N-terminus of E3. Dimer dissociation however requires a wider perturbation of the molecule. Additional analysis was initially hindered by the lack of crystal structures for archaeal MATs, a limitation that we overcame by construction of a 3D-homology model of Mj-MAT. This model predicts preservation of the chain topology and three-domain organization typical of this family, locates the least stable structural elements at the flat contact surface between monomers, and shows that alterations in all three domains are required for dimer dissociation.

In-trap decay spectroscopy for {beta}{beta} decays

Energy Technology Data Exchange (ETDEWEB)

Brunner, Thomas

2011-01-18

The presented work describes the implementation of a new technique to measure electron-capture (EC) branching ratios (BRs) of intermediate nuclei in {beta}{beta} decays. This technique has been developed at TRIUMF in Vancouver, Canada. It facilitates one of TRIUMF's Ion Traps for Atomic and Nuclear science (TITAN), the Electron Beam Ion Trap (EBIT) that is used as a spectroscopy Penning trap. Radioactive ions, produced at the radioactive isotope facility ISAC, are injected and stored in the spectroscopy Penning trap while their decays are observed. A key feature of this technique is the use of a strong magnetic field, required for trapping. It radially confines electrons from {beta} decays along the trap axis while X-rays, following an EC, are emitted isotropically. This provides spatial separation of X-ray and {beta} detection with almost no {beta}-induced background at the X-ray detector, allowing weak EC branches to be measured. Furthermore, the combination of several traps allows one to isobarically clean the sample prior to the in-trap decay spectroscopy measurement. This technique has been developed to measure ECBRs of transition nuclei in {beta}{beta} decays. Detailed knowledge of these electron capture branches is crucial for a better understanding of the underlying nuclear physics in {beta}{beta} decays. These branches are typically of the order of 10{sup -5} and therefore difficult to measure. Conventional measurements suffer from isobaric contamination and a dominating {beta} background at theX-ray detector. Additionally, X-rays are attenuated by the material where the radioactive sample is implanted. To overcome these limitations, the technique of in-trap decay spectroscopy has been developed. In this work, the EBIT was connected to the TITAN beam line and has been commissioned. Using the developed beam diagnostics, ions were injected into the Penning trap and systematic studies on injection and storage optimization were performed. Furthermore, Ge

A single amino acid difference between the intracellular domains of amyloid precursor protein and amyloid-like precursor protein 2 enables induction of synaptic depression and block of long-term potentiation.

Science.gov (United States)

Trillaud-Doppia, Emilie; Paradis-Isler, Nicolas; Boehm, Jannic

2016-07-01

Alzheimer disease (AD) is initially characterized as a disease of the synapse that affects synaptic transmission and synaptic plasticity. While amyloid-beta and tau have been traditionally implicated in causing AD, recent studies suggest that other factors, such as the intracellular domain of the amyloid-precursor protein (APP-ICD), can also play a role in the development of AD. Here, we show that the expression of APP-ICD induces synaptic depression, while the intracellular domain of its homolog amyloid-like precursor protein 2 (APLP2-ICD) does not. We are able to show that this effect by APP-ICD is due to a single alanine vs. proline difference between APP-ICD and APLP2-ICD. The alanine in APP-ICD and the proline in APLP2-ICD lie directly behind a conserved caspase cleavage site. Inhibition of caspase cleavage of APP-ICD prevents the induction of synaptic depression. Finally, we show that the expression of APP-ICD increases and facilitates long-term depression and blocks induction of long-term potentiation. The block in long-term potentiation can be overcome by mutating the aforementioned alanine in APP-ICD to the proline of APLP2. Based on our results, we propose the emergence of a new APP critical domain for the regulation of synaptic plasticity and in consequence for the development of AD. Copyright © 2016 Elsevier Inc. All rights reserved.

