bluuebunny/crossref_metadata_2025 huggingface dataset:
Biosynthesis and intracellular post-translational processing of normal and mutant platelet glycoprotein GPIb-IX
Authors: ULSEMER Philippe , STRASSEL Catherine , BAAS Marie-Jeanne , SALAMERO Jean , CHASSEROT-GOLAZ Sylvette , CAZENAVE Jean-Pierre , de LA SALLE Corinne , LANZA François
Published: August 2015

The multisubunit leucine-rich glycoprotein (GP) Ib–IX–V complex mediates von Willebrand factor-dependent platelet adhesion at sites of blood-vessel injury. Molecular defects of this receptor are reported to cause the Bernard–Soulier haemorrhagic disorder. To gain insight into the mechanisms controlling expression of normal and defective receptors, we performed pulse–chase metabolic studies and detailed analysis of intracellular processing in GPIb-IX-transfected Chinese-hamster ovary cells. In the native complex, after early subunit association, sugars N-linked to the three subunits are trimmed and sialylated in the Golgi compartment and GPIbα undergoes extensive O-glycosylation. Surface biotinylation during chase demonstrated that only fully processed complexes reach the cell surface. Tunicamycin treatment revealed that early N-glycosylation is not required for O-glycosylation of GPIbα and surface expression of the complex. Biosynthetic studies were then performed on a Bernard–Soulier variant based on previous description of abnormal GPIbα size and decreased surface expression. The mutant complex associated normally, but displayed defective processing of its N-linked sugars and abnormal O-glycosylation of GPIbα. Confocal immunofluorescence microscopy revealed that the mutant complexes could reach the cell surface but also accumulated intracellularly, while use of compartment specific markers showed strong co-localization in the endoplasmic reticulum (ER) and ER-to-Golgi intermediate compartments (‘ERGIC’) and only slight labelling of the cis-Golgi. Blockade before the Golgi was confirmed by brefeldin A treatment, which restored O-glycosylation and processing of N-linked sugars. The present study has shown that transfer from the ER to the Golgi represents an important step for controlling post-translational processing and surface expression of normal GPIb-IX-V complex.
Concurrent overexpression of ornithine decarboxylase and spermidine/spermine N1-acetyltransferase further accelerates the catabolism of hepatic polyamines in transgenic mice
Authors: SUPPOLA Suvikki , HEIKKINEN Sami , PARKKINEN Jyrki J. , UUSI-OUKARI Mikko , KORHONEN Veli-Pekka , KEINÄNEN Tuomo , ALHONEN Leena , JÄNNE Juhani
Published: August 2015

We have generated a hybrid transgenic mouse line overexpressing both ornithine decarboxylase (ODC) and spermidine/spermine N1-acetyltransferase (SSAT) under the control of the mouse metallothionein (MT) I promoter. In comparison with singly transgenic animals overexpressing SSAT, the doubly transgenic mice unexpectedly displayed much more striking signs of activated polyamine catabolism, as exemplified by a massive putrescine accumulation and an extreme reduction of hepatic spermidine and spermine pools. Interestingly, the profound depletion of the higher polyamines in the hybrid animals occurred in the presence of strikingly high ODC activity and tremendous putrescine accumulation. Polyamine catabolism in the doubly transgenic mice could be enhanced further by administration of zinc or the polyamine analogue N1,N11-diethylnorspermine. In tracer experiments with [14C]spermidine we found that, in comparison with syngenic animals, both MT-ODC and MT-SSAT mice possessed an enhanced efflux mechanism for hepatic spermidine. In the MT-ODC animals this mechanism apparently operated in the absence of measurable SSAT activity. In the hybrid animals, spermidine efflux was stimulated further in comparison with the singly transgenic animals. In spite of a dramatic accumulation of putrescine and a profound reduction of the spermidine and spermine pools, only marginal changes were seen in the level of ODC antizyme. Even though the hybrid animals showed no liver or other organ-specific overt toxicity, except an early and permanent loss of hair, their life span was greatly reduced. These results can be understood from the perspective that catabolism is the overriding regulatory mechanism in the metabolism of the polyamines and that, even under conditions of severe depletion of spermidine and spermine, extremely high tissue pools of putrescine are not driven further to replenish the pools of the higher polyamines.
Glucose exerts a permissive effect on the regulation of the initiation factor 4E binding protein 4E-BP1
Authors: PATEL Jigna , WANG Xuemin , PROUD Christopher G.
Published: August 2015

The eukaryotic initiation factor 4E (eIF4E) binding protein (4E-BP1) interacts directly with eIF4E and prevents it from forming initiation factor (eIF4F) complexes required for the initiation of cap-dependent mRNA translation. Insulin and other agents induce the phosphorylation of 4E-BP1 at multiple sites, resulting in its release from eIF4E, and this involves signalling through the mammalian target of rapamycin (mTOR). Here we show that d-glucose promotes the ability of insulin to bring about the phosphorylation of 4E-BP1 and the formation of eIF4F complexes. This appears to involve facilitation of the phosphorylation of at least three phosphorylation sites on 4E-BP1, i.e. Thr-36, Thr-45 and Thr-69. Non-metabolizable glucose analogues cannot substitute for d-glucose, but other hexoses can. This suggests that a product of hexose metabolism mediates the permissive effect of glucose. The effect of glucose was concentration-dependent within the range 1–5mM. In contrast with the situation for 4E-BP1, glucose does not allow full activation of the 70kDa ribosomal protein S6 kinase (p70 S6k; another target of mTOR signalling) or phosphorylation, in vivo, of its substrate, ribosomal protein S6. Taken together with earlier data showing that amino acids regulate 4E-BP1 and p70 S6k, the present findings show that 4E-BP1 in particular is regulated in response to the availability of both amino acids and sugars.
Local and spatial factors determining HIV-1 protease substrate recognition
Authors: HAZEBROUCK Stephane , MACHTELINCKX-DELMAS Valerie , KUPIEC Jean-Jacques , SONIGO Pierre
Published: August 2015

