bluuebunny/crossref_metadata_2025 huggingface dataset:
Proteins in unexpected locations.
Authors: Smalheiser N R
Published: August 2013

Members of all classes of proteins--cytoskeletal components, secreted growth factors, glycolytic enzymes, kinases, transcription factors, chaperones, transmembrane proteins, and extracellular matrix proteins--have been identified in cellular compartments other than their conventional sites of action. Some of these proteins are expressed as distinct compartment-specific isoforms, have novel mechanisms for intercompartmental translocation, have distinct endogenous biological actions within each compartment, and are regulated in a compartment-specific manner as a function of physiologic state. The possibility that many, if not most, proteins have distinct roles in more than one cellular compartment has implications for the evolution of cell organization and may be important for understanding pathological conditions such as Alzheimer's disease and cancer.
Syntaxin 4 in 3T3-L1 adipocytes: regulation by insulin and participation in insulin-dependent glucose transport.
Authors: Volchuk A , Wang Q , Ewart H S , Liu Z , He L , Bennett M K , Klip A
Published: August 2013

Syntaxins are thought to be membrane receptors that bind proteins of the synaptobrevin/vesicle-associated membrane protein (VAMP) family found on transport vesicles. Recently, we detected synaptobrevin II and cellubrevin on immunopurified vesicles containing the glucose transporter 4 (GLUT4) in insulin-responsive cells. In an effort to identify the plasma membrane receptors for these vesicles, we now examine the expression of syntaxins in the 3T3-L1 adipocyte cell line. Neither syntaxin 1A nor 1B was found, in keeping with the neuronal restriction of these isoforms. In contrast, syntaxins 2 and 4 were readily detectable. By subcellular fractionation and estimation of protein yields, 67% of syntaxin 4 was localized to the plasma membrane, 24% to the low-density microsomes, and 9% to the high-density microsomes. Interestingly, acute insulin treatment decreased the content of syntaxin 4 in low-density microsomes and caused a corresponding gain in the plasma membrane fraction, reminiscent of the recruitment of GLUT4 glucose transporters. In contrast, there was no change in the distribution of syntaxin 2, which was mostly associated in the plasma membrane. A fraction of the intracellular syntaxin 4 was recovered with immunopurified GLUT4-containing vesicles. Moreover, anti-syntaxin 4 antibodies introduced in permeabilized 3T3-L1 adipocytes significantly reduced the insulin-dependent stimulation of glucose transport, in contrast to the introduction of irrelevant immunoglobulin G, which was without consequence. We propose that either the plasma membrane and/or the vesicular syntaxin 4 are involved in docking and/or fusion of GLUT4 vesicles at the cell surface of 3T3-L1 adipocytes.
Activation of the osmo-sensitive chloride conductance involves P21rho and is accompanied by a transient reorganization of the F-actin cytoskeleton.
Authors: Tilly B C , Edixhoven M J , Tertoolen L G , Morii N , Saitoh Y , Narumiya S , de Jonge H R
Published: August 2013

Hypo-osmotic stimulation of human Intestine 407 cells rapidly activated compensatory CL- and K+ conductances that limited excessive cell swelling and, finally, restored the original cell volume. Osmotic cell swelling was accompanied by a rapid and transient reorganization of the F-actin cytoskeleton, affecting both stress fibers as well as apical ruffles. In addition, an increase in total cellular F-actin was observed. Pretreatment of the cells with recombinant Clostridium botulinum C3 exoenzyme, but not with mutant enzyme (C3-E173Q) devoid of ADP-ribosyltransferase activity, greatly reduced the activation of the osmo-sensitive anion efflux, suggesting a role for the ras-related GTPase p21rho. In contrast, introducing dominant negative N17-p21rac into the cells did not affect the volume-sensitive efflux. Cell swelling-induced reorganization of F-actin coincided with a transient, C3 exoenzyme-sensitive tyrosine phosphorylation of p125 focal adhesion kinase (p125FAK) as well as with an increase in phosphatidylinositol-3-kinase (PtdIns-3-kinase) activity. Pretreatment of the cells with wortmannin, a specific inhibitor of PtdIns-3-kinase, largely inhibited the volume-sensitive ion efflux. Taken together, our results indicate the involvement of a p21rho signaling cascade and actin filaments in the activation of volume-sensitive chloride channels.
An abundant nucleolar phosphoprotein is associated with ribosomal DNA in Tetrahymena macronuclei.
Authors: McGrath K E , Smothers J F , Dadd C A , Madireddi M T , Gorovsky M A , Allis C D
Published: August 2013

An abundant 52-kDa phosphoprotein was identified and characterized from macronuclei of the ciliated protozoan Tetrahymena thermophila. Immunoblot analyses combined with light and electron microscopic immunocytochemistry demonstrate that this polypeptide, termed Nopp52, is enriched in the nucleoli of transcriptionally active macronuclei and missing altogether from transcriptionally inert micronuclei. The cDNA sequence encoding Nopp52 predicts a polypeptide whose amino-terminal half consists of multiple acidic/serine-rich regions alternating with basic/proline-rich regions. Multiple serines located in these acidic stretches lie within casein kinase II consensus motifs, and Nopp52 is an excellent substrate for casein kinase II in vitro. The carboxyl-terminal half of Nopp52 contains two RNA recognition motifs and an extreme carboxyl-terminal domain rich in glycine, arginine, and phenylalanine, motifs common in many RNA processing proteins. A similar combination and order of motifs is found in vertebrate nucleolin and yeast NSR1, suggesting that Nopp52 is a member of a family of related nucleolar proteins. NSR1 and nucleolin have been implicated in transcriptional regulation of rDNA and rRNA processing. Consistent with a role in ribosomal gene metabolism, rDNA and Nopp52 colocalize in situ, as well as by cross-linking and immunoprecipitation experiments, demonstrating an association between Nopp52 and rDNA in vivo.
The nfkb1 promoter is controlled by proteins of the Ets family.
Authors: Lambert P F , Ludford-Menting M J , Deacon N J , Kola I , Doherty R R
Published: August 2013

