bluuebunny/crossref_metadata_2025 huggingface dataset:
Glucose-induced stimulation of human insulin-receptor mRNA and tyrosine kinase activity in cultured cells
Authors: Hauguel-de-Mouzon S , Mrejen C , Alengrin F , Van Obberghen E
Published: August 2015

The effects of high glucose on insulin-receptor tyrosine kinase activity and gene expression were investigated in 3T3-HIR cells. Cells incubated for 48 h in the presence of 25 mM glucose showed a 5-fold increase in the amount of insulin receptors per cell, receptor autophosphorylation and phosphorylation of the exogenous substrate poly(Glu/Tyr) compared with cells grown in the absence of glucose but in the presence of 25 mM fructose. These effects were associated with a 4-fold stimulation in steady-state levels of insulin-receptor mRNA. Significant cellular glucose utilization and lactate production were observed in the presence of high glucose in the culture medium, indicating a functional glycolytic pathway in glucose-treated cells, but not in cells treated with fructose. Such a differential response to hexoses favours the hypothesis of a carbohydrate regulation via a glycolytic intermediate. This was further supported by a similar glucose-induced increase in mRNA levels of the enzyme glyceraldehyde-3-phosphate dehydrogenase. To test the hypothesis that the stimulatory effect of glucose on amount of insulin receptors and phosphorylation state could result from post-transcriptional modifications, cells exposed to glucose were incubated with actinomycin D, a potent inhibitor of gene transcription. In cells challenged with high glucose plus inhibitor, insulin-receptor mRNA half-life was increased from 1 to 3 h, indicating that posttranscriptional mechanisms are involved in these processes of glucose regulation. Inhibition of protein synthesis by cycloheximide induced an overexpression of insulin-receptor mRNA levels in the presence of glucose, suggesting that labile repressor protein(s) could be implicated in the effects of glucose. We conclude that (1) long-term culture with high glucose increases the amount of insulin receptors and their tyrosine kinase activity and (2) the glucose-induced increase in insulin-receptor mRNA levels can be accounted for, at least in part, by posttranscriptional events.
Direct targeting of neutral endopeptidase (EC 3.4.24.11) to the apical cell surface of transfected LLC-PK1 cells and unpolarized secretion of its soluble form
Authors: Lanctôt C , Fournier H , Howell S , Boileau G , Crine P
Published: August 2015

LLC-PK1 cells were transfected with a cDNA encoding rabbit neutral endopeptidase (NEP; EC 3.4.24.11), an abundant enzyme of the kidney proximal brush border. Clones of cells expressing high levels of the protein were isolated. Selective biotinylation and radioimmunolabelling were used to determine that 85-95% of NEP was localized in the apical domain of filter-grown LLC-PK1 cells. Pulse-chase and selective biotinylation studies revealed that the majority (85%) of newly made NEP was directly targeted to the apical membrane. However, a soluble form of NEP was found to be secreted in approximately equal amounts from both sides of the monolayer when expressed in LLC-PK1 cells. Transfected pro-opiomelanocortin, a pituitary hormone precursor, was secreted almost exclusively into the basolateral medium, suggesting that the bulk flow is to the basolateral membrane. This behaviour contrasts with that observed in MDCK cells, where both the transmembrane and secreted forms of NEP are directly targeted to the apical membrane and where the secretion of pro-opiomelanocortin is unpolarized.
Effects of quin2 acetoxymethyl ester on H2O2-induced DNA single-strand breakage in mammalian cells: H2O2-concentration-dependent inhibition of damage and additive protective effect with the hydroxyl-radical scavenger dimethyl sulphoxide
Authors: Sandström B E
Published: August 2015