Insertional mutagenesis of the Escherichia coli thymidylate synthase (TS) was used to address substrate recognition of HIV-1 protease in a well characterized structural context. By modifying the TS conformation while maintaining its enzymic activity, we investigated the influence of protein folding on protease–substrate recognition. A slight destabilization of the TS structure permitted the cleavage of a target site, which was resistant in the native TS. This result supports a dynamic interpretation of HIV-1 protease specificity. Exposure time of the potential cleavage site, which depends on the stability of the global conformation, must be compatible with the cleavage kinetics, which are determined by the local sequence. Cleavage specificity has been described as the consequence of cumulative interactions, globally favourable, between at least six amino acids around the cleavage site. To investigate influence of local sequence, we introduced insertions of variable lengths in two exposed loops of the TS. In both environments, insertion of only two amino acids could determine specific cleavage. We then inserted libraries of dipeptides naturally cleaved by the HIV-1 protease in order to assess the limitations of established classifications of substrates in different conformational contexts.
Targeting of GLUT6 (formerly GLUT9) and GLUT8 in rat adipose cells
Authors: LISINSKI Ivonne , SCHÜRMANN Annette , JOOST Hans-Georg , CUSHMAN Samuel W. , AL-HASANI Hadi
Published: August 2015

The subcellular targeting of the two recently cloned novel mammalian glucose transporters, GLUT6 {previously referred to as GLUT9 [Doege, Bocianski, Joost and Schürmann (2000) Biochem. J. 350, 771–776]} and GLUT8, was analysed by expression of haemagglutinin (HA)-epitope-tagged GLUTs in transiently transfected primary rat adipose cells. Similar to HA-GLUT4, both transporters, HA-GLUT6 and HA-GLUT8, were retained in intracellular compartments in non-stimulated cells. In contrast, mutation of the N-terminal dileucine motifs in both constructs led to constitutive expression of the proteins on the plasma membrane. Likewise, when endocytosis was blocked by co-expression of a dominant-negative mutant of the dynamin GTPase, wild-type HA-GLUT6 and HA-GLUT8 accumulated on the cell surface. However, in contrast with HA-GLUT4, no translocation of HA-GLUT6 and HA-GLUT8 to the plasma membrane was observed when the cells were stimulated with insulin, phorbol ester or hyperosmolarity. Thus GLUT6 and GLUT8 appear to recycle in a dynamin-dependent manner between internal membranes and the plasma membrane in rat adipose cells, but are unresponsive to stimuli that induce translocation of GLUT4.
Age-related changes in the sulphation of the chondroitin sulphate linkage region from human articular cartilage aggrecan
Authors: LAUDER Robert M. , HUCKERBY Thomas N. , BROWN Gavin M. , BAYLISS Michael T. , NIEDUSZYNSKI Ian A.
Published: August 2015

The chondroitin sulphate (CS) linkage regions have been isolated from human articular cartilage aggrecan (from 10- to 72-year-olds) by chondroitin ABC endolyase digestion and size-exclusion chromatography. Linkage region hexasaccharides have been characterized and their abundance estimated by high-pH anion-exchange chromatography. The basic structure for the CS linkage region oligosaccharides identified from human aggrecan is as follows: ΔUA(β1–3)GalNAc[0S/4S/6S](β1–4)GlcA(β1–3)Gal[0S/6S](β1–3)Gal(β1–4)Xyl, where ΔUA represents 4,5-unsaturated hexuronic acid, 4S and 6S represent an O-ester sulphate group on C-4 and C-6 respectively, and 0S represents zero sulphation. There are significant age-related changes in the abundance of the various N-acetylgalactosamine (GalNAc) sulphation forms identified, occurring up to approx. 20 years old. During the period from 10 to 20 years old the level of GalNAc 6-sulphation at the linkage region increases from approx. 43% to approx. 75%, while there is a corresponding reduction in unsulphated (approx. 30% to approx. 20%) and 4-sulphated (approx. 25% to approx. 6%) GalNAc residues. There is also an increase in the incidence of linkage region galactose 6-sulphation (approx. 2% to approx. 10%) which was only observed in linkage regions with GalNAc 6-sulphation. Beyond 20 years old there are few changes in the relative abundance of these GalNAc sulphation variants; however, there is a slight increase in the abundance of 6-sulphation between approx. 20 years old and approx. 40 years old and a slight decrease in its abundance beyond approx. 40 years old. Our data show that in the majority of chains from tissues of all ages the GalNAc residue closest to the linkage region is 6-sulphated, but the level of GalNAc 6-sulphation within the linkage region is lower than the average level observed within the repeat region.
All three splice variants of the human sarco/endoplasmic reticulum Ca2+-ATPase 3 gene are translated to proteins: a study of their co-expression in platelets and lymphoid cells
Authors: KOVÁCS Tünde , FELFÖLDI Ferenc , PAPP Béla , PÁSZTY Katalin , BREDOUX Raymonde , ENYEDI Ágnes , ENOUF Jocelyne
Published: August 2015