The gene encoding NFKB1 is autoregulated, responding to NF-kappa B/Rel activation through NF-kappa B binding sites in its promoter, which also contains putative sites for Ets proteins. One of the Ets sites, which we refer to as EBS4, is located next to an NF-kappa B/Rel binding site, kB3, which is absolutely required for activity of the promoter in Jurkat T cells in response to activation by phorbol 12-myristate 13-acetate (PMA), PMA/ionomycin, or the Tax protein from human T cell leukemia virus type I. We show that EBS4 is, required for the full response of the nfkb1 promoter to PMA or PMA/ionomycin in Jurkat cells. EBS4 is bound by Ets-1, Elf-1, and other species. Overexpression of Ets-1 augments the response to PMA/ionomycin and this is reduced by mutation of EBS4. Elf-1 has less effect in conjunction with PMA/ionomycin, but by itself activates the promoter 12-fold. This activation is only partly affected by mutation of EBS4, and a mutant promoter that binds Ets-1, but not Elf-1, at the EBS4 site responds to PMA/ionomycin as efficiently as the wild-type. Ets proteins may be responsible for fine-tuning the activity of the nfkb1 gene in a cell-type-specific manner.
Bax- and Bak-induced cell death in the fission yeast Schizosaccharomyces pombe.
Authors: Jürgensmeier J M , Krajewski S , Armstrong R C , Wilson G M , Oltersdorf T , Fritz L C , Reed J C , Ottilie S
Published: August 2013

The effects of the expression of the human Bcl-2 family proteins Bax, Bak, Bcl-2, and Bcl-XL were examined in the fission yeast Schizosaccharomyces pombe and compared with Bax-induced cell death in mammalian cells. Expression of the proapoptotic proteins Bax and Bak conferred a lethal phenotype in this yeast, which was strongly suppressed by coexpression of the anti-apoptotic protein Bcl-XL. Bcl-2 also partially abrogated Bax-mediated cytotoxicity in S. pombe, whereas a mutant of Bcl-2 (Gly145Ala) that fails to heterodimerize with Bax or block apoptosis in mammalian cells was inactive. However, other features distinguished Bax- and Bak-induced death in S. pombe from animal cell apoptosis. Electron microscopic analysis of S. pombe cells dying in response to Bax or Bak expression demonstrated massive cytosolic vacuolization and multifocal nuclear chromatin condensation, thus distinguishing this form of cell death from the classical morphological features of apoptosis seen in animal cells. Unlike Bax-induced apoptosis in 293 cells that led to the induction of interleukin-1 beta-converting enzyme (ICE)/CED-3-like protease activity, Bax- and Bak-induced cell death in S. pombe was accompanied neither by internucleosomal DNA fragmentation nor by activation of proteases with specificities similar to the ICE/CED-3 family. In addition, the baculovirus protease inhibitor p35, which is a potent inhibitor of ICE/CED-3 family proteases and a blocker of apoptosis in animal cells, failed to prevent cell death induction by Bax or Bak in fission yeast, whereas p35 inhibited Bax-induced cell death in mammalian cells. Taken together, these findings suggest that Bcl-2 family proteins may retain an evolutionarily conserved ability to regulate cell survival and death but also indicate differences in the downstream events that are activated by overexpression of Bax or Bak in divergent cell types.
PF20 gene product contains WD repeats and localizes to the intermicrotubule bridges in Chlamydomonas flagella.
Authors: Smith E F , Lefebvre P A
Published: August 2013

The central pair of microtubules and their associated structures play a significant role in regulating flagellar motility. To begin a molecular analysis of these components, we generated central apparatus-defective mutants in Chlamydomonas reinhardtii using insertional mutagenesis. One paralyzed mutant recovered in our screen contains an allele of a previously identified mutation, pf20. Mutant cells have paralyzed flagella, and the entire central apparatus is missing in isolated axonemes. We have cloned the wild-type PF20 gene and confirmed its identity by rescuing the pf20 mutant phenotype upon transformation. Rescued transformants were wild type in motility and in axonemal ultrastructure. A cDNA clone containing a single, long open reading frame was obtained and sequenced. Database searches using the predicted 606-amino acid sequence of PF20 indicate that the protein contains five contiguous WD repeats. These repeats are found in a number of proteins with diverse cellular functions including beta-transducin and dynein intermediate chains. An antibody was raised against a fusion protein expressed from the cloned cDNA. Immunogold labeling of wild-type axonemes indicates that the PF20 protein is localized along the length of the C2 microtubule on the intermicrotubule bridges connecting the two central microtubules. We suggest that the PF20 gene product is a new member of the family of WD repeat proteins and is required for central microtubule assembly and/or stability and flagellar motility.
Serine phosphorylation site of the 46-kDa mannose 6-phosphate receptor is required for transport to the plasma membrane in Madin-Darby canine kidney and mouse fibroblast cells.
Authors: Breuer P , Körner C , Böker C , Herzog A , Pohlmann R , Braulke T
Published: August 2013