Delayed internalization and lack of recycling in a beta2-adrenergic receptor fused to the G protein alpha-subunit

Directory of Open Access Journals (Sweden)

Floridi Aristide

2008-10-01

Full Text Available Abstract Background Chimeric proteins obtained by the fusion of a G protein-coupled receptor (GPCR sequence to the N-terminus of the G protein α-subunit have been extensively used to investigate several aspects of GPCR signalling. Although both the receptor and the G protein generally maintain a fully functional state in such polypeptides, original observations made using a chimera between the β2-adrenergic receptor (β2AR and Gαs indicated that the fusion to the α-subunit resulted in a marked reduction of receptor desensitization and down-regulation. To further investigate this phenomenon, we have compared the rates of internalization and recycling between wild-type and Gαs-fused β2AR. Results The rate of agonist-induced internalization, measured as the disappearance of cell surface immunofluorescence in HEK293 cells permanently expressing N-terminus tagged receptors, was reduced three-fold by receptor-G protein fusion. However, both fused and non-fused receptors translocated to the same endocytic compartment, as determined by dual-label confocal analysis of cells co-expressing both proteins and transferrin co-localization. Receptor recycling, determined as the reversion of surface immunofluorescence following the addition of antagonist to cells that were previously exposed to agonist, markedly differed between wild-type and fused receptors. While most of the internalized β2AR returned rapidly to the plasma membrane, β2AR-Gαs did not recycle, and the observed slow recovery for the fusion protein immunofluorescence was entirely accounted for by protein synthesis. Conclusion The covalent linkage between β2AR and Gαs does not appear to alter the initial endocytic translocation of the two proteins, although there is reduced efficiency. It does, however, completely disrupt the process of receptor and G protein recycling. We conclude that the physical separation between receptor and Gα is not necessary for the transit to early endosomes

Preparation of superconductor precursor powders

Science.gov (United States)

Bhattacharya, Raghunath

1998-01-01

A process for the preparation of a precursor metallic powder composition for use in the subsequent formation of a superconductor. The process comprises the steps of providing an electrodeposition bath comprising an electrolyte medium and a cathode substrate electrode, and providing to the bath one or more soluble salts of one or more respective metals which are capable of exhibiting superconductor properties upon subsequent appropriate treatment. The bath is continually energized to cause the metallic and/or reduced particles formed at the electrode to drop as a powder from the electrode into the bath, and this powder, which is a precursor powder for superconductor production, is recovered from the bath for subsequent treatment. The process permits direct inclusion of all metals in the preparation of the precursor powder, and yields an amorphous product mixed on an atomic scale to thereby impart inherent high reactivity. Superconductors which can be formed from the precursor powder include pellet and powder-in-tube products.

Toward a theory of precursors

International Nuclear Information System (INIS)

Freivogel, Ben; Giddings, Steven B.; Lippert, Matthew

2002-01-01

To better understand the possible breakdown of locality in quantum gravitational systems, we pursue the identity of precursors in the context of the anti-de Sitter/conformal field theory correspondence. Holography implies a breakdown of standard bulk locality which we expect to occur only at extremely high energy. We consider precursors that encode bulk information causally disconnected from the boundary and whose measurement involves nonlocal bulk processes. We construct a toy model of holography which encapsulates the expected properties of precursors and compare it with previous such discussions. If these precursors can be identified in the gauge theory, they are almost certainly Wilson loops, perhaps with decorations, but the relevant information is encoded in the high-energy sector of the theory and should not be observable by low energy measurements. This would be in accord with the locality bound, which serves as a criterion for situations where breakdown of bulk locality is expected

BetaTPred: prediction of beta-TURNS in a protein using statistical algorithms.

Science.gov (United States)

Kaur, Harpreet; Raghava, G P S

2002-03-01

beta-turns play an important role from a structural and functional point of view. beta-turns are the most common type of non-repetitive structures in proteins and comprise on average, 25% of the residues. In the past numerous methods have been developed to predict beta-turns in a protein. Most of these prediction methods are based on statistical approaches. In order to utilize the full potential of these methods, there is a need to develop a web server. This paper describes a web server called BetaTPred, developed for predicting beta-TURNS in a protein from its amino acid sequence. BetaTPred allows the user to predict turns in a protein using existing statistical algorithms. It also allows to predict different types of beta-TURNS e.g. type I, I', II, II', VI, VIII and non-specific. This server assists the users in predicting the consensus beta-TURNS in a protein. The server is accessible from http://imtech.res.in/raghava/betatpred/