The molecular cloning of two previously unknown human sarco/endoplasmic reticulum Ca2+-ATPase 3 (SERCA3) 3′-end transcripts, 3b and 3c, has been recently published. Data were lacking, however, for the presence of these SERCA3 variants in different tissue or cell types at the protein level. Here we report the co-expression of three human SERCA3 protein isoforms in platelets and T lymphoid Jurkat cells. Isoform-specific polyclonal anti-peptide antibodies have been generated that recognize specifically the SERCA3a, 3b or 3c splice variants at their C-termini, and this has been confirmed by peptide-competition experiments as well. None of these antibodies cross-reacted with the housekeeping SERCA2b isoform co-expressed endogenously with SERCA3 proteins in non-muscle cells. Although all three SERCA3 isoforms could be detected in platelets, the 3a form was the most abundantly expressed species. Its size matched the apparent size of SERCA3a over-expressed in HEK-293 cells. Immunoprecipitation of the SERCA3 variants from platelet membranes using a PL/IM 430-affinity matrix provided evidence that the putative pan-anti-SERCA3 antibody, PL/IM 430, recognizes all SERCA3 protein isoforms. The epitope for the PL/IM 430 antibody could be localized in a 40kDa N-terminal tryptic fragment common to all three SERCA3 variants. Comparative Western-blot analysis showed that the expression level of the SERCA3a, 3b and 3c isoforms was more than 10 times lower in Jurkat cells than in platelets, whereas expression of the ubiquitous SERCA2b was nearly identical. This work highlights new Ca2+-transporting proteins of haematopoietic cells and provides specific antibodies for their detection.
Fatty acid and amino acid modulation of glucose cycling in isolated rat hepatocytes
Authors: GUSTAFSON Lori A. , NEEFT Mies , REIJNGOUD Dirk-Jan , KUIPERS Folkert , SAUERWEIN Hans P. , ROMIJN Johannes A. , HERLING Andreas W. , BURGER Hans-Joerg , MEIJER Alfred J.
Published: August 2015

We studied the influence of glucose/glucose 6-phosphate cycling on glycogen deposition from glucose in fasted-rat hepatocytes using S4048 and CP320626, specific inhibitors of glucose-6-phosphate translocase and glycogen phosphorylase respectively. The effect of amino acids and oleate was also examined. The following observations were made: (1) with glucose alone, net glycogen production was low. Inhibition of glucose-6-phosphate translocase increased intracellular glucose 6-phosphate (3-fold), glycogen accumulation (5-fold) without change in active (dephosphorylated) glycogen synthase (GSa) activity, and lactate production (4-fold). With both glucose 6-phosphate translocase and glycogen phosphorylase inhibited, glycogen deposition increased 8-fold and approached reported in vivo rates of glycogen deposition during the fasted → fed transition. Addition of a physiological mixture of amino acids in the presence of glucose increased glycogen accumulation (4-fold) through activation of GS and inhibition of glucose-6-phosphatase flux. Addition of oleate with glucose present decreased glycolytic flux and increased the flux through glucose 6-phosphatase with no change in glycogen deposition. With glucose 6-phosphate translocase inhibited by S4048, oleate increased intracellular glucose 6-phosphate (3-fold) and net glycogen production (1.5-fold), without a major change in GSa activity. It is concluded that glucose cycling in hepatocytes prevents the net accumulation of glycogen from glucose. Amino acids activate GS and inhibit flux through glucose-6-phosphatase, while oleate inhibits glycolysis and stimulates glucose-6-phosphatase flux. Variation in glucose 6-phosphate does not always result in activity changes of GSa. Activation of glucose 6-phosphatase flux by fatty acids may contribute to the increased hepatic glucose production as seen in Type 2 diabetes.
Human mucin gene MUC5AC: organization of its 5′-region and central repetitive region
Authors: ESCANDE Fabienne , AUBERT Jean-Pierre , PORCHET Nicole , BUISINE Marie-Pierre
Published: August 2015

Human mucin gene MUC5AC is clustered with MUC2, MUC5B and MUC6 on chromosome 11p15.5. We report here the full length cDNA sequence upstream of the repetitive region of human MUC5AC. We have also determined the sequence of its large central tandem repeat array. The 5′-region reveals high degree of sequence similarity with MUC2 and MUC5B and codes for 1336 amino acids organized into a signal peptide, four pro-von Willebrand factor-like D domains (D1, D2, D′ and D3) and a short domain which connects to the central repetitive region. In the central region, 17 major domains have been identified. Nine code for cysteine-rich domains (Cys-domains 1–9) and exhibit high sequence similarity to the cysteine-rich domains described in the central region of MUC2 and MUC5B. Cys-domains 1–5 are interspersed by domains enriched with serine, threonine, and proline residues. Cys-domains 1–9 are interspersed by four domains (TR1–TR4) composed of various numbers of MUC5AC-type repeats. Southern-blot analyses reveal allelic variations both in length and nucleotide sequence. The length polymorphism which is due to variable numbers of tandem repeats is located in TR1 and TR4, whereas a mutation polymorphism detected with TaqI is located in Cys-domain 6. In this study, the organization of MUC5AC has been entirely elucidated showing extensive similarity to the other chromosome 11p15 MUC genes, particularly MUC5B, and providing additional arguments for common evolution from a single ancestral gene.
GSK3 takes centre stage more than 20 years after its discovery
Authors: FRAME Sheelagh , COHEN Philip
Published: August 2015

