bluuebunny/crossref_metadata_2025 huggingface dataset:
The amino-terminal module of the C4b-binding protein α-chain is crucial for C4b binding and factor I-cofactor function
Authors: HÄRDIG Ylva , HILLARP Andreas , DAHLBÄCK Björn
Published: August 2015

C4b-binding protein (C4BP) regulates the classical pathway C3-convertase of the complement system. Human C4BP is composed of seven identical subunits (α-chains) and one unique one (β-chain). Both types of chains contain homologous repeats called complement control proteins (CCPs); the α-chain contains eight CCPs and the β-chain three. Each α-chain contains a binding site for C4b although the detailed localization of this binding site is not known. We have used three different chimeric proteins, originally designed to localize the protein S-binding site on C4BP, to demonstrate the importance of the amino-terminal part of the α-chain for the complement-regulatory functions of C4BP. These recombinant proteins were composed of C4BP α-chains with one, two or three of the amino-terminal CCPs replaced by corresponding CCPs from the C4BP β-chain. Furthermore, seven different monoclonal antibodies were raised against C4BP and characterized using the recombinant chimeric proteins. Whereas all three recombinant chimeras bind protein S with the same affinity as plasma-purified C4BP, none of them bound to C4b. Three of the antibodies, which were found to bind to α-chain CCP 1 and CCP 2, completely inhibited the binding of plasma-purified C4BP to immobilized C4b. In addition, two of these antibodies totally blocked the factor I-cofactor activity of C4BP in a C4b-degradation assay. The binding site for one of the monoclonal antibodies was also studied using electron microscopy where it was confirmed that this antibody bound to the amino-terminal tip of the α-chain. These results show that the amino-terminal CCP of the C4BP α-chain (CCP 1) is crucial for the C4b binding and factor I-cofactor activity.
Biosynthesis of a low-molecular-mass rat submandibular gland mucin glycoprotein in COS7 cells
Authors: NEHRKE Keith , TABAK Lawrence A.
Published: August 2015

We have examined the biosynthesis of a low-molecular-mass mucin from rat submandibular gland (RSMG) expressed recombinantly in COS7 tissue culture cells, focusing primarily on the addition of carbohydrate to the protein core of the mucin. We find evidence for N-linked glycosylation, but this modification is not required for secretion of the mucin. Similarly, although the recombinant RSMG mucin, like its native counterpart, contains large amounts of O-linked carbohydrate, chain extension beyond the initial O-linked GalNAc moiety is not required for secretion. We have identified partially glycosylated mucin by a combination of metabolic pulse–chase and lectin precipitations of the biosynthetic intermediates. Our results suggest that the addition of GalNAc to threonine and serine in the RSMG mucin does not occur simultaneously, as has been described for other O-glycosylated proteins.
Two different negative regulatory elements control the transcription of T-cell activation gene 3 in activated mast cells
Authors: OH Chad K. , NEURATH Markus , CHO Jeong-Je , SEMERE Tekli , METCALFE Dean D.
Published: August 2015

T-cell activation gene 3 (TCA3) encodes a β-chemokine that is transcriptionally regulated in mast cells; the gene has a functional NF-κB element at positions -194 to -185. The 5´-flanking region of this gene is also known to have a negative regulatory region between -2057 and -1342. To characterize the negative regulatory elements (NREs), this region was sequenced and then digested by HindIII enzyme into two fragments, NRE-1 (-2057 to -1493) and NRE-2 (-1492 to -1342). Both NRE-1 and NRE-2 in the 5´–3´ orientation inhibited chloramphenicol acetyltransferase (CAT)-protein synthesis by a TCA3–CAT construct transfected into mast cells that were then activated. Only NRE-1 inhibited CAT-protein synthesis in the 3´–5´ orientation. Further deletion of the 5´ region of NRE-1 partially abolished the inhibitory activity. Both NRE-1 and NRE-2 inhibited the activity of a CD20–CAT construct independent of cell activation. Electrophoretic mobility shift assays showed DNA–protein complex formation with subsequences (CCCCCATTCT) of NRE-1 (NRE-1a) and (CCATGA) of NRE-2 (NRE-2b). NRE-1a appears to be novel. NRE-2b is identical with a putative silencer motif in the αIIb integrin gene. Site-directed mutagenesis demonstrated that both NRE-1a and NRE-2b are important in the negative regulation of TCA3 promoter activity. In vivo ligation-mediated PCR footprinting of the NRE-2 region revealed protection between -1372 and -1354, which contains NRE-2b. The data thus demonstrate identity of a silencer motif, here termed NRE-2b, in both the αIIb integrin gene and the TCA3, and that this silencer region in mast cells is functional both in vivoand in vitro. Further, evidence is presented that the promoter for TCA3 contains a novel silencer motif, termed NRE-1a, characterized by a CT-rich sequence.
Phosphatidylinositol 3,5-bisphosphate defines a novel PI 3-kinase pathway in resting mouse fibroblasts
Authors: WHITEFORD Craig C. , BREARLEY Charles A. , ULUG Emin T.
Published: August 2015

PtdIns(3,5)P2 is identified as the product of an agonist-independent, wortmannin-sensitive pathway in resting mouse cells. Results are presented here to indicate that PtdIns(3,5)P2 is formed by phosphorylation of PtdIns3P at the D-5 position, and they suggest that relatively constant cellular levels of PtdIns3P and PtdIns(3,5)P2 are maintained by the concerted action of PtdIns3P 5-kinase and PtdIns(3,5)P2 5-phosphatase. These studies imply a novel mechanism for the action of PtdIns-specific phosphoinositide 3-hydroxykinases in mammalian cells.
Insulin regulation of mitogen-activated protein kinase kinase (MEK), mitogen-activated protein kinase and casein kinase in the cell nucleus: a possible role in the regulation of gene expression
Authors: KIM Sung-Jin , KAHN Ronald C.
Published: August 2015