Up to 4% of the human 46-kDa mannose 6-phosphate receptor (MPR46) expressed in Madin-Darby canine kidney (MDCK) cells are localized at the cell surface. At steady state, the expression of MPR46 on the apical surface of filter-grown MDCK cells is about sixfold lower than on the basolateral surface. The cytoplasmic domain of the MPR46 is phosphorylated on serine 56 at low stoichiometry. By expressing mutant MPR46 we have shown that the MPR46 phosphorylation site is required for delivery to the plasma membrane. In addition, mutant MPR46 expressed in MPR-deficient mouse embryonic fibroblasts were not detected at the cell surface and their ability to sort newly synthesized cathepsin D was not altered. Since the loss of MPR46 phosphorylation correlates with the lack of cell surface expression, phosphorylation of serine 56 may either function as a direct plasma membrane targeting signal or inhibit MPR46 recycling from endosomes to Golgi, resulting in trafficking to the cell surface.
Phosphatidylinositol 3-kinase is not required for recycling of mannose 6-phosphate receptors from late endosomes to the trans-Golgi network.
Authors: Nakajima Y , Pfeffer S R
Published: August 2013

Mannose 6-phosphate receptors carry newly synthesized lysosomal hydrolases from the trans-Golgi network to endosomes, then return to the trans-Golgi network for another round of enzyme delivery. Wortmannin, an inhibitor of phosphatidylinositol 3-kinase, interferes with the delivery of newly synthesized lysosomal enzymes to lysosomes. We used two independent assays of mannose 6-phosphate receptor trafficking to determine the precise step that is blocked by wortmannin. Using an assay that monitors resialylation of desialylated cell surface 300-kDa mannose 6-phosphate receptors, we found that receptor endocytosis and transport to the trans-Golgi network were not inhibited by 2 microM wortmannin. In addition, this concentration of drug had no effect on the transport of the mannose 6-phosphate receptor from late endosomes to the trans-Golgi network using a system that reconstitutes this transport process in cell extracts. Under the same conditions, wortmannin significantly inhibited the generation of mature cathepsin D. In addition, the structurally unrelated phosphatidylinositol 3-kinase inhibitor, LY294002, was also without effect when added to in vitro endosome-trans-Golgi network transport reactions. These experiments demonstrate that the interruption in lysosomal enzyme targeting is most likely due to a wortmannin-sensitive process required for the export of these receptors from the trans-Golgi network, consistent with the established role of phosphatidylinositol 3-kinase in the equivalent transport process in Saccharomyces cerevisiae.
An N-terminal fragment of titin coupled to green fluorescent protein localizes to the Z-bands in living muscle cells: overexpression leads to myofibril disassembly.
Authors: Turnacioglu K K , Mittal B , Dabiri G A , Sanger J M , Sanger J W
Published: August 2013

Cultures of nonmuscle cells, skeletal myotubes, and cardiomyocytes were transfected with a fusion construct (Z1.1GFP) consisting of a 1.1-kb cDNA (Z1.1) fragment from the Z-band region of titin linked to the cDNA for green fluorescent protein (GFP). The Z1.1 cDNA encodes only 362 amino acids of the approximately 2000 amino acids that make up the Z-band region of titin; nevertheless, the Z1.1GFP fusion protein targets the alpha-actinin-rich Z-bands of contracting myofibrils in vivo. This fluorescent fusion protein also localizes in the nascent and premyofibrils at the edges of spreading cardiomyocytes. Similarly, in transfected nonmuscle cells, the Z1.1GFP fusion protein localizes to the alpha-actinin-containing dense bodies of the stress fibers in vivo. A dominant negative phenotype was also observed in living cells expressing high levels of this Z1.1GFP fusion protein, with myofibril disassembly occurring as titin-GFP fragments accumulated. These data indicate that the Z-band region of titin plays an important role in maintaining and organizing the structure of the myofibril. The Z1.1 cDNA was derived from a chicken cardiac lambda gt11 expression library, screened with a zeugmatin antibody. Recent work has suggested that zeugmatin is actually part of the N-terminal region of the 81-kb titin cDNA. A reverse transcriptase polymerase chain reaction using a primer from the distal end (5' end) of the Z1.1 zeugmatin cDNA and a primer from the nearest known proximal (3' end) chicken titin (also called connectin) cDNA resulted in a predicted 0.3-kb polymerase chain reaction product linking the two known chicken titin cDNAs to each other. The linking region had a 79% identity at the amino acid level to human cardiac titin. This result and a Southern blot analysis of chicken genomic DNA hybridized with Z1.1 add further support to our original suggestion that zeugmatin is a proteolytic fragment from the N-terminal region of titin.
Cytoplasmic tails of beta 1, beta 2, and beta 7 integrins differentially regulate LFA-1 function in K562 cells.
Authors: Lub M , van Vliet S J , Oomen S P , Pieters R A , Robinson M , Figdor C G , van Kooyk Y
Published: August 2013

The beta 2 integrin lymphocyte function-associated antigen 1 (LFA-1) mediates activation-dependent adhesion of lymphocytes. To investigate whether lymphocyte-specific elements are essential for LFA-1 function, we expressed LFA-1 in the erythroleukemic cell line K562, which expresses only the integrin very late antigen 5. We observed that LFA-1-expressing K562 cannot bind to intercellular adhesion molecule 1-coated surfaces when stimulated by phorbol 12-myristate 13-acetate (PMA), whereas the LFA-1-activating antibody KIM185 markedly enhanced adhesion. Because the endogenously expressed beta 1 integrin very late antigen 5 is readily activated by PMA, we investigated the role of the cytoplasmic domain of distinct beta subunits in regulating LFA-1 function. Transfection of chimeric LFA-1 receptors in K562 cells reveals that replacement of the beta 2 cytoplasmic tail with the beta 1 but not the beta 7 cytoplasmic tail completely restores PMA responsiveness of LFA-1, whereas a beta 2 cytoplasmic deletion mutant of LFA-1 is constitutively active. Both deletion of the beta 2 cytoplasmic tail or replacement by the beta 1 cytoplasmic tail alters the localization of LFA-1 into clusters, thereby regulating LFA-1 activation and LFA-1-mediated adhesion to intercellular adhesion molecule 1. These data demonstrate that distinct signaling routes activate beta 1 and beta 2 integrins through the beta-chain and hint at the involvement of lymphocyte-specific signal transduction elements in beta 2 and beta 7 integrin activation that are absent in the nonlymphocytic cell line K562.
Role of Dictyostelium racE in cytokinesis: mutational analysis and localization studies by use of green fluorescent protein.
Authors: Larochelle D A , Vithalani K K , De Lozanne A
Published: August 2013