Identified originally as a regulator of glycogen metabolism, glycogen synthase kinase-3 (GSK3) is now a well-established component of the Wnt signalling pathway, which is essential for setting up the entire body pattern during embryonic development. It may also play important roles in protein synthesis, cell proliferation, cell differentiation, microtubule dynamics and cell motility by phosphorylating initiation factors, components of the cell-division cycle, transcription factors and proteins involved in microtubule function and cell adhesion. Generation of the mouse knockout of GSK3β, as well as studies in neurons, also suggest an important role in apoptosis. The substrate specificity of GSK3 is unusual in that efficient phosphorylation of many of its substrates requires the presence of another phosphorylated residue optimally located four amino acids C-terminal to the site of GSK3 phosphorylation. Recent experiments, including the elucidation of its three-dimensional structure, have enhanced our understanding of the molecular basis for the unique substrate specificity of GSK3. Insulin and growth factors inhibit GSK3 by triggering its phosphorylation, turning the N-terminus into a pseudosubstrate inhibitor that competes for binding with the ‘priming phosphate’ of substrates. In contrast, Wnt proteins inhibit GSK3 in a completely different way, by disrupting a multiprotein complex comprising GSK3 and its substrates in the Wnt signalling pathway, which do not appear to require a ‘priming phosphate’. These latest findings have generated an enormous amount of interest in the development of drugs that inhibit GSK3 and which may have therapeutic potential for the treatment of diabetes, stroke and Alzheimer's disease.
Characterization of unique amphipathic antimicrobial peptides from venom of the scorpion Pandinus imperator
Authors: CORZO Gerardo , ESCOUBAS Pierre , VILLEGAS Elba , BARNHAM Kevin J. , HE Weilan , NORTON Raymond S. , NAKAJIMA Terumi
Published: August 2015

Two novel antimicrobial peptides have been identified and characterized from venom of the African scorpion Pandinus imperator. The peptides, designated pandinin 1 and 2, are α-helical polycationic peptides, with pandinin 1 belonging to the group of antibacterial peptides previously described from scorpions, frogs and insects, and pandinin 2 to the group of short magainin-type helical peptides from frogs. Both peptides demonstrated high antimicrobial activity against a range of Gram-positive bacteria (2.4–5.2μM), but were less active against Gram-negative bacteria (2.4–38.2μM), and only pandinin 2 affected the yeast Candida albicans. Pandinin 2 also demonstrated strong haemolytic activity (11.1–44.5μM) against sheep erythrocytes, in contrast with pandinin 1, which was not haemolytic. CD studies and a high-resolution structure of pandinin 2 determined by NMR, showed that the two peptides are both essentially helical, but differ in their overall structure. Pandinin 2 is composed of a single α-helix with a predominantly hydrophobic N-terminal sequence, whereas pandinin 1 consists of two distinct α-helices separated by a coil region of higher flexibility. This is the first report of magainin-type polycationic antimicrobial peptides in scorpion venom. Their presence brings new insights into the mode of action of scorpion venom and also opens new avenues for the discovery of novel antibiotic molecules from arthropod venoms.
Chloride intracellular channel protein CLIC4 (p64H1) binds directly to brain dynamin I in a complex containing actin, tubulin and 14-3-3 isoforms
Authors: SUGINTA Wipa , KAROULIAS Nikolaos , AITKEN Alastair , ASHLEY Richard H.
Published: August 2015

Mammalian chloride intracellular channel (CLIC) (p64-related) proteins are widely expressed, with an unusual dual localization as both soluble and integral membrane proteins. The molecular basis for their cellular localization and ion channel activity remains unclear. To help in addressing these problems, we identified novel rat brain CLIC4 (p64H1) binding partners by affinity chromatography, mass spectrometric analysis and microsequencing. Brain CLIC4 binds dynamin I, α-tubulin, β-actin, creatine kinase and two 14-3-3 isoforms; the interactions are confirmed in vivo by immunoprecipitation. Gel overlay and reverse pull-down assays indicate that the binding of CLIC4 to dynamin I and 14-3-3∊ is direct. In HEK-293 cells, biochemical and immunofluorescence analyses show partial co-localization of recombinant CLIC4 with caveolin and with functional caveolae, which is consistent with a dynamin-associated role for CLIC4 in caveolar endocytosis. We speculate that brain CLIC4 might be involved in the dynamics of neuronal plasma membrane microdomains (micropatches) containing caveolin-like proteins and might also have other cellular roles related to membrane trafficking. Our results provide the basis for new hypotheses concerning novel ways in which CLIC proteins might be associated with cell membrane remodelling, the control of cell shape, and anion channel activity.
Role of connective tissue growth factor in the pathogenesis of diabetic nephropathy
Authors: WAHAB Nadia Abdel , YEVDOKIMOVA Natalia , WESTON Benjamin S. , ROBERTS Terry , LI Xin Jun , BRINKMAN Heike , MASON Roger M.
Published: August 2015

We characterized a rabbit polyclonal antibody raised against human recombinant connective tissue growth factor (CTGF). The antibody recognised a higher molecular mass form (approx. 56kDa) of CTGF in mesangial cell lysates as well as the monomeric (36–38kDa) and lower molecular mass forms (< 30kDa) reported previously. Immunohistochemistry detected CTGF protein in glomeruli of kidneys of non-obese diabetic mice 14 days after the onset of diabetes, and this was prominent by 70 days. CTGF protein is also present in glomeruli of human patients with diabetic nephropathy. No CTGF was detected in either normal murine or human glomeruli. Transient transfection of a transformed human mesangial cell line with a CTGF–V5 epitope fusion protein markedly increased fibronectin and plasminogen activator inhibitor-1 synthesis in cultures maintained in normal glucose (4mM) conditions; a CTGF-antisense construct reduced the elevated synthesis of these proteins in high glucose (30mM) cultures. Culture of primary human mesangial cells for 14 days in high glucose, or in low glucose supplemented with recombinant CTGF or transforming growth factor β1, markedly increased CTGF mRNA levels and fibronectin synthesis. However, whilst co-culture with a CTGF-antisense oligonucleotide reduced the CTGF mRNA pool by greater than 90% in high glucose, it only partially reduced fibronectin mRNA levels and synthesis. A chick anti-CTGF neutralizing antibody had a similar effect on fibronectin synthesis. Thus both CTGF and CTGF-independent pathways mediate increased fibronectin synthesis in high glucose. Nevertheless CTGF expression in diabetic kidneys is likely to be a key event in the development of glomerulosclerosis by affecting both matrix synthesis and, potentially through plasminogen activator inhibitor-1, its turnover.
Uptake and intracellular transport of the connective tissue growth factor: a potential mode of action
Authors: WAHAB Nadia Abdel , BRINKMAN Heike , MASON Roger M.
Published: August 2015