Existence of a solid solution from brucite to {beta}-Co(OH){sub 2}

Energy Technology Data Exchange (ETDEWEB)

Giovannelli, F., E-mail: fabien.giovannelli@univ-tours.fr [LEMA, UMR 6157 CNRS - CEA, Universite Francois Rabelais, 15 rue de la chocolaterie, 41000 Blois (France); Delorme, F.; Autret-Lambert, C. [LEMA, UMR 6157 CNRS - CEA, Universite Francois Rabelais, 15 rue de la chocolaterie, 41000 Blois (France); Seron, A.; Jean-Prost, V. [BRGM, 3 Avenue Claude Guillemin, BP 36009, 45060 Orleans Cedex 2 (France)

2012-05-15

Highlights: Black-Right-Pointing-Pointer A solid solution exist between Mg(OH){sub 2} and {beta}-Co(OH){sub 2}. Black-Right-Pointing-Pointer Synthesis has been performed through an easy and fast coprecipitation route. Black-Right-Pointing-Pointer No long range-ordering of the cations occurs. -- Abstract: This study shows that between brucite (Mg(OH){sub 2}) and {beta}-Co(OH){sub 2}, all the compositions are possible. The solid solution Mg{sub 1-x}Co{sub x}(OH){sub 2} has been synthesized by an easy and fast coprecipitation route and characterized by XRD and TEM. Single phase powders have been obtained. The particles exhibit platelets morphology with a size close to one hundred nanometers. XRD analysis shows an evolution of the cell parameters when x increases and demonstrates that no ordering of the cations occurs. However, extra reflections on TEM electron diffraction patterns seem to indicate that local ordering can exist. The compounds issued from this solid solution could be good candidates as precursors in order to obtain Mg-Co mixed oxide with all possible cationic ratios.

Roughing up Beta

DEFF Research Database (Denmark)

Bollerslev, Tim; Li, Sophia Zhengzi; Todorov, Viktor

-section. An investment strategy that goes long stocks with high jump betas and short stocks with low jump betas produces significant average excess returns. These higher risk premiums for the discontinuous and overnight market betas remain significant after controlling for a long list of other firm characteristics......Motivated by the implications from a stylized equilibrium pricing framework, we investigate empirically how individual equity prices respond to continuous, or \\smooth," and jumpy, or \\rough," market price moves, and how these different market price risks, or betas, are priced in the cross......-section of expected returns. Based on a novel highfrequency dataset of almost one-thousand individual stocks over two decades, we find that the two rough betas associated with intraday discontinuous and overnight returns entail significant risk premiums, while the intraday continuous beta is not priced in the cross...

[Beta]-Adrenergic Receptor Activation Rescues Theta Frequency Stimulation-Induced LTP Deficits in Mice Expressing C-Terminally Truncated NMDA Receptor GluN2A Subunits

Science.gov (United States)

Moody, Teena D.; Watabe, Ayako M.; Indersmitten, Tim; Komiyama, Noboru H.; Grant, Seth G. N.; O'Dell, Thomas J.

2011-01-01

Through protein interactions mediated by their cytoplasmic C termini the GluN2A and GluN2B subunits of NMDA receptors (NMDARs) have a key role in the formation of NMDAR signaling complexes at excitatory synapses. Although these signaling complexes are thought to have a crucial role in NMDAR-dependent forms of synaptic plasticity such as long-term…

Distribution of AMPA-type glutamate receptor subunits in the chick visual system

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Pires R.S.

1997-01-01

Full Text Available Several glutamate receptor (GluR subunits have been characterized during the past few years. In the present study, subunit-specific antisera were used to determine the distribution of the AMPA-type glutamate receptor subunits GluR1-4 in retinorecipient areas of the chick brain. Six white leghorn chicks (Gallus gallus, 7-15 days old, unknown sex were deeply anesthetized and perfused with 4% buffered paraformaldehyde and brain sections were stained using immunoperoxidase techniques. The AMPA-type glutamate receptor subunits GluR1, GluR2/3 and GluR4 were present in several retinorecipient areas, with varying degrees of colocalization. For example, perikarya in layers 2, 3, and 5 of the optic tectum contained GluR1, whereas GluR2/3 subunits appeared mainly in neurons of layer 13. The GluR4 subunit was only detected in a few cells of the tectal layer 13. GluR1 and GluR2/3 were observed in neurons of the nucleus geniculatus lateralis ventralis, whereas GluR4 was only present in its neuropil. Somata in the accessory optic nucleus appeared to contain GluR2/3 and GluR4, whereas GluR1 was the dominant subunit in the neuropil of this nucleus. These results suggest that different subpopulations of visual neurons might express different combinations of AMPA-type GluR subunits, which in turn might generate different synaptic responses to glutamate derived from retinal ganglion cell axons