After insulin receptor activation, many cytoplasmic enzymes, including mitogen-activated protein (MAP) kinase, MAP kinase kinase (MEK) and casein kinase II (CKII) are activated, but exactly how insulin signalling progresses to the nucleus remains poorly understood. In Chinese hamster ovary cells overexpressing human insulin receptors [CHO(Hirc)], MEK, CKII and the MAP kinases ERK I and ERK II can be detected by immunoblotting in the nucleus, as well as in the cytoplasm, in the unstimulated state. Nuclear localization of MAP kinase is also observed in 3T3-F442A adipocytes, NIH-3T3 cells and Fao hepatoma cells, whereas MEK is found in the nucleus only in Fao and CHO cells. Insulin treatment for 5–30 min induces a translocation of MEK from the cytoplasm to the nucleus, whereas the MAP kinases and CKII are not translocated into the nucleus in response to insulin during this period. However, nuclear MAP kinase and CKII activities increase by 2–3-fold within 1–10 min after stimulation with insulin. By using gel-shift assays, it has been shown that insulin also stimulates nuclear protein binding to an AP-1 site with kinetics similar to MEK translocation and MAP kinase and CKII activation. Treatment of the extracts in vitro with protein phosphatase 2A or treatment of the intact cells with 5,6-dichloro-1-β-d-ribofuranosylbenzimidazole, a cell-permeable inhibitor of CKII, almost completely blocks the insulin-induced DNA-binding activity, whereas incubation of cells with a MEK inhibitor produces only a slight decrease. These results suggest that insulin signalling results in the activation of serine kinases in the nucleus via two pathways: (1) insulin stimulates the nuclear translocation of some kinases, such as MEK, which might directly phosphorylate nuclear protein substrates or activate other nuclear kinases, and (2) insulin activates nuclear kinases without translocation. The latter is true of CKII, which seems to regulate the binding of nuclear proteins to the AP-1 site, possibly by phosphorylation of AP-1 transcription factors.
Mitogen-activated protein kinase translocates to the nucleus during ischaemia and is activated during reperfusion
Authors: MIZUKAMI Yoichi , YOSHIDA Ken-ichi
Published: August 2015

Growth factors and various cellular stresses are known to activate mitogen-activated protein (MAP) kinase, which plays a role in conveying signals from the cytosol to the nucleus. The phosphorylation of MAP kinase is thought to be a prerequisite for translocation. Here, we investigate the translocation and activation of MAP kinase during ischaemia and reperfusion in perfused rat heart. Ischaemia (0–40 min) induces the translocation of MAP kinase from the cytosol fraction to the nuclear fraction. Immunohistochemical observation shows that MAP kinase staining in the nucleus is enhanced after ischaemia for 40 min. Unexpectedly, tyrosine phosphorylation of MAP kinase is unchanged in the nuclear fraction during ischaemia, indicating that unphosphorylated MAP kinase translocates from the cytosol to the nucleus. During reperfusion (0–30 min), after ischaemia for 20 min, tyrosine phosphorylation of MAP kinase in the nuclear fraction is increased with a peak at 10 min of reperfusion. The activation is confirmed by MAP kinase activity with similar kinetics to the tyrosine phosphorylation. However, the amount of MAP kinase in the fraction is almost constant during reperfusion for 10 min. Although an upstream kinase for MAP kinase, MAP kinase/extracellular signal-regulated kinase kinase (MEK)-1, remains in the cytosol throughout ischaemia and reperfusion, MEK-2, another upstream kinase for MAP kinase, is constantly present in the nucleus as well as in the cytoplasm, based on analyses by fractionation and immunohistochemistry. Furthermore, MEK-2 activity in the nuclear fraction is rapidly increased during post-ischaemic reperfusion. These findings demonstrate that nuclear MAP kinase is activated by tyrosine phosphorylation during reperfusion, probably by MEK-2.
Transcriptional activation of human 12-lipoxygenase gene promoter is mediated through Sp1 consensus sites in A431 cells
Authors: LIU Yi-Wen , ARAKAWA Toshiya , YAMAMOTO Shozo , CHANG Wen-Chang
Published: August 2015

The functional 5′ flanking region of the human 12-lipoxygenase in epidermoid carcinoma A431 cells was characterized. By a primer extension method, the transcription initiation sites were mapped at -47 adenosine, -48 guanosine and -55 guanosine upstream of the ATG translation start codon. Transient transfection with a series of 5′ and 3′ deletion constructs showed that the 5′ flanking region spanning from -224 to -100 bp was important for the basal expression of 12-lipoxygenase gene. Gel mobility shift assays with antibodies of transcription factors showed that both Sp1 and Sp3 required highly GC-rich Sp1 sites within this region for binding. Disruption of two Sp1 recognition motifs residing at -158 to -150 bp and -123 to -114 bp by site-directed mutagenesis markedly reduced the basal 12-lipoxygenase promoter activity and abolished the retarded bands in a gel-shift assay, indicating that these two Sp1-binding sites were essential for gene expression. The same two Sp1-binding sites in this promoter region were also responsible for epidermal growth factor (EGF)-induced expression of 12-lipoxygenase gene. Moreover, EGF also induced the transcriptional activation of luciferase driven by SV40 early promoter, which contained rich Sp1-binding sites. Taken together, the results suggest that two specific Sp1 consensus sites are involved in the mediation of the basal promoter activity as well as EGF induction of the 12-lipoxygenase gene and that Sp1 and Sp3 transcription factors might have a role in their regulation.
von Willebrand factor binds to native collagen VI primarily via its A1 domain
Authors: HOYLAERTS Marc F. , YAMAMOTO Hiroshi , NUYTS Katarine , VREYS Ingrid , DECKMYN Hans , VERMYLEN Jos
Published: August 2015