Connective tissue growth factor (CTGF) is a secreted cysteine-rich protein now considered as an important effector molecule in both physiological and pathological processes. An increasing amount of evidence indicates that CTGF plays a key role in the pathogenesis of different fibrotic disorders including diabetic nephropathy. However, the molecular mechanisms by which CTGF exerts its effects are not known. Here we provide the first evidence for the existence of an intracellular transport pathway for the growth factor in human mesangial cells. Our results demonstrate that CTGF is internalized from the cell surface in endosomes and accumulates in a juxtanuclear organelle from which the growth factor is then translocated into the cytosol. In the cytosol CTGF is phosphorylated by protein kinase C and PMA treatment can enhance this phosphorylation. Phosphorylated CTGF may have an important role in the cytosol, but it is also translocated into the nucleus where it may directly affect transcription.
Structure of the 5′ region of the Hst70 gene transcription unit: presence of an intron and multiple transcription initiation sites
Authors: ŚCIEGLIŃSKA Dorota , WID̄ŁAK Wiesława , KONOPKA Witold , POUTANEN Matti , RAHMAN Nafis , HUHTANIEMI Ilpo , KRAWCZYK Zdzisław
Published: August 2015

The rat Hst70 gene and its mouse counterpart Hsp70.2 belong to the family of Hsp70 heat shock genes and are specifically expressed in male germ cells. Previous studies regarding the structure of the 5′ region of the transcription unit of these genes as well as localization of the ‘cis’ elements conferring their testis-specific expression gave contradictory results [Widłak, Markkula, Krawczyk, Kananen and Huhtaniemi (1995) Biochim. Biophys. Acta 1264, 191–200; Dix, Rosario-Herrle, Gotoh, Mori, Goulding, Barret and Eddy (1996) Dev. Biol. 174, 310–321]. In the present paper we solve these controversies and show that the 5′ untranslated region (UTR) of the Hst70 gene contains an intron which is localized similar to that of the mouse Hsp70.2 gene. Reverse transcriptase-mediated PCR, Northern blotting and RNase protection analysis revealed that the transcription initiation of both genes starts at two main distant sites, and one of them is localized within the intron. As a result two populations of Hst70 gene transcripts with similar sizes but different 5′ UTR structures can be detected in total testicular RNA. Functional analysis of the Hst70 gene promoter in transgenic mice and transient transfection assays proved that the DNA fragment of approx. 360bp localized upstream of the ATG transcription start codon is the minimal promoter required for testis-specific expression of the HST70/chloramphenicol acetyltransferase transgene. These experiments also suggest that the expression of the gene may depend on ‘cis’ regulatory elements localized within exon 1 and the intron sequences.
A precursor form of vascular endothelial growth factor arises by initiation from an upstream in-frame CUG codon
Authors: TEE Meng Kian , JAFFE Robert B.
Published: August 2015

Vascular endothelial growth factor (VEGF) is a mitogen in physiological and pathological angiogenesis. Understanding the expression of different VEGF isoforms might be important for distinguishing angiogenesis in tissue development, vascular remodelling and tumour formation. We examined its expression and noted the presence of the isoforms VEGF121 and VEGF165 (121 and 165 residues long respectively) in fetal heart, lung, ovary, spleen, placenta and ovarian tumours. Unexpectedly, a 47kDa species predominated in fetal intestine and muscle. The presumed initiation site in VEGF is an AUG codon (AUG1039), 1039nt from its main transcriptional start site. AUG1039 is preceded in the 5′ untranslated region by an in-frame CUG at nt 499 (CUG499), which could produce the 47kDa form with a 180-residue N-terminal extension. We therefore assessed whether CUG499 functions as an initiator. CUG499 initiation produced the 47kDa VEGF165 precursor, which was processed at two sites to yield VEGF and three N-terminal fragments. When CTG499 was mutated to CGC, the precursor and N-terminal fragments were barely detectable. Although the precursor form was predominant in VEGF165, both CUG499 and AUG1039 forms were found in VEGF121. VEGF precursor induced neither the proliferation of human umbilical vein endothelial cells nor the expression of angiopoietin 2, which can be induced by, and act with, VEGF to induce tumour angiogenesis. The precursor also adheres to the extracellular matrix (ECM), suggesting that it might be a storage form for generating active VEGF in the cell or ECM. Alternate CUG499 and AUG1039 initiation and processing of the inactive precursor and its products might be important in regulating angiogenesis.
Lysophosphatidic acid promotes phorbol-ester-induced apoptosis in TF-1 cells by interfering with adhesion
Authors: LAI Jin-Mei , LU Ching-Yi , YANG-YEN Hsin-Fang , CHANG Zee-Fen
Published: August 2015

When exposed to PMA, the erythroblastic cell line TF-1 and its cytokine-independent variant D2 cells can be induced to undergo differentiation and apoptosis. In this study we investigated the mechanism responsible for the differential responses to PMA induction and show that serum present in the medium has a major role in promoting PMA-induced apoptosis in TF-1 and D2 cells. Interestingly, lysophosphatidic acid (LPA) could replace serum to co-operate with PMA in inducing apoptosis via the Rho-dependent pathway. The expression of a constitutively active form of RhoA also increased PMA-induced apoptosis. However, by inhibiting adhesion, most cells underwent PMA-induced apoptosis even in the absence of LPA or serum, indicating that adhesion is required for PMA-induced differentiation. Given that LPA could prevent adhesion for cells maintained in the serum-free medium, here we propose that RhoA has a switching role in determining whether TF-1 and D2 cells undergo differentiation or apoptosis in response to PMA, by modulating cell adhesion.
Metabolism of exogenous sn-1-alkyl-sn-2-lyso-glucosaminyl-phosphatidylinositol in HeLa D cells: accumulation of glucosaminyl(acyl)phosphatidylinositol in a metabolically inert compartment
Authors: WONGKAJORNSILP Adisak , SEVLEVER Daniel , ROSENBERRY Terrone L.
Published: August 2015