The small GTPase racE is essential for cytokinesis in Dictyostelium but its precise role in cell division is not known. To determine the molecular mechanism of racE function, we undertook a mutational analysis of racE. The exogenous expression of either wild-type racE or a constitutively active V20racE mutant effectively rescues the cytokinesis deficiency of racE null cells. In contrast, a constitutively inactive N25racE mutant fails to rescue the cytokinesis deficiency. Thus, cytokinesis requires only the activation of racE by GTP and not the inactivation of racE by hydrolysis of GTP. To determine the spatial distribution of racE, we created a fusion protein with GFP at the amino terminus of racE. Remarkably, GFP-racE fusion protein was fully competent to rescue the phenotype of racE null cells and, therefore, must reside in the same location as native racE. We found that GFP-racE localized to the plasma membrane of the cell throughout the entire cell cycle. Furthermore, constitutively active and inactive GFP-racE fusion proteins also localized to the plasma membrane. We mapped the domain required for plasma membrane localization to the carboxyl-terminal 40 amino acids of racE. This domain, however, is not sufficient to confer racE function onto a closely related GTPase. Taken together, these results suggest that racE functions at the cell cortex but it is not involved in determining the timing or placement of the contractile ring.
The role of the Src homology domains in morphological transformation by v-src.
Authors: Tian M , Martin G S
Published: August 2013

The Src homology (SH2 and SH3) domains of v-Src are required for transformation of Rat-2 cells and for wild-type (morphr) transformation of chicken embryo fibroblasts (CEFs). We report herein that the N-terminal domains of v-Src, when expressed in trans, cannot complement the transformation defect of a deletion mutant lacking the "unique," SH3, and SH2 regions. However, the same regions of Src can promote transformation when translocated to the C terminus of v-Src, although the transformation of CEFs is somewhat slower. We conclude that the SH3 and SH2 domains must be present in cis to the catalytic domain to promote transformation but that transformation is not dependent on the precise intramolecular location of these domains. In CEFSs and in Rat-2 cells, the expression of wild-type v-Src results in tyrosine phosphorylation of proteins that bind to the v-Src SH3 and SH2 domains in vitro; mutations in the SH2 or SH3 and SH2 domains prevent the phosphorylation of these proteins. These findings are most consistent with models in which the SH3 and SH2 domains of v-Src directly or indirectly target the catalytic domain to substrates involved in transformation. However, the N-terminal domains of v-Src can promote tyrosine phosphorylation of certain proteins, in particular p130Cas, even when expressed in the absence of the catalytic domain, indicating that the N-terminal domains of v-Src have effects that are independent of the catalytic domain.
A novel a-factor-related peptide of Saccharomyces cerevisiae that exits the cell by a Ste6p-independent mechanism.
Authors: Chen P , Choi J D , Wang R , Cotter R J , Michaelis S
Published: August 2013

Many secreted signaling molecules are synthesized as precursors that undergo multiple maturation steps to generate their mature forms. The Saccharomyces cerevisiae mating pheromone a-factor is a C-terminally isoprenylated and carboxylmethylated dodecapeptide that is initially synthesized as a larger precursor containing 36 or 38 amino acids. We have previously shown that the maturation of a-factor occurs by an ordered biogenesis pathway involving 1) three C-terminal modification steps, 2) two N-terminal proteolytic processing events, and 3) a nonclassical export mechanism mediated by the ATP-binding-cassette (ABC) transporter Ste6p. In the present study, we demonstrate that an unexpected and abundant a-factor-related peptide (AFRP) exists in the culture fluid of MATa cells and that its biogenesis is integrally related to that of mature a-factor itself. We show by purification followed by mass spectrometry that AFRP corresponds to the C-terminal 7 amino acids (VFWDPAC) of mature a-factor (YIIKGVFWDPAC), including both the farnesyl- and carboxylmethylcysteine modifications. The formation and export of AFRP displays three striking features. First, we show that AFRP is produced intracellularly and that mutants (ste24 and axl1) that cannot produce mature a-factor due to an N-terminal processing defect are nevertheless normal for AFRP production. Thus, AFRP is not derived from mature a-factor but, instead, from the P1 form of the a-factor precursor. Second, fusion constructs with foreign amino acids substituted for authentic a-factor residues still yield AFRP-sized molecules; however, the composition of these corresponds to the altered residues instead of to AFRP residues. Thus, AFRP may be generated by a sequence-dependent but length-specific proteolytic activity. Third, a-factor and AFRP use distinct cellular machinery for their secretion. Whereas a-factor export is Ste6p-dependent, AFRP is secreted normally even in a ste6 deletion mutant. Thus, AFRP may exit the cell by another ATP-binding-cassette transporter, a different type of transporter altogether, or possibly by diffusion. Taken together, these studies indicate that the biogenesis of AFRP involves novel mechanisms and machinery, distinct from those used to generate mature a-factor. Because AFRP neither stimulates nor inhibits mating or a-factor halo activity, its function remains an intriguing question.
Evidence for a recycling role for Rab7 in regulating a late step in endocytosis and in retention of lysosomal enzymes in Dictyostelium discoideum.
Authors: Buczynski G , Bush J , Zhang L , Rodriguez-Paris J , Cardelli J
Published: August 2013