Collagen VI is abundant in the arterial subendothelium. To investigate its mechanism of interaction with von Willebrand factor (vWF), collagen VI was isolated from human placenta and from the extracellular matrix of the human lung fibroblast cell line MRC-5. Purified vWF bound to non-digested collagen VI with moderately high affinity (EC50 ≈ 5 nM) and could be inhibited by the Hirudo medicinalis collagen inhibitor calin. The anti-(human vWF A1 domain) monoclonal antibody (AJvW-2), as well as aurin tricarboxylic acid (ATA), at concentrations that saturate the vWF A1 domain, also inhibited this binding. In contrast, the monoclonal anti-(human vWF A3 domain) antibody (82D6A3) inhibited vWF binding to collagens I, III and IV, but had no effect on vWF binding to collagen VI. Likewise, vWF binding to collagen VI was not inhibited by the recombinant vWF domain D4. Polyclonal anti-(collagen VI) antibodies, specifically neutralizing the binding of vWF to collagen VI, confirmed that in the intact endothelial cell extracellular matrix, collagen VI was accessible for interaction with vWF. This binding was only marginally affected by 82D6A3 but was dose-dependently inhibited by AJvW-2, ATA and the A1 domain analogue VCL (recombinant A1 domain of vWF), with IC50 values comparable to those found for the inhibition of vWF binding to isolated collagen VI. The weak interaction of isolated human platelets with collagen VI was mediated via the platelet collagen receptor (GPIa/IIa) and was competitively inhibited by vWF but not by VCL, suggesting that vWF and GPIa/IIa bind to neighbouring but distinct sites on collagen VI. We conclude that vWF binds to collagen VI primarily via its A1 domain, which distinguishes it from the vWF A3 domain-mediated binding to fibrillar collagens.
Functional studies in 3T3L1 cells support a role for SNARE proteins in insulin stimulation of GLUT4 translocation
Authors: MACAULAY S. Lance , HEWISH Dean R. , GOUGH Keith H. , STOICHEVSKA Violet , MACPHERSON Susan F. , JAGADISH Mittur , WARD Colin W.
Published: August 2015

Insulin stimulation of glucose transport in the major insulin-responsive tissues results predominantly from the translocation to the cell surface of a particular glucose transporter isoform, GLUT4, residing normally under basal conditions in intracellular vesicular structures. Recent studies have identified the presence of vesicle-associated membrane protein (VAMP) 2, a protein involved in vesicular trafficking in secretory cell types, in the vesicles of insulin-sensitive cells that contain GLUT4. The plasma membranes of insulin-responsive cells have also been shown to contain syntaxin 4 and the 25 kDa synaptosome-associated protein (SNAP-25), two proteins that form a complex with VAMP 2. The potential functional involvement of VAMP 2, SNAP-25 and syntaxin 4 in the trafficking of GLUT4 was assessed in the present study by determining the effect on GLUT4 translocation of microinjection of toxins that specifically cleave VAMPs or SNAP-25, or microinjection of specific peptides from VAMP 2 and syntaxin 4. Microinjection of tetanus toxin light chain or botulinum D toxin light chain resulted in an 80 and 61% inhibition respectively of insulin stimulation of GLUT4 translocation in 3T3L1 cells assessed using the plasma-membrane lawn assay. Botulinum A toxin light chain, which cleaves SNAP-25, was without effect. Microinjection of an N-terminal VAMP 2 peptide (residues 1–26) inhibited insulin stimulation of GLUT4 translocation by 54%. A syntaxin 4 peptide (residues 106–122) inhibited insulin stimulation of GLUT4 translocation by 40% whereas a syntaxin 1c peptide (residues 226–260) was without effect. These data taken together strongly suggest a role for VAMP 2 in GLUT4 trafficking and also for syntaxin 4. They further indicate that the isoforms of SNAP-25 isolated to date that are sensitive to cleavage by botulinum A toxin light chain do not appear to be involved in GLUT4 translocation.
Interaction between cAMP-dependent and insulin-dependent signal pathways in tyrosine phosphorylation in primary cultures of rat hepatocytes
Authors: ITO Yoshiaki , UCHIJIMA Yasunobu , ARIGA Miyako , SEKI Taiichiro , TAKENAKA Asako , HAKUNO Fumihiko , TAKAHASHI Shin-Ichiro , ARIGA Toyohiko , NOGUCHI Tadashi
Published: August 2015

The present studies were undertaken to determine whether the interaction between cAMP-dependent and insulin-dependent pathways in primary cultures of rat hepatocytes affects biological functions and tyrosine phosphorylation. Quiescent hepatocytes were pretreated with dibutyryl cAMP or cAMP-generating agents such as glucagon, and then treated or not with insulin. Preincubation for 6 h with dibutyryl cAMP or glucagon enhanced the effect of insulin on DNA synthesis, but not the effect of insulin on amino acid transport or glycogen and protein synthesis. Tyrosine phosphorylation of intracellular proteins was determined by immunoblot analysis using an anti-phosphotyrosine antibody. Maximum tyrosine phosphorylation of a 195 kDa protein, which may be a substrate of insulin receptor kinase, of 175–180 kDa proteins, including insulin receptor substrate (IRS)-1, and of 90–95 kDa proteins, including the insulin receptor β-subunit, was reached within 30 s of incubation with insulin. Pretreatment for about 3 h with dibutyryl cAMP or cAMP-generating agents clearly increased insulin-dependent tyrosine phosphorylation of the 195 kDa protein, but not IRS-1, IRS-2 or the insulin receptor β-subunit. Because dibutyryl cAMP and cAMP-generating agents did not increase insulin receptor number or its kinase activity, the effect of cAMP on this potentiation of tyrosine phosphorylation is assumed to be exerted at a step distal to insulin receptor kinase activation. The potentiation by cAMP pretreatment of insulin-stimulated tyrosine phosphorylation may in part be secondary to inhibition of phosphotyrosine phosphatase activity, because cAMP pretreatment blunted the effect of Na3VO4 on the net tyrosine phosphorylation of the 195 kDa protein as compared with cells pretreated with no additive. In summary, the interactions between cAMP-dependent and insulin-dependent pathways that lead to augmentation of DNA synthesis appear to parallel the changes in tyrosine phosphorylation. Further studies will be required to determine whether there is a causal relationship between these phenomena.
Expression of an exogenous isopentenyl diphosphate isomerase gene enhances isoprenoid biosynthesis in Escherichia coli
Authors: KAJIWARA Susumu , FRASER Paul D. , KONDO Keiji , MISAWA Norihiko
Published: August 2015