The cell-membrane-permeable calcium probe quin2 acetoxymethyl ester (quin2 AM) was ineffective, in comparison with o-phenanthroline, in protecting cells against H2O2-induced DNA single-strand breakage at H2O2 concentrations of about, and higher than, 0.5 mM. The present study shows that quin2 actually potentiated intracellular DNA damage at high H2O2 concentrations. H2O2-induced DNA breakage appeared within 5 min after exposure, and quin2 affected the induction of DNA breaks at both 0 degree C and 37 degrees C. Aurintricarboxylic acid, an endonuclease inhibitor, or a decrease in extracellular Ca2+, did not reduce DNA damage. These facts strongly suggest that the breaks were not produced by a Ca(2+)-dependent nuclease. We showed previously that, in the presence of Fe3+ and H2O2, quin2 strongly potentiated the formation of oxidizing species as well as plasmid DNA breakage, and, as could be expected for a transition-metal chelator, quin2 inhibited the Fenton reaction when Cu2+ was tested instead of Fe3+ [Sandström, Granström and Marklund (1994) Free Radicals Biol. Med. 16, 177-185]. In the present work with cultured cells, titration with quin2 AM showed that, despite the fact that Cu2+ has a three-to-four-orders-of-magnitude higher affinity for quin2 than has Fe3+, both inhibition and potentiation of H2O2-induced DNA damage occurred at quin2 AM concentrations of about 100 nM. Thus inhibition appeared not to involve Cu2+. The combination of quin2 AM and dimethyl sulphoxide (DMSO) gave an additive effect on H2O2-induced DNA damage compared with the effect of quin2 AM or DMSO alone, whereas the combination of o-phenanthroline and DMSO gave about the same effect as o-phenanthroline alone. In conclusion, our results do not support a role for Ca2+ in the inhibiting effect of quin2 on H2O2-induced DNA damage. Instead, it is likely that inhibition and potentiation by quin2 involves interaction with Fe ions.
Characterization of a winged bean (Psophocarpus tetragonolobus) protein kinase with calmodulin-like domain: regulation by autophosphorylation
Authors: Saha P , Singh M
Published: August 2015

A soluble protein kinase purified from winged bean (Psophocarpus tetragonolobus) shoots, has been assessed as a monomeric enzyme with an approximate M(r) of 60,000 in spite of the presence of two polypeptides of 61 and 58 kDa determined by SDS/PAGE. Immunoblot analyses using either of the two antisera raised individually against the polypeptides, detect both of them in purified preparations and a single larger polypeptide (62 kDa) in freshly prepared tissue homogenates, clearly indicating the likelihood of the doublet being formed from the larger one by proteolysis. Histone H1, syntide 2 and a synthetic myosin light chain-related peptide (MLC-peptide) have been identified as exogenous substrates of the enzyme. Complete Ca(2+)-dependence for substrate phosphorylation, a drastic inhibition of the reaction by a calmodulin (CaM) antagonist which can be partially reversed by a heterologous CaM and direct 45Ca(2+)-binding on blot, form compelling evidence in favour of a CaM-like domain of the enzyme. Both the polypeptides of the purified enzyme undergo intramolecular autophosphorylation on serine residue(s). Unlike the substrate phosphorylation reaction, autophosphorylation is Ca(2+)-independent and is not inhibited by the CaM antagonist. Down-regulation of substrate phosphorylation by auto-phosphorylation, and stimulation of the autophosphorylation by histone H1 and MLC-peptide, are novel regulatory features of the enzyme.
Cloning and structure-function analysis of the Leishmania donovani kinetoplastid membrane protein-11
Authors: Jardim A , Hanson S , Ullman B , McCubbin W D , Kay C M , Olafson R W
Published: August 2015

This report describes the complete translated gene sequence, predicted secondary structure and lipid bilayer association of a novel kinetoplastid membrane protein (KMP-11) from Leishmania donovani promastigotes. KMP-11 was previously referred to as the lipophosphoglycan-associated protein (LPGAP). The isolation, species distribution and chemical characterization, including a partial protein sequence analysis and post-translational modifications, of this major membrane component have been described [Jardim, Funk, Caprioli and Olafson (1995) Biochem. J. 305, 307-313]. C.d. measurements of KMP-11 indicated a very high helical content estimated to be approximately 86% in trifluoroethanol. This was in agreement with computer-based secondary-structure analyses which predicted KMP-11 to be almost exclusively alpha-helical, with the protein adopting a helix-loop-helix motif. Arrangement of the residues located in the putative helical regions on an Edmundson helical wheel showed that this molecule could have a strongly amphipathic conformation and provided an explanation for how such a highly charged protein might be inserted into the plasma membrane. Evidence in support of KMP-11 association with lipid bilayers was provided by showing that KMP-11 could mediate carboxyfluorescein release from liposomes. These findings suggested that KMP-11 may function in part to increase bilayer pressure, stabilizing molecules such as lipophosphoglycan within the parasite pellicular membrane.
Tissue-specific and developmentally regulated alternative splicing in mouse skeletal muscle ryanodine receptor mRNA
Authors: Futatsugi A , Kuwajima G , Mikoshiba K
Published: August 2015