The somatic genetic defect in paroxysmal nocturnal haemoglobinuria (PNH) involves a block in the transfer of GlcNAc from UDP-GlcNAc to phosphatidylinositol (PI), the first step in the biosynthetic pathway for glycosylphosphatidylinositols (GPIs). We asked whether an exogenous lipid corresponding to an early intermediate in this pathway can be taken up by cells in culture and proceed through the GPI pathway. This approach could offer a strategy to bypass the block in PNH. To address this question we incubated HeLa D cells with sn-1-alkyl-sn-2-lyso-GlcN-[3H]PI (lyso-alkyl-GlcN-[3H]PI) for 24h and analysed the cellular lipids. We found three lipid products: unaltered lyso-alkyl-GlcN-[3H]PI, GlcN-[3H]PI and GlcN(acyl)[3H]PI (GlcN-PI with a fatty acid acyl group on inositol). Since the latter two lipids are intermediates in the GPI biosynthetic pathway, this observation demonstrates that an exogenous lipid can enter and proceed partially through this pathway. However, the conversion of GlcN(acyl)PI to downstream mannosylated GPI intermediates in the GPI pathway was inefficient, both for GlcN(acyl)PI produced from the exogenous lipid as well as from that obtained by metabolic labelling with [3H]inositol. We investigated this poor conversion by examining whether GlcN(acyl)PI, radioactively labelled sequentially by [14C]inositol and [3H]inositol, resided in one compartment and could be readily metabolized to downstream intermediates. Isotope ratios indicated that the turnover of GlcN(acyl)PI was slower than those of either downstream mannosylated GPIs or even GPI anchors on proteins, the final products of GPI pathway. This result is incompatible with the one-compartment model and indicates that GlcN(acyl)PI in HeLa D cells accumulates largely in a compartment that is inert to subsequent mannosylation.
Endothelin-1 induces phosphorylation of GATA-4 transcription factor in the HL-1 atrial-muscle cell line
Authors: KITTA Kazumi , CLÉMENT Sophie A. , REMEIKA Jane , BLUMBERG Jeffrey B. , SUZUKI Yuichiro J.
Published: August 2015

The transcription factor GATA-4 plays a central role in the regulation of cardiac-muscle gene transcription. The present study demonstrates that endothelin-1 (ET-1) induces GATA-4 activation and phosphorylation. The treatment of HL-1 adult mouse atrial-muscle cells with ET-1 (30nM) caused a rapid increase in the DNA binding activity of GATA-4 within 3min. The activation was associated with an upward mobility shift of the GATA-4 band on native PAGE in an electrophoretic- mobility-shift assay. The upward shift of the GATA-4 band also occurred on SDS/PAGE as monitored by immunoblotting. The in vitro treatment of nuclear extracts with λ-protein phosphatase abolished the upward shift, indicating that GATA-4 was phosphorylated. ET-1 activated the p44/42 mitogen-activated protein kinase (MAPK) and the MAPK kinase (MEK) within 3min, and PD98059 (a specific inhibitor of MEK) abolished the ET-1-induced GATA-4 phosphorylation. PMA also caused the rapid activation of MAPK and the phosphorylation of GATA-4. In contrast, the activation of MAPK by phenylephrine or H2O2 was weak and did not lead to GATA-4 phosphorylation. Thus ET-1 induces a GATA-4 phosphorylation by activating a MEK–MAPK pathway.
Mammalian class Sigma glutathione S-transferases: catalytic properties and tissue-specific expression of human and rat GSH-dependent prostaglandin D2 synthases
Authors: JOWSEY Ian R. , THOMSON Anne M. , FLANAGAN Jack U. , MURDOCK Paul R. , MOORE Gary B. T. , MEYER David J. , MURPHY Gregory J. , SMITH Stephen A. , HAYES John D.
Published: August 2015

GSH-dependent prostaglandin D2 synthase (PGDS) enzymes represent the only vertebrate members of class Sigma glutathione S-transferases (GSTs) identified to date. Complementary DNA clones encoding the orthologous human and rat GSH-dependent PGDS (hPGDS and rPGDS, respectively) have been expressed in Escherichia coli, and the recombinant proteins isolated by affinity chromatography. The purified enzymes were both shown to catalyse specifically the isomerization of prostaglandin (PG) H2 to PGD2. Each transferase also exhibited GSH-conjugating and GSH-peroxidase activities. The ability of hPGDS to catalyse the conjugation of aryl halides and isothiocyanates with GSH was found to be less than that of the rat enzyme. Whilst there is no difference between the enzymes with respect to their Km values for 1-chloro-2,4-dinitrobenzene, marked differences were found to exist with respect to their Km for GSH (8mM versus 0.3mM for hPGDS and rPGDS, respectively). Using molecular modelling techniques, amino acid substitutions have been identified in the N-terminal domain of these enzymes that lie outside the proposed GSH-binding site, which may explain these catalytic differences. The tissue-specific expression of PGDS also varies significantly between human and rat; amongst the tissues examined, variation in expression between the two species was most apparent in spleen and bone marrow. Differences in catalytic properties and tissue-specific expression of hPGDS and rPGDS appears to reflect distinct physiological roles for class Sigma GST between species. The evolution of divergent functions for the hPGDS and rPGDS is discussed in the context of the orthologous enzyme from chicken.
Structure and differential expression of the four members of the Arabidopsis thaliana ferritin gene family
Authors: PETIT Jean-Michel , BRIAT Jean-François , LOBRÉAUX Stéphane
Published: August 2015