Escherichia coli expressing the Erwinia carotenoid biosynthesis genes, crtE, crtB, crtI and crtY, form yellow-coloured colonies due to the presence of β-carotene. This host was used as a visible marker for evaluating regulatory systems operating in isoprenoid biosynthesis of E. coli. cDNAs enhancing carotenoid levels were isolated from the yeast Phaffia rhodozyma and the green alga Haematococcus pluvialis. Nucleotide sequence analysis indicated that they coded for proteins similar to isopentenyl diphosphate (IPP) isomerase of the yeast Saccharomyces cerevisiae. Determination of enzymic activity confirmed the identity of the gene products as IPP isomerases. The corresponding gene was isolated from the genomic library of S. cerevisiae based on its nucleotide sequence, and was confirmed to have the same effect as the above two IPP isomerase genes when introduced into the E. coli transformant accumulating β-carotene. In the three E. coli strains carrying the individual exogenous IPP isomerase genes, the increases in carotenoid levels are comparable to the increases in IPP isomerase enzyme activity with reference to control strains possessing the endogenous gene alone. These results imply that IPP isomerase forms an influential step in isoprenoid biosynthesis of the prokaryote E. coli, with potential for the efficient production of industrially useful isoprenoids by metabolic engineering.
The vacuolar H+-ATPase: a universal proton pump of eukaryotes
Authors: FINBOW Malcolm E. , HARRISON Michael A.
Published: August 2015

The vacuolar H+-ATPase (V-ATPase) is a universal component of eukaryotic organisms. It is present in the membranes of many organelles, where its proton-pumping action creates the low intra-vacuolar pH found, for example, in lysosomes. In addition, there are a number of differentiated cell types that have V-ATPases on their surface that contribute to the physiological functions of these cells. The V-ATPase is a multi-subunit enzyme composed of a membrane sector and a cytosolic catalytic sector. It is related to the familiar FoF1 ATP synthase (F-ATPase), having the same basic architectural construction, and many of the subunits from the two display identity with one another. All the core subunits of the V-ATPase have now been identified and much is known about the assembly, regulation and pharmacology of the enzyme. Recent genetic analysis has shown the V-ATPase to be a vital component of higher eukaryotes. At least one of the subunits, i.e. subunit c (ductin), may have multifunctional roles in membrane transport, providing a possible pathway of communication between cells. The structure of the membrane sector is known in some detail, and it is possible to begin to suggest how proton pumping is coupled to ATP hydrolysis.
Interferon α2 recombinant and epidermal growth factor modulate proliferation and hypusine synthesis in human epidermoid cancer KB cells
Authors: CARAGLIA Michele , PASSEGGIO Amelia , BENINATI Simone , LEARDI Annalisa , NICOLINI Laura , IMPROTA Salvatore , PINTO Antonio , BIANCO Raffaele A. , TAGLIAFERRI Piersandro , ABBRUZZESE Alberto
Published: August 2015

We previously found that interferon α2 recombinant (IFNα) increases the expression of epidermal growth factor receptor (EGF-R) in the human epidermoid cancer KB cell line. Here we report the effects of IFNα and epidermal growth factor (EGF) on KB cell cycle kinetics. IFNα (1000 i.u./ml) for 48 h decreased the S-phase fraction and diminished the expression of Ki67 and proliferating cell nuclear antigen on KB cells. Incubation of IFNα-treated KB cells with 10 nM EGF for 12 h reversed these effects. We then studied several biochemical markers of cell proliferation. Ornithine decarboxylase activity was decreased to about one-tenth by IFNα and partly restored by EGF. Hypusine is contained only in eukaryotic initiation factor 5A and its levels are correlated with cell proliferation. IFNα decreased hypusine synthesis by 75%; exposure of cells to EGF for 12 h restored hypusine synthesis almost completely. We also studied the effects of IFNα on the cytotoxicity of the recombinant toxin TP40, which inhibits elongation factor 2 through EGF-R binding and internalization. IFNα greatly enhanced the TP40-induced inhibition of protein synthesis in KB cells. In conclusion, IFNα, which affects protein synthesis machinery and increases EGF-R expression, enhances the tumoricidal activity of TP40 and hence could be useful in the setting of anti-cancer therapy.
Validation and steady-state analysis of a power-law model of purine metabolism in man
Authors: CURTO Raul , VOIT Eberhard O. , SORRIBAS Albert , CASCANTE Marta
Published: August 2015