The ryanodine receptor is a channel for Ca2+ release from intracellular stores. By PCR analysis, we identified two alternatively spliced regions in mRNA of the mouse skeletal muscle ryanodine receptor (sRyR). The splice variants were characterized by the presence or absence of 15 bp (ASI) and 18 bp (ASII) exons. The exclusion of these exons results in the absence of the regions corresponding to Ala3481-Gln3485 and Val3865-Asn3870, respectively, of rabbit sRyR; these amino acid sequences exist in the modulatory region, where sites for phosphorylation and binding of Ca2+, calmodulin and ATP are postulated to be. We also detected sRyR in brain and heart as well as in skeletal muscle, and the splicing patterns were found to be tissue-specific. Only the ASII-lacking isoform was detected in heart, whereas in other tissues the ASII-containing isoform was predominant. The splicing patterns were also found to change during development. In skeletal muscle, the ASI-containing isoform increased gradually from embryo to adult. The ASII-lacking isoform abruptly increased upon birth, but the ASII-containing isoform increased steadily afterwards. In cerebrum, the ratio of the ASII-containing isoform to the ASII-lacking one increased abruptly during embryonic days 14 and 18. These findings suggest that the alternative splicing of ASI and ASII, by affecting the modulatory region, generates functionally different sRyR isoforms in a tissue-specific and developmentally regulated manner.
Post-transcriptional regulation of the content of spermidine/spermine N1-acetyltransferase by N1N12-bis(ethyl)spermine
Authors: Parry L , Balaña Fouce R , Pegg A E
Published: August 2015

Spermidine/spermine N1-acetyltransferase (SSAT) is the rate-limiting enzyme for the degradation and excretion of polyamines in mammalian cells, and its activity is known to be increased enormously on exposure to polyamines and polyamine analogues. The mechanism by which such an analogue, BESM [N1N12-bis(ethyl)spermine], increases the content of SSAT was investigated by transfecting COS-7 cells with plasmids containing SSAT cDNA in the pEUK expression vector. Despite a large increase in mRNA production, there was only a very small increase in SSAT activity in the transfected cells. When BESM was added at 36 h after transfection, there was a large and very rapid increase in SSAT protein amounting to 380-fold in 12 h without any increase in the mRNA. SSAT protein turned over very rapidly, with a half-life of about 20 min. In the presence of BESM, this turnover was greatly reduced, and the half-life increased to more than 13 h. However, this increase was not sufficient to account for all of the increase in SSAT protein, suggesting that there is also regulation of the translation of the mRNA by BESM. Further evidence for such translation regulation was obtained by studying the polysomal distribution of the SSAT mRNA. In the absence of BESM, most of the mRNA was present in fractions which sedimented more slowly than the monoribosome peak. In BESM-treated cells, a significant proportion of the SSAT mRNA was moved into the small-polysome region of the gradient. The expression of SSAT and the effects of BESM on the polysomal distribution of SSAT mRNA were not affected by the 5′- or 3′-untranslated regions of the mRNA, since constructs which lacked all of these regions gave similar results to constructs containing the entire mRNA sequence. These results show that the increased transcription of the SSAT gene that occurs in the presence of polyamine analogues such as BESM is not sufficient for SSAT expression and that post-transcriptional regulation is critical for the control of SSAT content.
Regulation of membrane-associated tyrosine phosphatases in UMR 106.06 osteoblast-like cells
Authors: Southey M C , Findlay D M , Kemp B E
Published: August 2015