Plasma beta-endorphin, beta-lipotropin and corticotropin in polycystic ovarian disease.

Science.gov (United States)

Laatikainen, T; Salminen, K; Virtanen, T; Apter, D

1987-04-01

In 9 women with polycystic ovarian disease (PCOD) and in 11 control subjects at the follicular phase of the normal cycle, blood samples were collected at 15-min intervals during a 2 h period of bed rest for the assay of beta-endorphin, beta-lipotropin, corticotropin, cortisol and prolactin. During the study period, the plasma levels of these hormones decreased more significantly in the PCOD than in the control group, suggesting that the PCOD patients had a more significant stress response to the puncture of the vein than the control subjects. The second hour of the study period was considered to represent resting levels of hormones. The mean resting levels (+/- S.E.) of the hormones between the PCOD and control groups, respectively, were as follows: beta-E, 2.0 +/- 0.4 vs. 1.1 +/- 0.1 pmol/l, p less than 0.05; beta-LPH, 3.4 +/- 0.6 vs. 2.1 +/- 0.5 pmol/l, N.S.; corticotropin, 2.0 +/- 0.3 vs. 1.1 +/- 0.5 pmol/l, p less than 0.05; cortisol, 176 +/- 24 vs. 128 +/- 16, N.S.; and prolactin; 3.9 +/- 0.6 vs. 5.6 +/- 1.2 ng/ml, N.S. These results confirm the previous findings on increased circulating levels of beta-E in PCOD. A concomitant increase of the plasma level of corticotropin suggests that the basal secretion of both beta-E and corticotropin from the anterior pituitary gland is increased in women with PCOD.

Adult Human Pancreatic Islet Beta-Cells Display Limited Turnover and Long Lifespan as Determined by In-Vivo Thymidine Analog Incorporation and Radiocarbon Dating

Energy Technology Data Exchange (ETDEWEB)

Perl, S; Kushner, J A; Buchholz, B A; Meeker, A K; Stein, G M; Hsieh, M; Kirby, M; Pechhold, S; Liu, E H; Harlan, D M; Tisdale, J F

2010-03-15

Diabetes mellitus results from an absolute or relative deficiency of insulin producing pancreatic beta-cells. The adult human beta-cell's turnover rate remains unknown. We employed novel techniques to examine adult human islet beta-cell turnover and longevity in vivo. Subjects enrolled in NIH clinical trials received thymidine analogues [iododeoxyuridine (IdU) or bromodeoxyuridine (BrdU)] 8-days to 4-years prior to death. Archival autopsy samples from ten patients (aged 17-74 years) were employed to assess beta-cell turnover by scoring nuclear analog labeling within insulin staining cells. Human adult beta-cell longevity was determined by estimating the cells genomic DNA integration of atmospheric carbon-14 ({sup 14}C). DNA was purified from pancreatic islets isolated from cadaveric donors; whole islet prep DNA was obtained from a 15 year old donor, and purified beta-cell DNA was obtained from two donors (age 48 and 80 years). {sup 14}C levels were then determined using accelerator mass spectrometry (AMS). Cellular 'birth date' was determined by comparing the subject's DNA {sup 14}C content relative to a well-established {sup 14}C atmospheric prevalence curve. In the two subjects less than age 20 years, 1-2% of the beta-cell nuclei co-stained for BrdU/IdU. No beta-cell nuclei co-stained in the eight patients more than 30 years old. Consistent with the BrdU/IdU turnover data, beta-cell DNA {sup 14}C content indicated the cells 'birth date' occurred within the subject's first 30 years of life. Under typical circumstances, adult human beta-cells and their cellular precursors are established by young adulthood.