The paper introduces a model of human purine metabolism in situ. Chosen from among several alternative system descriptions, the model is formulated as a Generalized Mass Action system within Biochemical Systems Theory and validated with analyses of steady-state and dynamic characteristics. Eigenvalue and sensitivity analyses indicate that the model has a stable and robust steady-state. The model quite accurately reproduces numerous biochemical and clinical observations in healthy subjects as well as in patients with disorders of purine metabolism. These results suggest that the model can be used to assess biochemical and clinical aspects of human purine metabolism. It provides a means of exploring effects of enzyme deficiencies and is a potential tool for identifying steps of the pathway that could be the target of therapeutical intervention. Numerous quantitative comparisons with data are given. The model can be used for biomathematical exploration of relationships between enzymic deficiencies and clinically manifested diseases.
Mechanism of apoptotic cell death of human gastric carcinoma cells mediated by transforming growth factor β
Authors: OHTA Shigeki , YANAGIHARA Kazuyoshi , NAGATA Kiyoshi
Published: August 2015

Human gastric carcinoma cell line HSC-39 has been shown to undergo apoptotic cell death in response to treatment with transforming growth factor β1 (TGF-β1). To understand better the cell death mechanism in this TGF-β1-mediated apoptosis, we investigated the effect of the expression of TGF-β-stimulated clone 22 (TSC-22) on cell death events. TGF-β1 induced TSC-22 gene expression in HSC-39 cells only when the cells had previously been adapted to the serum-free culture conditions required to undergo TGF-β1-mediated apoptosis. HSC-39 cells transfected with a TSC-22 expression vector showed a significant decrease in cell viability compared with those transfected with a control vector. The cellular events characteristic of apoptosis, chromatin condensation and DNA fragmentation were observed only in cells transfected with a TSC-22 expression vector. On immunostaining of the transfected cells, almost every cell that expressed TSC-22 tagged with influenza virus haemagglutinin exhibited the morphology of an apoptotic cell. Partial protection from the cell death effect of TGF-β1 on HSC-39 cells was observed when cells were treated with acetyl-l-aspartyl-l-glutamyl-l-valyl-l-aspart-1-al (Ac-DEVD-CHO, an inhibitor specific for CPP32-type protease). Protection against cell death by the transfection of a TSC-22 expression vector was also offered by Ac-DEVD-CHO addition. These results suggest that TSC-22 elicits the apoptotic cell death of human gastric carcinoma cells through the activation of CPP32-like protease and mediates the TGF-β1 signalling pathway to apoptosis.
α-Difluoromethylornithine-resistant cell lines obtained after one-step selection of Leishmania mexicana promastigote cultures
Authors: SÁNCHEZ Cecilia P. , MUCCI Juan , GONZÁLEZ Nélida S. , OCHOA Alberto , ZAKIN Mario M. , ALGRANATI Israel D.
Published: August 2015

Proliferation of Leishmania mexicana promastigotes in synthetic medium can be blocked by the depletion of intracellular polyamine pools induced by the presence of d,l-α-difluoromethylornithine (DFMO), a specific and irreversible inhibitor of ornithine decarboxylase (ODC). Here we report that DFMO-resistant cell lines growing normally at DFMO levels of 10 mM have been obtained from non-proliferating cultures after a single-step selection in the presence of high concentrations of the drug. The DFMO-resistant promastigotes underwent a morphological transformation into an ‘amastigote-like’ form after incubation for several hours at gradually increasing temperatures up to 35 °C. The uptake of DFMO was not significantly altered in the drug-resistant cell lines but in both cases (promastigote and ‘amastigote-like’ forms) the ODC specific activity was increased approx. 15-fold over the normal enzymic levels found in the wild-type Leishmania. The enzyme affinities for its substrate and for DFMO gave very similar values in the drug-resistant promastigotes and the wild-type parasites. In contrast, ODC from the ‘amastigote-like’Leishmania showed a higher affinity for ornithine and a decreased capacity for the binding of DFMO. An 80-fold amplification of the ODC gene and a corresponding increase in its transcripts have been detected in both DFMO-resistant Leishmania cell lines. The drug-resistant phenotypes with their characteristic morphologies, the increased levels of ODC activity and the amplification of the ODC gene have been stable for at least 6 months in the absence of selective pressure.
Members of the nuclear factor 1 family and hepatocyte nuclear factor 4 bind to overlapping sequences of the L-II element on the rat pyruvate kinase L gene promoter and regulate its expression
Authors: YAMADA Kazuya , TANAKA Takashi , NOGUCHI Tamio
Published: August 2015

The L-II element (-149 to -126 bp) in the enhancer unit of the rat pyruvate kinase L (PKL) gene is required for cell-type-specific transcription and induction by carbohydrates. This element was found to bind multiple nuclear proteins with different heat stabilities. A heat-labile factor was shown to be hepatocyte nuclear factor (HNF) 4 by the electrophoretic mobility-shift assay (EMSA) using various competitor DNAs and anti-HNF4 serum. A heat-stable factor was purified from rat liver nuclear extract and was resolved as two protein bands migrating at about 33 kDa on SDS/polyacrylamide gels. Peptide sequence analysis revealed that these proteins were nuclear factor (NF) 1-L and NF1/Red1. The heat-stable factor was also identified as a member of the NF1 family by using various competitor DNAs and anti-NF1 serum in an EMSA. In addition, we found that a factor bound to the accessory site of the rat S14 gene, which is necessary for carbohydrate responsiveness of this gene, was also a member of the NF1 family, raising the possibility that the NF1 family is involved in the carbohydrate regulation of gene transcription by interactions with other proteins. The NF1 family members and HNF4 interacted with overlapping sequences of the L-II element, wherein the 5′ half-site was more critical for NF1 binding, and the 3′ site was more important for HNF4 binding. Co-transfection of a vector expressing either NF1-L or NF1/Red1 repressed the transcription of the PKL enhancer unit–chloramphenicol acetyltransferase (CAT) fusion gene in HepG2 cells, whereas co-transfection of a vector expressing HNF4 activated the transcription of the same reporter gene. Furthermore NF1 family members antagonized the effect of HNF4 on PKL enhancer unit–CAT fusion gene expression when both expression plasmids were co-transfected. We conclude that NF1 family members and HNF4 regulate transcription of the PKL gene in an opposing manner by binding overlapping sequences of the L-II element.
Role of ascorbate in the activation of NF-κB by tumour necrosis factor-α in T-cells
Authors: MUÑOZ Eduardo , BLÁZQUEZ Valle M. , ORTIZ Carmen , GOMEZ-DÍAZ Consuelo , NAVAS Plácido
Published: August 2015