The mammalian small molecular weight GTPase Rab7 (Ypt7 in yeast) has been implicated in regulating membrane traffic at postinternalization steps along the endosomal pathway. A cDNA encoding a protein 85% identical at the amino acid level to mammalian Rab7 has been cloned from Dictyostelium discoideum. Subcellular fractionation and immunofluorescence microscopy indicated that Rab7 was enriched in lysosomes, postlysosomes, and maturing phagosomes. Cell lines were generated that overexposed Rab7 wild-type (WT), Rab7 Q67L (constitutively active form), and Rab7 T22N (dominant negative form) proteins. The Rab7 T22N cell line internalized fluid phase markers and latex beads (phagocytosis) at one-third the rate of control cells, whereas Rab7 WT and Rab7 Q67L cell lines were normal in uptake rates but exocytosed fluid phase faster than control cells. In contrast, fluid phase markers resided in acidic compartments for longer periods of time and were more slowly exocytosed from Rab7 T22N cells as compared with control cells. Light microscopy indicated that Rab7-expressing cell lines contained morphologically altered endosomal compartments. Compared with control cells, Rab7 WT- and Rab7 Q67L-expressing cells contained a reduced number of vesicles, the size of postlysosomes (> 2.5 microns) and an increased number of smaller vesicles, many of which were nonacidic; in control cells, > 90% of the smaller vesicles were acidic. In contrast, Rab7 T22N cells contained an increased proportion of large acidic vesicles relative to nonacidic vesicles. Radiolabel pulse-chase experiments indicated that all of the cell lines processed and targeted lysosomal alpha-mannosidase normally, indicating the lack of a significant role for Rab7 in the targeting pathway; however, retention of mature lysosomal hydrolases was affected in Rab7 WT and Rab7 T22N cell lines. Contrary to the results observed for the fluid phase efflux experiments, Rab7 T22N cells oversecreted alpha-mannosidase, whereas Rab7 WT cells retained this hydrolase as compared with control cells. These data support a model that Rab7 may regulate retrograde transport of lysosomal enzymes and the V-type H(+)-ATPase from postlysosomes to lysosomes coupled with the efficient release of fluid phase from cells.
The carboxyl-terminal valine residues of proTGF alpha are required for its efficient maturation and intracellular routing.
Authors: Briley G P , Hissong M A , Chiu M L , Lee D C
Published: August 2013

Soluble forms of transforming growth factor-alpha (TGF alpha) are derived by proteolytic processing of an integral membrane glycoprotein precursor (pro TGF alpha). Previous studies indicated that phorbol ester-induced cleavage of pro TGF alpha in CHO cells is dependent on the presence of a valine residue located at the carboxyl terminus of the precursor's cytoplasmic domain. We reassessed this requirement with epitope-tagged constructs introduced into transformed rat liver epithelial cells that normally express and process TGF alpha. We found that pro TGF alpha mutants lacking the terminal valine residues showed greatly reduced maturation to the fully glycosylated form. Additionally, they were present at substantially reduced levels on the cell surface and, instead, accumulated in the endoplasmic reticulum. Consistent with these results, enzyme-linked immunosorbent assay (ELISA) and Western blot analyses revealed little or no soluble TGF alpha in medium conditioned by cells expressing the mutant constructs. Finally, a truncated pro TGF alpha mutant lacking most of the cytoplasmic domain but retaining a carboxyl-terminal valine was processed and cleaved in a near-normal manner. These results, some of which were reproduced in CHO cells, indicate that the predominant effect of the carboxyl-terminal valines is to ensure normal maturation and routing of the precursor.
G alpha 3 regulates the cAMP signaling system in Dictyostelium.
Authors: Brandon M A , Podgorski G J
Published: August 2013

The Dictyostelium discoideum developmental program is initiated by starvation and its progress depends on G-protein-regulated transmembrane signaling. Disruption of the Dictyostelium G-protein alpha-subunit G alpha 3 (g alpha 3-) blocks development unless the mutant is starved in the presence of artificial cAMP pulses. The function of G alpha 3 was investigated by examining the expression of several components of the cAMP transmembrane signaling system in the g alpha 3- mutant. cAMP receptor 1 protein, cyclic nucleotide phosphodiesterase, phosphodiesterase inhibitor, and aggregation-stage adenylyl cyclase mRNA expression were absent or greatly reduced when cells were starved without exogenously applied pulses of cAMP. However, cAMP receptor 1 protein and aggregation-stage adenylyl cyclase mRNA expression were restored by starving the g alpha 3- cells in the presence of exogenous cAMP pulses. Adenylyl cyclase activity was also reduced in g alpha 3- cells starved without exogenous cAMP pulses compared with similarly treated wild-type cells but was elevated to a level twofold greater than wild-type cells in g alpha 3- cells starved in the presence of exogenous cAMP pulses. These results suggest that G alpha 3 is essential in early development because it controls the expression of components of the transmembrane signaling system.
The laminin-binding activity of the alpha 7 integrin receptor is defined by developmentally regulated splicing in the extracellular domain.
Authors: Ziober B L , Chen Y , Kramer R H
Published: August 2013

The expression pattern of the laminin-binding alpha 7 beta 1 integrin is developmentally regulated in skeletal, cardiac, and smooth muscle. The X1/X2 alternative splicing in the extracellular domain of alpha 7 is found in the variable region between conserved alpha-chain homology repeat domains III and IV, a site implicated in ligand binding. To assess differences in X1/X2 isoform activity, we generated MCF-7 cell lines transfected with alpha 7-X1/X2 cDNAs. Transfectants expressing the alpha 7-X2 variant adhered rapidly to laminin 1, whereas those expressing alpha 7-X1 failed to attach. That alpha 7-X1 exists in an inactive state was established in assays using an activating beta 1 antibody that induced X1-dependent cell adhesion and spreading. Furthermore, the activation of alpha 7-X1 was cell type specific, and when expressed in HT1080 cells, the integrin was converted into a fully functional receptor capable of promoting adhesion. Thus, the expression of the alpha 7-X1/X2 integrin is a novel mechanism that regulates receptor affinity states in a cell-specific context and may modulate integrin-dependent events during muscle development and repair.
An isoform of the Golgi t-SNARE, syntaxin 5, with an endoplasmic reticulum retrieval signal.
Authors: Hui N , Nakamura N , Sönnichsen B , Shima D T , Nilsson T , Warren G
Published: August 2013