Effect of high and low molecular weight glutenin subunits, and subunits of gliadin on physicochemical parameters of different wheat genotypes

Directory of Open Access Journals (Sweden)

Mariana Souza Costa

2013-02-01

Full Text Available Identification of functional properties of wheat flour by specific tests allows genotypes with appropriate characteristics to be selected for specific industrial uses. The objective of wheat breeding programs is to improve the quality of germplasm bank in order to be able to develop wheat with suitable gluten strength and extensibility for bread making. The aim of this study was to evaluate 16 wheat genotypes by correlating both glutenin subunits of high and low molecular weight and gliadin subunits with the physicochemical characteristics of the grain. Protein content, sedimentation volume, sedimentation index, and falling number values were analyzed after the grains were milled. Hectoliter weight and mass of 1000 seeds were also determined. The glutenin and gliadin subunits were separated using polyacrylamide gel in the presence of sodium dodecyl sulfate. The data were evaluated using variance analysis, Pearson's correlation, principal component analysis, and cluster analysis. The IPR 85, IPR Catuara TM, T 091015, and T 091069 genotypes stood out from the others, which indicate their possibly superior grain quality with higher sedimentation volume, higher sedimentation index, and higher mass of 1000 seeds; these genotypes possessed the subunits 1 (Glu-A1, 5 + 10 (Glu-D1, c (Glu-A3, and b (Glu-B3, with exception of T 091069 genotype that possessed the g allele instead of b in the Glu-B3.

E. s. r. of free radicals in irradiated uracil-. beta. -D-arabinofuranoside

Energy Technology Data Exchange (ETDEWEB)

Bergene, R [Oslo Univ. (Norway). Fysisk Institutt; Vaughan, R A

1976-02-01

Electron-spin-resonance measurements have been made on single crystals of uracil-..beta..-D-arabinofuranoside, which were irradiated by 4.0 MeV electrons at 77 K. At low temperatures, two radicals have been identified, one attributed to a hydrogen abstraction of 05' in the sugar moiety and the other to a radical anion located on the pyrimidine ring. The former was very unstable and seemed to act as a precursor to other unidentified radical species stable at 77 K. At room temperature, the main resonance was due to hydrogen addition to C5 and was probably produced by protonation of the anion. This same radical was also produced by irradiation at room temperature.

Influvac, a trivalent inactivated subunit influenza vaccine.

Science.gov (United States)

Zuccotti, Gian Vincenzo; Fabiano, Valentina

2011-01-01

Influenza represents a major sanitary and socio-economic burden and vaccination is universally considered the most effective strategy for preventing the disease and its complications. Traditional influenza vaccines have been on the market since the late 1940s, with million of doses administered annually worldwide, and demonstrated a substantial efficacy and safety. The trivalent inactivated subunit vaccine has been available for more than 25 years and has been studied in healthy children, adults and the elderly and in people affected by underlying chronic medical conditions. We describe vaccine technology focusing on subunit vaccine production procedures and mode of action and provide updated information on efficacy and safety available data. A review of efficacy and safety data in healthy subjects and in high risk populations from major sponsor- and investigator-driven studies. The vaccine showed a good immunogenicity and a favorable safety profile in all target groups. In the panorama of actually available influenza vaccines, trivalent inactivated subunit vaccine represents a well-established tool for preventing flu and the associated complications.

Concomitant alpha7 and beta2 nicotinic AChR subunit deficiency leads to impaired energy homeostasis and increased physical activity in mice.