The first product of ascorbate oxidation, the ascorbate free radical (AFR), acts in biological systems mainly as an oxidant, and through its role in the plasma membrane redox system exerts different effects on the cell. We have investigated the role of ascorbate, AFR and dehydroascorbate (DHA) in the activation of the NF-κB transcription factor in Jurkat T-cells stimulated by tumour necrosis factor-α (TNF-α). Here we show, by electrophoretic mobility shift assays, that ascorbate increases the binding of NF-κB to DNA in TNF-α-stimulated Jurkat cells. The ability of ascorbate to enhance cytoplasmic inhibitory IkBα protein degradation correlates completely with its capacity to induce NF-κB binding to DNA and to potentiate NF-κB-mediated transactivation of the HIV-1 long terminal repeat promoter in TNF-α-stimulated Jurkat cells but not in cells stimulated with PMA plus ionomycin. AFR behaves like ascorbate, while DHA and ascorbate phosphate do not affect TNF-α-mediated NF-κB activation. These results provide new evidence for a possible relationship between the activation of the electron-transport system at the plasma membrane by ascorbate or its free radical and redox-dependent gene transcription in T-cells.
Analysis of E-box DNA binding during myeloid differentiation reveals complexes that contain Mad but not Max
Authors: RYAN Kevin M. , BIRNIE George D.
Published: August 2015

It has been shown that during myeloid differentiation the levels of mad1 mRNA are induced before the loss of c-Myc protein. This suggests that inactivation of the differentiation-blocking activity of c-Myc might not occur primarily through the loss of Myc protein, but through an increase in the levels of its antagonist, Mad1. To investigate this question we have analysed the levels of mad1 mRNA during differentiation of myeloid leukaemic HL60 cells. Although levels of mad1 mRNA were moderately increased after induction with phorbol ester, we also found that differentiation could be achieved with other inducers without any concomitant up-regulation of mad1 mRNA. In addition, analysis of E-box DNA binding revealed that, although Myc–Max complexes were lost rapidly after differentiation induction, formation of Mad1-containing complexes only occurred during the later stages of the differentiation programme. Further analysis of these Mad-containing complexes revealed that they were also unlikely to have the capacity to antagonize c-Myc function, as they did not contain Max. Therefore these data suggest that an increase in the levels of mad1 mRNA or the formation of a Mad–Max complex are unlikely to be essential or determining events for myeloid differentiation. In addition, the discovery of DNA-binding complexes that contain Mad1, but not Max, opens up this transcription factor network to include other Max-like proteins or proteins of an unrelated nature.
Tissue-specific expression and promoter analyses of the human tissue kallikrein gene in transgenic mice
Authors: XIONG William , WANG Jing , CHAO Lee , CHAO Julie
Published: August 2015

The expression of the tissue kallikrein gene is tissue-specific and exhibits a complex pattern of transcriptional and post-translational regulation. Information concerning the mechanism of its tissue-specific expression has been limited owing to the lack of suitable cell lines for the expression study. We approached this problem by introducing human tissue kallikrein gene constructs into mouse embryos, creating transgenic lines carrying its coding sequence with varying lengths of the promoter region. One construct (PHK) contained 801 bp in the 5′-flanking region and two deletion constructs contained either 302 bp (D300) or 202 bp (D200) of the promoter region. The expression of human tissue kallikrein in these transgenic mice was monitored by Northern blot, reverse transcriptase–PCR followed by Southern blot, and radioimmunoassay. In all three lines, human tissue kallikrein was expressed predominantly in the pancreas and at lower levels in other tissues, including salivary gland, kidney and spleen. This pattern was similar to that of tissue kallikrein expression in human tissues. The D300 line has higher levels of transgene expression than the D200 and PHK lines. The results indicate that the 202 bp segment immediately upstream of the translation starting site is sufficient to direct a tissue-specific expression pattern of the human tissue kallikrein gene, and that regulatory elements might exist between -801 and -202.
Glutathione and the rate of cellular proliferation determine tumour cell sensitivity to tumour necrosis factor in vivo
Authors: OBRADOR Elena , NAVARRO José , MOMPO Juan , ASENSI Miguel , PELLICER José A. , ESTRELA José M.
Published: August 2015

Low rates of cellular proliferation are associated with low GSH content and enhanced sensitivity of Ehrlich ascites-tumour (EAT) cells to the cytotoxic effects of recombinant human tumour necrosis factor (rhTNF-α). Buthionine sulphoximine, a selective inhibitor of GSH synthesis, inhibited tumour growth and increased rhTNF-α cytoxicityin vitro. Administration of sublethal doses (106 units/kg per day) of rhTNF-α to EAT-bearing mice promoted oxidative stress (as measured by increases in intracellular peroxide levels, O2-• generation and mitochondrial GSSG) and resulted in a slight reduction (19%) in tumour cell number when controls showed the highest rate of cellular proliferation. ATP (1 mmol/kg per day)-induced selective GSH depletion, when combined with rhTNF-α administration, afforded a 61% inhibition of tumour growth and resulted in a significant extension of host survival. Administration of N-acetylcysteine (1 mmol/kg per day) or GSH ester (5 mmol/kg per day) abolished the rhTNF-α- and ATP-induced effects on tumour growth by maintaining high GSH levels in the cancer cells. Our results demonstrate that the sensitivity of tumour cells to rhTNF-αin vivo depends on their GSH content and their rate of proliferation.
A rapid quantitative assay for the detection of mammalian heparanase activity
Authors: FREEMAN Craig , PARISH Christopher R.
Published: August 2015