Protein tyrosine phosphatases play an important role in cell metabolism. Three distinct protein tyrosine phosphatase activities have been identified in an osteoblast-like cell line, UMR 106.06. These activities comprised two membrane-associated phosphatases and one cytosolic phosphatase of apparent molecular mass > 153 kDa, 80 kDa and 40 kDa respectively, estimated by gel filtration. On the basis of differences in apparent molecular mass, proteolytic-digestion profiles, substrate specificities and responses to a range of extracellular influences and inhibitor molecules, the two membrane-associated tyrosine phosphatases are distinct proteins. Tyrosine phosphatase activity in UMR 106.06 cells was sensitive to cell density. Cells at confluence contained membrane protein tyrosine phosphatase with specific activity 9-fold higher than cells at medium or low cell density. This elevation in membrane tyrosine phosphatase activity was due specifically to an increase in the high-molecular-mass enzyme. This phosphatase was also responsive to extracellular matrix components. This activity was elevated in cells grown on a collagen type-I matrix independently of cell density. Membrane and cytosolic protein tyrosine phosphatases were differentially regulated by a variety of agents including phorbol 12-myristate 13-acetate, parathyroid hormone, epidermal growth factor, okadaic acid and transforming growth factor beta. These observations suggest that regulatory influences control tyrosine phosphorylation in UMR 106.06 cells including cell-cell contact, cell-matrix contact and signal transduction involving tyrosine and serine/threonine phosphorylation events.
Comparison of the activities of a multiple inositol polyphosphate phosphatase obtained from several sources: a search for heterogeneity in this enzyme
Authors: Craxton A , Ali N , Shears S B
Published: August 2015

A multiple inositol polyphosphate phosphatase (formerly known as inositol 1,3,4,5-tetrakisphosphate 3-phosphatase) was purified approx. 22,000-fold from rat liver. The final preparation migrated on SDS/PAGE as a doublet with a mean apparent molecular mass of 47 kDa. Upon size-exclusion chromatography, the enzyme was eluted with an apparent molecular mass of 36 kDa. This enzyme was approximately evenly distributed between the ‘rough’ and ‘smooth’ subfractions of endoplasmic reticulum. There was a 20-fold range of specific activities of this phosphatase in CHAPS-solubilized particulate fractions prepared from the following rat tissues: liver, heart, kidney, testis and brain. However, each of these extracts contained different amounts of endogenous inhibitors of enzyme activity. After removal of these inhibitors by MonoQ anion-exchange chromatography, there was only a 2.5-fold range of specific activities; kidney contained the most and brain contained the least. We prepared and characterized polyclonal antiserum to the hepatic phosphatase, which immunoprecipitated 85-100% of both particulate and soluble phosphatase activities. The antiserum also immunoprecipitated, with equivalent efficacy, CHAPS-solubilized phosphatase activities from heart, kidney, testis, brain and erythrocytes (all prepared from rat). Our data strengthen the case that the function of the mammalian phosphatase is unrelated to the metabolism of Ca(2+)-mobilizing cellular signals. The CHAPS-solubilized phosphatase from turkey erythrocytes was not immunoprecipitated by the polyclonal antiserum, and is therefore an isoform that is structurally distinct, and possibly functionally unique.
Trypanosomatid cysteine protease activity may be enhanced by a kininogen-like moiety from host serum
Authors: Lonsdale-Eccles J D , Mpimbaza G W N , Nkhungulu Z R M , Olobo J , Smith L , Tosomba O M , Grab D J
Published: August 2015

African trypanosomes contain cysteine proteases (trypanopains) the activity of which can be measured by in vitro digestion of fibrinogen, after electrophoresis in fibrinogen-containing SDS/polyacrylamide gels. When assessed by this procedure, trypanopain from Trypanosoma brucei (trypanopain-Tb) is estimated to have a molecular mass of 28 kDa. However, two additional bands of trypanopain activity (87 kDa and 105 kDa) are observed if serum is added to the trypanopain before electrophoresis. Formation of the 87 and 105 kDa bands is frequently accompanied by a reduction in the intensity of the 28 kDa activity which suggests that the extra bands are complexes of the 28 kDa trypanopain-Tb and a molecule from rat serum called rat trypanopain moledulator (rTM). The rTM-induced activation of cysteine proteases is not restricted to T. brucei as it is also observed with proteases from other protozoan parasites such as bloodstream forms of Trypanosoma congolense and the mammalian-infective in vitro-derived promastigote forms of Leishmania donovani and Leishmania major. The physical properties of rTM resemble those of the kininogen family of cysteine protease inhibitors. rTM is an acidic (pI 4.7) heat-stable 68 kDa glycoprotein with 15 kDa protease-susceptible domains. This resemblance between rTM and kininogens was confirmed by the positive, albeit weak, immunoreactivity between anti-(human low-molecular-mass kininogen) antibody and rTM as well as anti-rTM antibody and human low-molecular-mass kininogen. Furthermore, commercial preparations of human-low-molecular-mass kininogen and chicken egg white cystatin mimicked rTM by forming extra bands of proteolytic activity in the presence of trypanopain-Tb. In some instances, low-molecular-mass kininogen was also observed to increase the rate of hydrolysis of 7-(benzyloxycarbonyl-phenylalanyl-arginyl-amido)-4- methylcoumarin by live T. brucei. Although this effect was rather erratic, in no instance was significant inhibition observed when this putative cysteine protease inhibitor was used under these conditions. The activation of parasite cysteine proteases by commonly accepted cysteine protease inhibitors is unexpected and may have important pathological repercussions.
Tyrosine phosphorylation and association of Syk with FcγRII in monocytic THP-1 cells
Authors: Ghazizadeh S , Bolen J B , Fleit H B
Published: August 2015