Science.gov (United States)

Somm, Emmanuel; Guérardel, Audrey; Maouche, Kamel; Toulotte, Audrey; Veyrat-Durebex, Christelle; Rohner-Jeanrenaud, Françoise; Maskos, Uwe; Hüppi, Petra S; Schwitzgebel, Valérie M

2014-05-01

Nicotinic acetylcholine receptors (nAChRs) are pentameric ligand-gated cation channels well characterized in neuronal signal transmission. Moreover, recent studies have revealed nAChR expression in nonneuronal cell types throughout the body, including tissues involved in metabolism. In the present study, we screen gene expression of nAChR subunits in pancreatic islets and adipose tissues. Mice pancreatic islets present predominant expression of α7 and β2 nAChR subunits but at a lower level than in central structures. Characterization of glucose and energy homeostasis in α7β2nAChR(-/-) mice revealed no major defect in insulin secretion and sensitivity but decreased glycemia apparently unrelated to gluconeogenesis or glycogenolysis. α7β2nAChR(-/-) mice presented an increase in lean and bone body mass and a decrease in fat storage with normal body weight. These observations were associated with elevated spontaneous physical activity in α7β2nAChR(-/-) mice, mainly due to elevation in fine vertical (rearing) activity while their horizontal (ambulatory) activity remained unchanged. In contrast to α7nAChR(-/-) mice presenting glucose intolerance and insulin resistance associated to excessive inflammation of adipose tissue, the present metabolic phenotyping of α7β2nAChR(-/-) mice revealed a metabolic improvement possibly linked to the increase in spontaneous physical activity related to central β2nAChR deficiency. Copyright © 2014 Elsevier Inc. All rights reserved.

On Fuzzy {beta}-I-open sets and Fuzzy {beta}-I-continuous functions

Energy Technology Data Exchange (ETDEWEB)

Keskin, Aynur [Department of Mathematics, Faculty of Science and Arts, Selcuk University, Campus, 42075 Konya (Turkey)], E-mail: akeskin@selcuk.edu.tr

2009-11-15

In this paper, first of all we obtain some properties and characterizations of fuzzy {beta}-I-open sets. After that, we also define the notion of {beta}-I-closed sets and obtain some properties. Lastly, we introduce the notions of fuzzy {beta}-I-continuity with the help of fuzzy {beta}-I-open sets to obtain decomposition of fuzzy continuity.

Clusters of conserved beta cell marker genes for assessment of beta cell phenotype

DEFF Research Database (Denmark)

Martens, Geert A; Jiang, Lei; Hellemans, Karine H

2011-01-01

The aim of this study was to establish a gene expression blueprint of pancreatic beta cells conserved from rodents to humans and to evaluate its applicability to assess shifts in the beta cell differentiated state. Genome-wide mRNA expression profiles of isolated beta cells were compared to those...... of a large panel of other tissue and cell types, and transcripts with beta cell-abundant and -selective expression were identified. Iteration of this analysis in mouse, rat and human tissues generated a panel of conserved beta cell biomarkers. This panel was then used to compare isolated versus laser capture...... microdissected beta cells, monitor adaptations of the beta cell phenotype to fasting, and retrieve possible conserved transcriptional regulators....

The interrelationships of mathematical precursors in kindergarten.

Science.gov (United States)

Cirino, Paul T

2011-04-01

This study evaluated the interrelations among cognitive precursors across quantitative, linguistic, and spatial attention domains that have been implicated for math achievement in young children. The dimensionality of the quantity precursors was evaluated in 286 kindergarteners via latent variable techniques, and the contribution of precursors from each domain was established for small sums addition. Results showed a five-factor structure for the quantity precursors, with the major distinction being between nonsymbolic and symbolic tasks. The overall model demonstrated good fit and strong predictive power (R(2)=55%) for addition number combinations. Linguistic and spatial attention domains showed indirect relationships with outcomes, with their effects mediated by symbolic quantity measures. These results have implications for the measurement of mathematical precursors and yield promise for predicting future math performance. Copyright © 2010 Elsevier Inc. All rights reserved.

N-linked glycans are required on epithelial Na+ channel subunits for maturation and surface expression.

Science.gov (United States)

Kashlan, Ossama B; Kinlough, Carol L; Myerburg, Michael M; Shi, Shujie; Chen, Jingxin; Blobner, Brandon M; Buck, Teresa M; Brodsky, Jeffrey L; Hughey, Rebecca P; Kleyman, Thomas R