Heparan sulphate (HS) is an important component of the extracellular matrix and the vasculature basal laminar which functions as a barrier to the extravasation of metastatic and inflammatory cells. Cleavage of HS by endoglycosidase or heparanase activity produced by invading cells may assist in the disassembly of the extracellular matrix and basal laminar, and thereby facilitate cell migration. Heparanase activity has previously been shown to be related to the metastatic potential of murine and human melanoma cell lines [Nakajima, Irimura and Nicolson (1988) J. Cell. Biochem. 36, 157–167]. To determine heparanase activity, porcine mucosal HS was partially de-N-acetylated and re-N-acetylated with [3H]acetic anhydride to yield a radiolabelled substrate. This procedure prevented the masking of, or possible formation of, new heparanase-sensitive cleavage sites as has been observed with previous methods of radiolabelling. Heparanase activity in a variety of tissues and cell homogenates including human platelets, colonic carcinoma cells, umbilical vein endothelial cells and rat mammary adenocarcinoma cells (both metastatic and non-metastatic variants) and liver homogenates all degraded the substrate in a stepwise fashion from 18.5 to approximately 13, 8 and finally to 4.5 kDa fragments, as assessed by gel-filtration analysis, confirming the substrate as suitable for the detection of heparanase activity present in a variety of cells and tissues. A rapid quantitative assay was developed with the HS substrate using a novel method for separating degradation products from the substrate by taking advantage of the decreased affinity of the heparanase-cleaved products for the HS-binding plasma protein chicken histidine-rich glycoprotein (cHRG). Incubation mixtures were applied to cHRG–Sepharose columns, with unbound material corresponding to heparanase-degradation products. Heparanase activity was determined for a variety of human, rat and murine cell and tissue homogenates. The highly metastatic rat mammary adenocarcinoma and murine lung carcinoma cell lines had four to ten times the heparanase activity of non-metastatic variants, confirming the correlation of heparanase activity with metastatic potential. Human cancer patients had twice the serum heparanase levels of normal healthy adults. The assay will be valuable for the determination of heparanase activity from a variety of tissue and cell sources, as a diagnostic tool for the determination of heparanase potential, and for the development of specific inhibitors of heparanase activity and metastasis.
Identification and characterization of the MUC2 (human intestinal mucin) gene 5′-flanking region: promoter activity in cultured cells
Authors: GUM James R. , HICKS James W. , KIM Young S.
Published: August 2015

The initiation point for MUC2 gene transcription is located within a 7000-base GC-rich region of the mucin gene cluster found on chromosome 11p15.5. The promoter activity of the 5′-flanking region of the MUC2 gene was examined following its cloning into the luciferase-producing pGL2-Basic reporter vector. A short segment comprising bases -91 to -73 relative to the start of transcription was found to be important for basal promoter activity in all cell lines tested. Electrophoretic mobility shift assays demonstrated nuclear protein binding to this region, which contains the consensus CACCC motif (5′-GCCACACCC). This element has been shown to be functionally important in several promoters that are active in diverse cell types. Competition experiments using an Sp1 oligonucleotide and antibody supershift experiments indicated that both Sp1 and other Sp1 family members bind to this element. Inclusion of the region between bases -228 and -171 in pGL2-Basic constructs increased normalized luciferase reporter activity by almost 3-fold in C1a cells, which produce relatively high levels of MUC2 mRNA. Significantly lower levels of normalized luciferase activity resulted when the same construct was transfected into cultured cell lines that express low or undetectable levels of MUC2, suggesting a possible role for this region in conferring cell-type specificity of expression. We also demonstrate, using actinomycin D, that the MUC2 mRNA is long-lived, at least in cultured cells. Moreover, no evidence was found that the MUC2 mRNA turned over more rapidly in LS174T cells, which produce relatively low levels of MUC2 mRNA, as compared with C1a cells, which produce high levels of mRNA. Thus a long mRNA half-life appears to be an important mechanism involved in achieving elevated levels of MUC2 mRNA.
Evidence that multifunctional protein 2, and not multifunctional protein 1, is involved in the peroxisomal β-oxidation of pristanic acid
Authors: DIEUAIDE-NOUBHANI Martine , ASSELBERGHS Stanny , MANNAERTS Guy P. , VAN VELDHOVEN Paul P.
Published: August 2015

The second (enoyl-CoA hydratase) and third (3-hydroxyacyl-CoA dehydrogenase) steps of peroxisomal β-oxidation are catalysed by two separate multifunctional proteins (MFPs), MFP-1 being involved in the degradation of straight-chain fatty acids and MFP-2 in the β-oxidation of the side chain of cholesterol (bile acid synthesis). In the present study we determined which of the two MFPs is involved in the peroxisomal degradation of pristanic acid by using the synthetic analogue 2-methylpalmitic acid. The four stereoisomers of 3-hydroxy-2-methylpalmitoyl-CoA were separated by gas chromatography after hydrolysis, methylation and derivatization of the hydroxy group with (S)-2-phenylpropionic acid, and the stereoisomers were designated I–IV according to their order of elution from the column. Purified MFP-1 dehydrated stereoisomer IV but dehydrogenated stereoisomer III, so by itself MFP-1 is not capable of converting a branched enoyl-CoA into a 3-ketoacyl-CoA. In contrast, MFP-2 dehydrated and dehydrogenated the same stereoisomer (II), so it is highly probable that MFP-2 is involved in the peroxisomal degradation of branched fatty acids and that stereoisomer II is the physiological intermediate in branched fatty acid oxidation. By analogy with the results obtained with the four stereoisomers of the bile acid intermediate varanoyl-CoA, stereoisomer II can be assigned the 3R-hydroxy, 2R-methyl configuration.
Fatty acyl-CoA–acyl-CoA-binding protein complexes activate the Ca2+ release channel of skeletal muscle sarcoplasmic reticulum
Authors: FULCERI Rosella , KNUDSEN Jens , GIUNTI Roberta , VOLPE Pompeo , NORI Alessandra , BENEDETTI Angelo
Published: August 2015