Four ferritin genes are found within the complete sequence of the Arabidopsis thaliana genome. All of them are expressed and their corresponding cDNA species have been cloned. The polypeptide sequences deduced from these four genes confirm all the properties of the ferritin subunits described so far, non-exhaustively, from various plant species. All are predicted to be targeted to the plastids, which is consistent with the existence of a putative transit peptide at their N-terminal extremity. They also all possess a conserved extension peptide in the mature subunit. Specific residues for ferroxidase activity and iron nucleation, which are found respectively in H-type or L-type ferritin subunits in animals, are both conserved within each of the four A. thaliana ferritin polypeptides. In addition, the hydrophilic nature of the plant ferritin E-helix is conserved in the four A. thaliana ferritin subunits. Besides this strong structural conservation, the four genes are differentially expressed in response to various environmental signals, and during the course of plant growth and development. AtFer1 and AtFer3 are the two major genes expressed in response to treatment with an iron overload. Under our experimental conditions, AtFer4 is expressed with different kinetics and AtFer2 is not responsive to iron. H2O2 activates the expression of AtFer1 and, to a smaller extent, AtFer3. Abscisic acid promotes the expression of only AtFer2, which is consistent with the observation that this is the only gene of the four to be expressed in seeds, whereas AtFer1, AtFer4 and AtFer3 are expressed in various vegetative organs but not in seeds.
AtPIP5K1, an Arabidopsis thaliana phosphatidylinositol phosphate kinase, synthesizes PtdIns(3,4)P2 and PtdIns(4,5)P2 in vitro and is inhibited by phosphorylation
Authors: WESTERGREN Tomas , DOVE Stephen K. , SOMMARIN Marianne , PICAL Christophe
Published: August 2015

PtdIns phosphate kinases (PIPkins), which generate PtdInsP2 isomers, have been classified into three subfamilies that differ in their substrate specificities. We demonstrate here that the previously identified AtPIP5K1 gene from Arabidopsis thaliana encodes a PIPkin with dual substrate specificity in vitro, capable of phosphorylating PtdIns3P and PtdIns4P to PtdIns(3,4)P2 and PtdIns(4,5)P2 respectively. We also show that recombinant AtPIP5K1 is phosphorylated by protein kinase A and a soluble protein kinase from A. thaliana. Phosphorylation of AtPIP5K1 by protein kinase A is accompanied by a 40% inhibition of its catalytic activity. Full activity is recovered by treating phosphorylated AtPIP5K1 with alkaline phosphatase.
Characterization of the MN/CA 9 promoter proximal region: a role for specificity protein (SP) and activator protein 1 (AP1) factors
Authors: KALUZOVÁ Milota , PASTOREKOVÁ Silvia , SVASTOVÁ Eliska , PASTOREK Jaromír , STANBRIDGE Eric J. , KALUZ Stefan
Published: August 2015

MN/CA IX (MN) is a tumour-associated isoenzyme of the carbonic anhydrase family. Previous deletion analysis of the MN promoter established that protected regions (PRs) 1 and 2 are crucial for its transcriptional activity. Computer-assisted searching indicated putative binding sites for activator protein (AP) 2 and specificity protein (SP) 1 transcription factors, plus a CACCC box in PR1 and an AP1 site in PR2. PR1 produced four complexes in electrophoretic mobility-shift assay (EMSA) with HeLa nuclear extracts. Of these, three were completely competed with the SP1 and transforming growth factor-β retinoblastoma control-element CACCC box (RCE) probes, whereas the AP2 probe competed against the same three complexes partially. Supershift EMSA identified SP1 in the complex 1 and SP3 in the complexes 2 and 4. Point mutations in the SP1 site abrogated the PR1 function, while mutations affecting the overlapping CACCC box/AP2 site in PR1had minor effect on MN promoter activity. Block-replaced MN promoter mutants that had a consensus binding site (SP1 or AP2) or the RCE in place of PR1 demonstrated the stringent selectivity of the PR1 position as only the SP1 mutant reconstituted the MN promoter activity. The consensus SP1 probe generated the same SP1 and SP3 complexes as PR1 in EMSA; therefore we conclude that SP activity is both necessary and sufficient in the PR1 position. The critical role of AP1 in the PR2 position was confirmed by supershift of the PR2 complex with c-Fos antibody and markedly decreased activity of the construct with a mutated AP1 site. Detailed deletion analysis proved that PR1+PR2 account for 90% of the MN promoter activity, while neither PR1 nor PR2 on their own are sufficient for transactivation. Thus, synergistic co-operation between SP and AP1 factors bound to the adjacent PR1 and PR2, respectively, is necessary for MN transcriptional activity. The PR1+PR2 module also stimulated transcription from a heterologous promoter. The modulation of AP1 activity with PMA stimulated MN expression and activated the MN promoter, whereas inhibition of protein kinase C activity had no effect on MN expression in HeLa cells.
Roles of calcium ions in the membrane binding of C2 domains
Authors: STAHELIN Robert V. , CHO Wonhwa
Published: August 2015