Although the cytoplasmic portion of the low-affinity receptor for immunoglobulin G, Fc gamma RII, does not contain a kinase domain, rapid tyrosine phosphorylation of intracellular substrates occurs in response to aggregation of the receptor. The use of specific tyrosine kinase inhibitors has suggested that these phosphorylations are required for subsequent cellular responses. We previously demonstrated the coprecipitation of a tyrosine kinase activity with Fc gamma RII, suggesting that non-receptor tyrosine kinases might associate with the cytoplasmic domain of Fc gamma RII. Anti-receptor immune complex kinase assays revealed the coprecipitation of several phosphoproteins, most notably p56/53lyn, an Src-family protein tyrosine kinase (PTK), and a 72 kDa phosphoprotein. Here we identify the 72 kDa Fc gamma RII-associated protein as p72syk (Syk), a member of a newly described family of non-receptor PTKs. A rapid and transient tyrosine phosphorylation of Syk was observed following Fc gamma RII activation. Syk was also tyrosyl-phosphorylated following aggregation of the high-affinity Fc gamma receptor, Fc gamma RI. The Fc gamma RI activation did not result in association of Syk with Fc gamma RII, implying that distinct pools of Syk are activated upon aggregation of each receptor in a localized manner. These results demonstrate a physical association between Syk and Fc gamma RII and suggest that the molecules involved in Fc gamma RII signalling are very similar to the ones utilized by multichain immune recognition receptors such as the B-cell antigen receptor and the high-affinity IgE receptor.
A gelsolin-related protein from lobster muscle: cloning, sequence analysis and expression
Authors: Lück A , D'Haese J , Hinssen H
Published: August 2015

The tail muscle of the lobster Homarus americanus contains an actin-binding protein with an apparent molecular mass of 105 kDa determined by SDS/PAGE and gelsolin-like properties. We isolated this protein and peptide sequences were obtained after limited proteolysis with chymotrypsin. A tail-muscle-specific cDNA library was constructed in a lambda expression vector and a full-length clone was obtained by screening with a polyclonal anti-(crustacean gelsolin) antibody. The cDNA insert of approx. 3.2 kb length was sequenced. The cDNA contained an open reading frame of 2.265 kb, and the deduced amino acid sequence of 754 residues (83,469 Da) identified the protein as a cytoplasmic member of the gelsolin/villin protein family. Comparison of the lobster gelsolin amino acid sequence with other members of this protein family revealed the characteristic 6-fold repeated segmental structure as well as the three conserved sequence motifs typical of each segment [Way and Weeds (1988) J. Mol. Biol. 203, 1127-1133]. Strong homologies were found with Drosophila gelsolin, human gelsolin, villin core, Dictyostelium severin and Physarum fragmin. In addition, the gelsolin-like protein from lobster muscle revealed motifs that were clearly similar to the actin-bundling region of human villin headpiece although it did not itself contain a distinct headpiece domain. The recombinant lobster gelsolin-like protein, expressed in Escherichia coli as a fusion protein, was purified from inclusion bodies and renatured as a functional protein. There were no significant differences in the biological activity tested between the recombinant and the native protein isolated from lobster muscle.
Platelet-derived growth factor and angiotensin II stimulate the mitogen-activated protein kinase cascade in renal mesangial cells: comparison of hypertrophic and hyperplastic agonists
Authors: Huwiler A , Stabel S , Fabbro D , Pfeilschifter J
Published: August 2015