The early Golgi t-SNARE (target-membrane-associated soluble-N-ethylmaleimide-sensitive factor attachment protein receptor) syntaxin 5 is thought to specify the docking site for both COPI and COPII coated vesicles originating from the endoplasmic reticulum (ER) and COPI vesicles on the retrograde pathway. We now show that there are two forms of syntaxin 5 that appear to be generated from the same mRNA by alternative initiation of translation. The short form (35 kDa) corresponds to the published sequence. The long form (42 kDa) has an N-terminal cytoplasmic extension containing a predicted type II ER retrieval signal. When grafted onto a reporter molecule, this signal localized the construct to the ER. Biochemical fractionation and immunofluorescence microscopy showed that there was less of the long form in the Golgi apparatus and more in peripheral punctate structures, some of which colocalized with markers of the intermediate compartment. The predicted absence of the long form in budding yeast points to a function unique to higher organisms.
Accounting for the Poor
Authors: Townsend Robert M.
Published: August 2013

Economists and other social scientists have long tried to understand the nature of poverty and how poor people make decisions. For example, T.W. Schultz, a Nobel Laureate, former professor of economics at the University of Chicago, and former president of the American Economic Association, spent his career working in development and agricultural economics. In his 1980 Nobel Prize acceptance speech, Schultz suggests that there is some accounting for the behavior of the poor in agriculture. “Farmers, the world over, in dealing with costs, returns, and risks are calculating economic agents. Within their small, individual, allocative domain they are fine‐tuning entrepreneurs, tuning so subtly that many experts fail to recognize how efficient they are” (Schultz 1980).
ROBNCA: robust network component analysis for recovering transcription factor activities
Authors: Noor Amina , Ahmad Aitzaz , Serpedin Erchin , Nounou Mohamed , Nounou Hazem
Published: August 2013

Motivation: Network component analysis (NCA) is an efficient method of reconstructing the transcription factor activity (TFA), which makes use of the gene expression data and prior information available about transcription factor (TF)–gene regulations. Most of the contemporary algorithms either exhibit the drawback of inconsistency and poor reliability, or suffer from prohibitive computational complexity. In addition, the existing algorithms do not possess the ability to counteract the presence of outliers in the microarray data. Hence, robust and computationally efficient algorithms are needed to enable practical applications.

Results: We propose ROBust Network Component Analysis (ROBNCA), a novel iterative algorithm that explicitly models the possible outliers in the microarray data. An attractive feature of the ROBNCA algorithm is the derivation of a closed form solution for estimating the connectivity matrix, which was not available in prior contributions. The ROBNCA algorithm is compared with FastNCA and the non-iterative NCA (NI-NCA). ROBNCA estimates the TF activity profiles as well as the TF–gene control strength matrix with a much higher degree of accuracy than FastNCA and NI-NCA, irrespective of varying noise, correlation and/or amount of outliers in case of synthetic data. The ROBNCA algorithm is also tested on Saccharomyces cerevisiae data and Escherichia coli data, and it is observed to outperform the existing algorithms. The run time of the ROBNCA algorithm is comparable with that of FastNCA, and is hundreds of times faster than NI-NCA.

Availability: The ROBNCA software is available at http://people.tamu.edu/∼amina/ROBNCA

Contact:  serpedin@ece.tamu.edu

Supplementary information:  Supplementary data are available at Bioinformatics online.
Single‐Kernel Near‐Infrared Analysis for Evaluating Wheat Samples for Fusarium Head Blight Resistance
Authors: Peiris Kamaranga H. S. , Dong Yanhong , Bockus William W. , Dowell Floyd E.
Published: August 2013

This report describes a method to estimate the bulk deoxynivalenol (DON) content of wheat grain samples with the single‐kernel DON levels estimated by a single‐kernel near‐infrared (SKNIR) system combined with single‐kernel weights. The described method estimated the bulk DON levels in 90% of 160 grain samples to within 6.7 ppm of DON when compared with the DON content determined with the gas chromatography–mass spectrometry method. The single‐kernel DON analysis showed that the DON content among DON‐containing kernels (DCKs) varied considerably. The analysis of the distribution of DON levels among all kernels and among the DCKs of grain samples is helpful for the in‐depth evaluation of the effect of varieties or fungicides on Fusarium head blight (FHB) reactions. The SKNIR DON analysis and estimation of the single‐kernel DON distribution patterns demonstrated in this study may be helpful for wheat breeders to evaluate the FHB resistance of varieties in relation to their resistance to the spread of the disease and resistance to DON accumulation.
Similarities and Differences in Physiological Responses to ‘Candidatus Liberibacter solanacearum’ Infection Among Different Potato Cultivars
Authors: Wallis C. M. , Rashed A. , Wallingford A. K. , Paetzold L. , Workneh F. , Rush C. M.
Published: August 2013