We previously reported that fatty acyl-CoA esters activate ryanodine receptor/Ca2+ release channels in a terminal cisternae fraction from rabbit skeletal muscle [Fulceri, Nori, Gamberucci, Volpe, Giunti and Benedetti (1994) Cell Calcium 15, 109–116]. Skeletal muscle cytosol contains a high-affinity fatty acyl-CoA-binding protein (ACBP) [Knudsen, Hojrup, Hansen, H. O., Hansen, H. F. and Roepstorff (1989) Biochem. J. 262, 513–519]. We show here that palmitoyl-CoA (PCoA) in a complex with a molar excess of bovine ACBP causes a discrete Ca2+ efflux or allows Ca2+ release from the Ca2+-preloaded terminal cisternae fraction by sub-optimal caffeine concentrations. Both effects were abolished by elevating the free [Mg2+] in the system, which inhibits the Ca2+ release channel activity. Sensitization towards caffeine was a function of both the concentration of the complex and the [PCoA]-to-[ACBP] ratio. In all experimental conditions the calculated free [PCoA] was no more than 50 nM, and such concentrations by themselves were inactive on Ca2+ release channels. The KD for PCoA binding was approx. 2 nM for bovine and yeast ACBP, and slightly higher (8 nM) for rat ACBP. The PCoA–rat ACBP complex behaved in the same manner as the PCoA–bovine ACBP complex, whereas the ester complexed with yeast ACBP was more active in activating/sensitizing Ca2+ efflux. A non-hydrolysable analogue of PCoA bound to (bovine) ACBP also sensitized the Ca2+ release channel towards caffeine. These findings indicate that fatty acyl-CoA–ACBP complexes either interact directly with one or more components in the terminal cisternae membranes or, through interaction with the component(s), donate the fatty acyl-CoA esters to high-affinity binding sites of the membrane, thus affecting (and possibly regulating) Ca2+ release channel activity.
Perturbation of the antigen-binding site and staphylococcal Protein A-binding site of IgG before significant changes in global conformation during denaturation: an equilibrium study
Authors: WANG Xi-De , LUO Jie , GUO Zhen-Quan , ZHOU Jun-Mei , TSOU Chen-Lu
Published: August 2015

Although conformational perturbation of the active sites of many enzymes has been reported to precede global molecular conformational changes [Tsou (1993) Science 262, 380–381], little effort has been made to compare the susceptibility of the ligand-binding site of proteins and the protein molecules as a whole to perturbation by denaturants. Immunoglobulin is chosen in this study to address this problem. It is found that the variable and constant regions (Fv and Fc) of a monoclonal antibody of an IgG subclass against adenylate kinase lose their abilities to bind antigen and staphylococcal Protein A after treatment with guanidinium chloride concentrations considerably lower than those required to change the global conformation of the antibody as a whole, as detected by fluorescence and second-derivative UV absorption spectroscopy. These results indicate that both ligand-binding sites of the antibody concerned are more fragile than the molecule as a whole and that the Fv and Fc regions of the antibody molecule unfold sequentially during denaturation.
Identification and characterization of two distinct intracellular GLUT4 pools in rat skeletal muscle: evidence for an endosomal and an insulin-sensitive GLUT4 compartment
Authors: ALEDO J. Carlos , LAVOIE Louis , VOLCHUK Allen , KELLER Susanna R. , KLIP Amira , HUNDAL Harinder S.
Published: August 2015

In skeletal muscle, acute insulin treatment results in the recruitment of the GLUT4 glucose transporter from intracellular vesicular structures to the plasma membrane. The precise nature of these intracellular GLUT4 stores has, however, remained poorly defined. Using an established skeletal-muscle fractionation procedure we present evidence for the existence of two distinct intracellular GLUT4 compartments. We have shown that after fractionation of crude muscle membranes on a discontinuous sucrose gradient the majority of the GLUT4 immunoreactivity was largely present in two sucrose fractions (30 and 35%, w/w, sucrose; denoted F30 and F35 respectively) containing intracellular membranes of different buoyant densities. Here we show that these fractions contained 44±6 and 49±7% of the crude membrane GLUT4 reactivity respectively, and could be further discriminated on the basis of their immunoreactivity against specific subcellular antigen markers. Membranes from the F30 fraction were highly enriched in transferrin receptor (TfR) and annexin II, two markers of the early endosome compartment, whereas they were significantly depleted of both GLUT1 and the α1-subunit of (Na++K+)-ATPase, two cell-surface markers. Insulin treatment resulted in a significant reduction in GLUT4 content in membranes from the F35 fraction, whereas the amount of GLUT4 in the less dense (F30) fraction remained unaffected by insulin. Immunoprecipitation of GLUT4-containing vesicles from both intracellular fractions revealed that TfR was present in GLUT4 vesicles isolated from membranes from the F30 fraction. In contrast, GLUT4 vesicles from the F35 fraction were devoid of TfR. The aminopeptidase, vp165, was present in GLUT4 vesicles from both F30 and F35; however, vesicles isolated from F30 contained over twice as much vp165 per unit of GLUT4 than those isolated from F35. The biochemical co-localization of vp165/GLUT4 was further substantiated by double-immunogold labelling of ultrathin muscle sections. Overall, our data indicate the presence of at least two internal GLUT4 pools: one possibly derived from an endosomal recycling compartment, and the other representing a specialized insulin-sensitive GLUT4 storage pool. Both pools contain vp165.