Exposure of mesangial cells to platelet-derived growth factor (PDGF) BB caused a significant stimulation of cell proliferation and protein synthesis, as measured by [3H]thymidine incorporation and [3H]leucine incorporation respectively. In contrast, cells treated with angiotensin II had no significant increase in [3H]thymidine incorporation, but demonstrated a marked increase in [3H]leucine incorporation. Furthermore, angiotensin II significantly increased total protein content per cell. These data show that, whereas PDGF-BB is a mitogen and stimulates mesangial-cell hyperplasia, angiotensin II causes hypertrophy of the cells without hyperplasia. Treatment of mesangial cells with PDGF and angiotensin II rapidly and dose-dependently stimulated mitogen-activated protein (MAP) kinase activity, as shown by an assay for activity in vitro using myelin basic protein as a substrate, and by immunoprecipitation of 32P-labelled cells with specific antibodies against the 42 kDa and 44 kDa mitogen-activated protein kinases p42mapk and p44mapk, respectively. Whereas stimulation with PDGF-BB caused a potent and sustained (for more than 30 min) phosphorylation and activation of p42mapk and p44mapk, as well as of the upstream activators MAP kinase kinase and c-Raf, the effect of angiotensin II was less potent, reaching a peak at 5-10 min and thereafter declining rapidly. In summary, these results suggest that PDGF-BB and angiotensin II differ in their potency and duration of activation of the MAP kinase cascade, which may explain why PDGF-BB is a potent mitogen for mesangial cells, whereas angiotensin II only triggers mesangial-cell hypertrophy.
Cooperative interaction of oestrogen receptor ‘zinc finger’ domain polypeptides on DNA binding
Authors: Predki P F , Sarkar B
Published: August 2015

The consensus oestrogen response element (ERE) contains two inverted copies of an AGGTCA consensus hexameric half-site, spaced by three base pairs. It differs from many other hormone response elements, such as consensus thyroid (TREp) and retinoic acid (DR-5 RARE) response elements, only in the relative spacing and orientation of these sequences. In the present study we report values for cooperativity (omega) of an oestrogen receptor DNA-binding domain polypeptide upon binding to these sequences. The polypeptide binds with negative cooperativity, or without cooperativity to retinoic acid and thyroid response elements respectively, but with high cooperativity to the ERE. We have also examined cooperativity upon binding of the polypeptide to an ERE variant. Since naturally occurring EREs commonly contain one hexamer which is considerably more degenerate than the other, we designed a hybrid response element in which one hexamer is a consensus ERE, while specific mutations were introduced into the other. We chose to mutate the second half-site to a glucocorticoid response element (GRE) half-site sequence (AGAACA), since normally no binding of the DNA-binding domain polypeptide to a GRE hexamer alone can be detected. In the hybrid response element, however, the GRE half-site is recognized with relatively high affinity, although binding to this sequence is dependent on the previous binding of a polypeptide to the ERE hexamer. Thus, cooperative interactions are capable of mediating the recognition of ERE sequence degeneracy. The ability of protein-protein interactions to mediate recognition of DNA sequence degeneracy may also have implications for transcription factors in general.
Tissue differences in the response of the pyruvate dehydrogenase complex to a glucose load during the development of obesity in gold-thioglucose-obese mice
Authors: Bryson J M , Cooney G J , Wensley V R , Phuyal J L , Caterson I D
Published: August 2015

The activity of pyruvate dehydrogenase (PDHC), a key enzyme complex in the oxidative disposal of glucose, was measured after an oral glucose load in the heart, liver, quadriceps muscle, white adipose tissue (WAT) and brown adipose tissue (BAT) of gold-thioglucose (GTG)-obese mice at different stages during the development of obesity and in age-matched controls. Significant responses to the glucose load were seen 30 min post-gavage in heart, WAT and BAT of control mice but no change was observed in quadriceps muscle. The increase in activity of the active form of PDHC (PDHCa) in response to glucose in heart was reduced 2 weeks after the induction of GTG-obesity with no response in 5 or 10 week obese mice. A 2-3-fold increase in the PDHCa response in both WAT and BAT of 2 week obese mice was absent in 5 and 10 week obese animals. Basal PDHCa activity in quadriceps muscle was increased in 2 week obese mice but subsequently returned to control levels as obesity progressed. The glucose load produced no change in the activity of PDHCa in quadriceps muscle of obese mice. These results demonstrate that changes in the capacity for oxidative glucose disposal in different tissues, as indicated by changes in PDHCa activity, may contribute to glucose-intolerance and insulin-resistance in GTG-obese mice and that the response of the PDHC to insulin during the development of obesity varies in different tissues.
Hepatic molecular conversion and detoxification of ferritin iron in adult lampreys (Geotria australis), following natural and induced iron loading
Authors: Harris L R , Macey D J , Potter I C , Cake M H
Published: August 2015