Zebra chip disease (ZC), putatively caused by the fastidious bacterium ‘Candidatus Liberibacter solanacearum’, is a threat to potato growers worldwide. However, little is known about biochemical shifts in different potato genotypes in response to ‘Ca. L. solanacearum’ infection. To address this, ‘Red La Soda’, ‘Russet Norkotah’, and ‘FL 1867’ potato were infected with ‘Ca. L. solanacearum’ 4, 3, 2, and 1 weeks before harvest to observe variability in cultivar responses to ‘Ca. L. solanacearum’ infection. ZC symptoms, ‘Ca. L. solanacearum’ titers, and tuber biochemistry were assessed. Red La Soda tubers exhibited greater symptoms when infected for 4 weeks than Russet Norkotah or FL 1867 tubers. ‘Ca. L. solanacearum’ titers did not vary among cultivars. Tuber levels of amino acids, carbohydrates, and phenolics varied among cultivars but no consistent trends were observed. Individual amino acids and phenolics were greater in FL 1867 than Red La Soda, whereas others were greater in Red La Soda or Russet Norkotah than FL 1867. Most amino acids, carbohydrates, and phenolics were positively associated with infection duration and symptoms regardless of cultivar. Associations between most of the evaluated compounds and ‘Ca. L. solanacearum’ titer were positive in Red La Soda. However, no associations between ‘Ca. L. solanacearum’ quantity and compounds were observed in FL 1867 and Russet Norkotah.
Lipid Rescue Reverses the Bupivacaine-induced Block of the Fast Na+ Current (INa) in Cardiomyocytes of the Rat Left Ventricle
Authors: Wagner Michael , Zausig York A. , Ruf Stefan , Rudakova Elena , Gruber Michael , Graf Bernhard M. , Volk Tilmann
Published: August 2013

Background:

Cardiovascular resuscitation upon intoxication with lipophilic ion channel–blocking agents has proven most difficult. Recently, favorable results have been reported when lipid rescue therapy is performed, i.e., the infusion of a triglyceride-rich lipid emulsion during resuscitation. However, the mechanism of action is poorly understood.

Methods:

The authors investigate the effects of a clinically used lipid emulsion (Lipovenös® MCT 20%; Fresenius Kabi AG, Bad Homburg, Germany) on the block of the fast Na+ current (INa) induced by the lipophilic local anesthetic bupivacaine in adult rat left ventricular myocytes by using the whole cell patch clamp technique.

Results:

Bupivacaine at 10 µm decreased INa by 54% (−19.3 ± 1.9 pApF−1
vs. −42.3 ± 4.3 pApF−1; n = 17; P < 0.001; VPip = −40 mV, 1 Hz). Addition of 10% lipid emulsion in the presence of bupivacaine produced a 37% increase in INa (−26.4 ± 2.8 pApF−1; n = 17; P < 0.001 vs. bupivacaine alone). To test whether these results could be explained by a reduction in the free bupivacaine concentration by the lipid (lipid-sink effect), the authors removed the lipid phase from the bupivacaine–lipid mixture by ultracentrifugation. Also, the resulting water phase led to an increase in INa (+19%; n = 17; P < 0.001 vs. bupivacaine), demonstrating that part of the bupivacaine had been removed during ultracentrifugation. The substantially less lipophilic mepivacaine (40 µm) reduced INa by 27% (n = 24; P < 0.001). The mepivacaine–lipid mixture caused a significant increase in INa (+17%; n = 24; P < 0.001). For mepivacaine, only a small lipid-sink effect could be demonstrated (+8%; n = 23; P < 0.01), reflecting its poor lipid solubility.

Conclusion:

The authors demonstrate lipid rescue on the single-cell level and provide evidence for a lipid-sink mechanism.
Efficacy of Pegylated Interferon α‐2b and Ribavirin in Chronic Hepatitis C Virus (Genotypes 1 and 4) Infection
Authors: Wiśniewska‐Ligier Malgorzata , Pawłowska Malgorzata , Pilarczyk Malgorzata , Halota Waldemar , Woźniakowska‐Gęsicka Teresa
Published: August 2013

Background and Objective:

The course of chronic hepatitis C in children is often mild or asymptomatic, but may lead to liver cirrhosis and neoplasm. The aim of our study was retrospective evaluation of treatment efficacy using pegylated interferon (IFN)‐α2b with ribavirin in children and adolescents with chronic hepatitis C, both treatment naïve and re‐treated.

Methods:

The study comprised 79 patients with chronic hepatitis C ages 8 to 18 years (43 patients re‐treated; 54 infected with genotype 1 hepatitis C virus and 25 with genotype 4), treated with pegylated IFN‐α2b (1.5 μg · kg−1 · week−1) plus ribavirin (15 mg · kg−1 · day−1) for 48 weeks. The primary endpoint was sustained virologic response (SVR).

Results:

Early viral response (EVR) was observed in 43.1% and end‐of‐treatment response in 47.9% of patients. In 44.3% of patients, SVR was achieved, which was maintained for at least the next 6 months. Patients not treated before significantly more frequently attained EVR, end‐of‐treatment response, and SVR (64%, 65.6%, and 63.9%, respectively) as compared with re‐treated patients (30%, 33.3%, and 27.9%, respectively). Among 28 patients who attained EVR, 23 achieved SVR. In 2 patients, despite lack of EVR, SVR was observed. There were numerous adverse effects. They were not so severe as to discontinue therapy.

Conclusions:

Combined therapy with pegylated IFN‐α2b and ribavirin in patients with chronic hepatitis C, infected with hepatitis C virus genotypes 1 and 4, was more effective in treatment‐naïve patients (63.9%) as compared with re‐therapy cases (27.9%). SVR was maintained for at least the next 6 months in all of the patients. The applied treatment has limited efficacy and evokes numerous adverse effects; thus, search for new methods of treatment is mandatory.
Effects of Surgical Correction of Neuromuscular Scoliosis on Gastric Myoelectrical Activity, Emptying, and Upper Gastrointestinal Symptoms
Authors: Jalanko Tuomas , Helenius Ilkka , Pakarinen Mikko , Puisto Ville , Salminen Päivi , Peltonen Jari , Rintala Risto , Koivusalo Antti
Published: August 2013

Objectives:

Gastrointestinal complications are frequent after surgical correction of neuromuscular scoliosis, but the effects of scoliosis surgery on gastric function and upper gastrointestinal symptoms over the long term are unknown.