2018-03-01

Epithelial Na + channel (ENaC) subunits undergo N-linked glycosylation in the endoplasmic reticulum where they assemble into an αβγ complex. Six, 13, and 5 consensus sites (Asn-X-Ser/Thr) for N-glycosylation reside in the extracellular domains of the mouse α-, β-, and γ-subunits, respectively. Because the importance of ENaC N-linked glycans has not been fully addressed, we examined the effect of preventing N-glycosylation of specific subunits on channel function, expression, maturation, and folding. Heterologous expression in Xenopus oocytes or Fischer rat thyroid cells with αβγ-ENaC lacking N-linked glycans on a single subunit reduced ENaC activity as well as the inhibitory response to extracellular Na + . The lack of N-linked glycans on the β-subunit also precluded channel activation by trypsin. However, channel activation by shear stress was N-linked glycan independent, regardless of which subunit was modified. We also discovered that the lack of N-linked glycans on any one subunit reduced the total and surface levels of cognate subunits. The lack of N-linked glycans on the β-subunit had the largest effect on total levels, with the lack of N-linked glycans on the γ- and α-subunits having intermediate and modest effects, respectively. Finally, channels with wild-type β-subunits were more sensitive to limited trypsin proteolysis than channels lacking N-linked glycans on the β-subunit. Our results indicate that N-linked glycans on each subunit are required for proper folding, maturation, surface expression, and function of the channel.

Dopamine beta-hydroxylase deficiency

Directory of Open Access Journals (Sweden)

Senard Jean-Michel

2006-03-01

Full Text Available Abstract Dopamine beta-hydroxylase (DβH deficiency is a very rare form of primary autonomic failure characterized by a complete absence of noradrenaline and adrenaline in plasma together with increased dopamine plasma levels. The prevalence of DβH deficiency is unknown. Only a limited number of cases with this disease have been reported. DβH deficiency is mainly characterized by cardiovascular disorders and severe orthostatic hypotension. First symptoms often start during a complicated perinatal period with hypotension, muscle hypotonia, hypothermia and hypoglycemia. Children with DβH deficiency exhibit reduced ability to exercise because of blood pressure inadaptation with exertion and syncope. Symptoms usually worsen progressively during late adolescence and early adulthood with severe orthostatic hypotension, eyelid ptosis, nasal stuffiness and sexual disorders. Limitation in standing tolerance, limited ability to exercise and traumatic morbidity related to falls and syncope may represent later evolution. The syndrome is caused by heterogeneous molecular alterations of the DBH gene and is inherited in an autosomal recessive manner. Restoration of plasma noradrenaline to the normal range can be achieved by therapy with the synthetic precursor of noradrenaline, L-threo-dihydroxyphenylserine (DOPS. Oral administration of 100 to 500 mg DOPS, twice or three times daily, increases blood pressure and reverses the orthostatic intolerance.

Beta Emission and Bremsstrahlung

Energy Technology Data Exchange (ETDEWEB)

Karpius, Peter Joseph [Los Alamos National Lab. (LANL), Los Alamos, NM (United States)

2017-11-13

Bremsstrahlung is continuous radiation produced by beta particles decelerating in matter; different beta emitters have different endpoint energies; high-energy betas interacting with high-Z materials will more likely produce bremsstrahlung; depending on the data, sometimes all you can say is that a beta emitter is present.

Dynamic properties of motor proteins with two subunits

International Nuclear Information System (INIS)

Kolomeisky, Anatoly B; III, Hubert Phillips

2005-01-01

The dynamics of motor protein molecules consisting of two subunits is investigated using simple discrete stochastic models. Exact steady-state analytical expressions are obtained for velocities and dispersions for any number of intermediate states and conformations between the corresponding binding states of proteins. These models enable us to provide a detailed description and comparison of two different mechanisms of the motion of motor proteins along the linear tracks: the hand-over-hand mechanism, when the motion of subunits alternate; and the inchworm mechanism, when one subunit is always trailing another one. It is shown that the proteins in the hand-over-hand mechanism move faster and fluctuate more than the molecules in the inchworm mechanism. The effect of external forces on dynamic properties of motor proteins is also discussed. Finally, a quantitative method, based on experimental observations for single motor proteins, is proposed for distinguishing between two mechanisms of motion

Bridging the gap between protein carboxyl methylation and phospholipid methylation to understand glucose-stimulated insulin secretion from the pancreatic beta cell.

Science.gov (United States)

Kowluru, Anjaneyulu

2008-01-15