The C2 domain is a membrane-targeting domain found in many cellular proteins involved in signal transduction or membrane trafficking. The majority of C2 domains co-ordinate multiple Ca2+ ions and bind the membrane in a Ca2+-dependent manner. To understand the mechanisms by which Ca2+ mediates the membrane binding of C2 domains, we measured the membrane binding of the C2 domains of group IV cytosolic phospholipase A2 (cPLA2) and protein kinase C-α (PKC-α) by surface plasmon resonance and lipid monolayer analyses. Ca2+ ions mainly slow the membrane dissociation of cPLA2-C2, while modulating both membrane association and dissociation rates for PKC-α-C2. Further studies with selected mutants showed that for cPLA2 a Ca2+ ion bound to the C2 domain of cPLA2 induces the intra-domain conformational change that leads to the membrane penetration of the C2 domain whereas the other Ca2+ is not directly involved in membrane binding. For PKC-α, a Ca2+ ion induces the inter-domain conformational changes of the protein and the membrane penetration of non-C2 residues. The other Ca2+ ion of PKC-α-C2 is involved in more complex interactions with the membrane, including both non-specific and specific electrostatic interactions. Together, these studies of isolated C2 domains and their parent proteins allow for the determination of the distinct and specific roles of each Ca2+ ion bound to different C2 domains.
Conserved tryptophan in cytochrome c: importance of the unique side-chain features of the indole moiety
Authors: BLACK Karen M. , CLARK-LEWIS Ian , WALLACE Carmichael J. A.
Published: August 2015

The absolute conservation of tryptophan at position 59 in cytochrome c is related to the unique chemical nature of its indole moiety. The indole side chain of Trp-59 possesses three salient features: bulk, hydrophobicity and the ability of its indole nitrogen to act as a hydrogen-bond donor. Crystallographic evidence identifies the indole nitrogen of Trp-59 as having a stabilizing hydrogen-bonding interaction with the buried carboxylate group of haem propionate 7. Side-chain bulk is also likely to be important because a Phe or Leu residue can replace Trp to give an at least partly functional protein, whereas the smaller Gly or Ser cannot. Semisynthetic analogues were designed to test the importance of the side-chain features of tryptophan by using a recently developed method for stereoselective fragment religation in yeast cytochrome c. Three yeast iso-1 cytochrome c analogues were produced in which Trp-59 was replaced by a non-coded amino acid: p-iodophenylalanine, β-(3-pyridyl)-alanine or β-(2-naphthyl)-alanine. Replacement of Trp-59 with these non-coded amino acids allows the reasons for its conservation to be analysed, because they vary with respect to size, hydrophobicity and hydrogen-bond potential. Our results show that decreasing the bulk and hydrophobicity of the side chain at position 59 has a profound but different impact on physicochemical and biological parameters from those of abolishing hydrogen-bond donor potential. This suggests that Trp-59 has both a local and a global stability effect by solvating a buried charge and by having a key role in the packing of the cytochrome c hydrophobic core.
The shorter zinc finger protein ZNF230 gene message is transcribed in fertile male testes and may be related to human spermatogenesis
Authors: ZHANG Sizhong , QIU Weimin , WU Hui , ZHANG Ge , HUANG Mingkong , XIAO Cuiying , YANG Jun , KAMP Christine , HUANG Xinli , HUELLEN Karin , YUE Ying , PAN Agen , LEBO Roger , MILUNSKY Aubrey , VOGT Peter H.
Published: August 2015

The zinc finger gene family represents one of the largest in the mammalian genome, with several of these genes reported to be involved in spermatogenesis. A newly discovered gene has been identified that is expressed abundantly in the testicular tissue of fertile men as determined by mRNA differential display. The gene encodes a C3HC4-type zinc finger protein motif (ring finger motif) consistent with a role in pre-meiotic or post-meiotic sperm development. The gene was named ZNF230 and mapped to the short arm of chromosome 11 (11p15). ZNF230 has two transcripts, of 1kb and 4.4kb in length. The shorter 1kb transcript was only detected in testicular tissue whereas the longer 4.4kb transcript was not detected in testis but was found in several other tissues. The lack of detectable ZNF230 expression in azoospermic patients by reverse transcriptase-mediated PCR analysis is interpreted to mean that this gene is involved in maintaining normal human male fertility.
Regulation of Ca2+-release-activated Ca2+ current (Icrac) by ryanodine receptors in inositol 1,4,5-trisphosphate-receptor-deficient DT40 cells
Authors: KISELYOV Kirill , SHIN Dong Min , SHCHEYNIKOV Nikolay , KUROSAKI Tomohiro , MUALLEM Shmuel
Published: August 2015

Persistence of capacitative Ca2+ influx in inositol 1,4,5-trisphosphate (IP3) receptor (IP3R)-deficient DT40 cells (DT40IP3R-/−) raises the question of whether gating of Ca2+-release activated Ca2+ current (Icrac) by conformational coupling to Ca2+-release channels is a general mechanism of gating of these channels. In the present work we examined the properties and mechanism of activation of Icrac Ca2+ current in wild-type and DT40IP3R-/− cells. In both cell types passive depletion of internal Ca2+ stores by infusion of EGTA activated a Ca2+ current with similar characteristics and time course. The current was highly Ca2+-selective and showed strong inward rectification, all typical of Icrac. The activator of ryanodine receptor (RyR), cADP-ribose (cADPR), facilitated activation of Icrac, and the inhibitors of the RyRs, 8-N-cADPR, ryanodine and Ruthenium Red, all inhibited Icrac activation in DT40IP3R-/− cells, even after complete depletion of intracellular Ca2+ stores by ionomycin. Wild-type and DT40IP3R-/− cells express RyR isoforms 1 and 3. RyR levels were adapted in DT40IP3R-/− cells to a lower RyR3/RyR1 ratio than in wild-type cells. These results suggest that IP3Rs and RyRs can efficiently gate Icrac in DT40 cells and explain the persistence of Icrac gating by internal stores in the absence of IP3Rs.