Methods:

Thirty‐one children (16 spastic, 15 flaccid patients) who underwent surgical correction of neuromuscular scoliosis were included in a prospective follow‐up study. Median (range) age at surgery was 14.9 (5–20) years and follow‐up time 4.3 (2–8) years. Electrogastrography (n = 28), gastric emptying scintigraphy (n = 17), and structured upper gastrointestinal symptoms questionnaire (n = 26) were evaluated before and after surgery. The results were related to patients' clinical state, type and extent of corrective spinal surgery, and gastrointestinal complications.

Results:

The median main scoliosis curve of 81 degrees (51–129 degrees) was corrected to 25 degrees (1.0–85 degrees) after surgery. In electrogastrogram, power ratio increased from preoperative 1.4 (0.30–11) to postoperative 6.2 (1.2–26) in the spastic group (P = 0.008), whereas in the flaccid group, power ratio remained unchanged at 2.2 (0.1–17). Patients with prolonged postoperative paralytic ileus had the most substantial increase in gastric power ratio (P = 0.038). Correction of sagittal spinal balance correlated with increased postprandial normogastric activity after surgery (R2 = 0.459; P = 0.004). The gastric emptying results, upper gastrointestinal symptoms, and body mass index were not significantly altered after scoliosis surgery.

Conclusions:

Gastric myoelectrical power increased after surgical correction of spastic neuromuscular scoliosis and was associated with prolonged postoperative paralytic ileus. Correction of poor, stooped spinal balance improved gastric myoelectrical activity. The net effect of scoliosis surgery on gastric emptying, upper gastrointestinal symptoms, and clinical nutritional state was minimal.
Effects of Boric Acid and 2‐Aminoethoxydiphenyl Borate on Necrotizing Enterocolitis
Authors: Yazc Selçuk , Akşit Hasan , Korkut Oğuzhan , Sunay Bahar , Çelik Tanju
Published: August 2013

Objective:

The aim was to study the effects of boric acid (BA) and 2‐aminoethoxydiphenyl borate (2‐APB) on oxidative stress and inflammation in an experimental necrotizing enterocolitis (NEC) rat model.

Methods:

Experimental NEC was induced in 40 newborn Sprague‐Dawley rats by asphyxia and hypothermia applied in 3 consecutive days. Rats were subdivided into 4 subgroups as NEC, NEC + BA, NEC + 2‐APB, and controls. BA and 2‐APB were applied daily before the procedure. Serum total antioxidant status, superoxide dismutase (SOD), tumor necrosis factor (TNF)‐α, interleukin (IL)‐6, and erythrocyte glutathione (GSH) levels were measured. Pathological changes for NEC in intestinal architecture were evaluated by a grading system.

Results:

Pretreatment with BA and 2‐APB resulted in a decrease in NEC incidence. In all of the NEC groups, decreased serum levels of GSH and SOD were measured. Boron limited GSH consumption but had no effect on SOD levels. Total antioxidant status levels were not statistically different among groups. In our experimental NEC model, BA, but not 2‐APB, prevented the increase of TNF‐α. Pretreatment with BA and 2‐APB downregulated the activity levels of IL‐6 in NEC.

Conclusions:

In the experimental NEC model, BA and 2‐APB partly prevent NEC formation, modulate the oxidative stress parameters, bring a significant decrease in GSH consumption, and enhance the antioxidant defense mechanism, but have no effect on total antioxidant status. BA inhibits the hypoxia and hypothermia‐induced increase in both IL‐6 and TNF‐a, but 2‐APB only in IL‐6. Boron may be beneficial in preventing NEC.
Overexpression of LDOC1 in Human Biliary Epithelial Cells Inhibits Apoptosis Through NF‐κB Signaling
Authors: Song Zai , Dong Rui , Zhao Rui , Zheng Shan
Published: August 2013

Objectives:

Biliary atresia (BA) is a devastating pediatric cholestatic liver disease. Increasing evidence indicates that nuclear factor (NF)‐κB signaling plays a key role in the pathogenesis of BA. Leucine zipper downregulated in cancer 1 (LDOC1) may control the expression of NF‐κB. The aim of this study was to evaluate the relation between LDOC1 and inflammation/apoptosis mediated by NF‐κB in the human intrahepatic biliary epithelial cells (HIBECs).

Methods:

HIBECs were divided into 3 treatment groups: control, mock transfection group, and LDOC1 transfection. Immunofluorescence, reverse transcription polymerase chain reaction, Western blot, and flow cytometry analysis were used to investigate the effectiveness of LDOC1‐transfected HIBECs and the expression of NF‐κB. Apoptosis was detected by Hochest/ propidium iodide staining. Interleukin (IL)‐2 and tumor necrosis factor (TNF)‐α levels were evaluated by enzyme‐linked immunosorbent assay.

Results:

The expression of NF‐κB was higher in the LDOC1‐transfected group when compared with the control and mock‐transfected groups as evaluated by immunofluorescence, reverese transcription polymerase chain reaction, and Western blot analysis. The rate of apoptosis was significantly lower in the LDOC1‐transfected group when compared with the control and mock‐transfected groups. The levels of IL‐2 and TNF‐α were significantly higher in the LDOC1‐transfected group when compared with the control and mock‐transfected groups.

Conclusions:

Upregulation of LDOC1 in HIBEC increases the expression of NF‐κB, which may promote the activation of IL‐2 and TNF‐α secretion and inhibit cell apoptosis.