Weekly intramuscular injections of 3 mg of iron as horse spleen ferritin into adult Geotria australis over 10 weeks, resulted in a progressive increase in that form of iron in the serum. However, as with control animals, the ferritin in the liver of injected lampreys consisted of one subunit type, whose M(r) (20,300) differed from those of the two subunit types of horse spleen ferritin. Thus, lampreys had converted horse spleen ferritin iron into endogenous ferritin iron, presumably in their liver. Marked rises in hepatic non-haem iron during the first 2 weeks and between weeks 8 and 10 of iron injections were accompanied by pronounced increases in superoxide dismutase (SOD) activity. This rise, which parallels the rise in SOD activity that occurs as iron increases during the very protracted upstream migration of G. australis, is consistent with the view that SOD protects against iron-mediated damage by removing the superoxide radical, which facilitates the formation of the highly toxic hydroxyl radical. A levelling off of the iron concentration between weeks 2 and 8 was accompanied by a decline in SOD activity, even though nonhaem iron levels were well above those of control animals. Enhanced SOD activity may therefore only be required when there is an elevated flux of iron in the liver through low-molecular-mass intermediates. A small amount of ferritin iron was converted into the more inert haemosiderin iron.
A single-chain insulin-like growth factor I/insulin hybrid binds with high affinity to the insulin receptor
Authors: Kristensen C , Andersen A S , Hach M , Wiberg F C , Schäffer L , Kjeldsen T
Published: August 2015

1. To investigate the structure/function relationship of the interaction between ligand and receptor in the insulin-like growth factor I (IGF-I) and insulin receptor systems we have prepared and characterized a single-chain insulin/IGF-I hybrid. The single-chain hybrid consists of the insulin molecule combined with the C domain of IGF-I. The single-chain hybrid was found to bind with high affinity to both truncated soluble insulin receptors and membrane-bound holoreceptors. The affinity for interacting with the soluble truncated insulin receptors was 55-94% relative to insulin, and affinity for membrane-bound insulin receptors was 113% of that of insulin. Furthermore we found that the affinity of the single-chain hybrid molecule for IGF-I receptors was 19-28% relative to IGF-I. 2. The affinity of the single-chain hybrid for chimeric insulin/IGF-I receptors exceeded that of either natural ligand. This indicates that coordinately changing domains of the receptors and the ligands can induce higher affinity of ligand for receptor, supporting the idea that these receptors have a common ligand-binding site [Kjeldsen, Andersen, Wiberg, Rasmussen, Schäffer, Balschmidt, Møller and Møller (1991) Proc. Natl. Acad. Sci. U.S.A. 88, 4404-4408]. 3. In contrast with what was generally assumed about the ligand structure required for binding to the insulin receptor we demonstrate the first single-chain insulin analogue that can bind with high affinity to the insulin receptor.
Reversal of the Ca2+ pump of blood platelets
Authors: Benech J C , Wolosker H , de Meis L
Published: August 2015

In this study, the endoplasmic Ca2+ transport ATPase of blood platelets was compared with the Ca2+ ATPase of sarcoplasmic reticulum skeletal muscle. Similar to the muscle enzyme, the Ca2+ ATPase from platelets was found to catalyse an ATP<-->P(i) exchange both in the presence and in the absence of a transmembrane Ca2+ gradient. When platelet vesicles are loaded with Ca2+ and diluted in medium containing ADP, P(i) and EGTA, the ATPase catalyses Ca2+ efflux coupled to synthesis of ATP. The stoichiometry between Ca2+ ion released and ATP synthesized by platelet Ca2+ ATPase is 1, while that of skeletal muscle is 2. Thapsigargin, a specific inhibitor of sarcoplasmic/endoplasmic reticulum Ca2+ ATPases, inhibited both the Ca(2+)-dependent ATPase activity and the reversal of the platelet Ca2+ pump. The possibility is discussed that the differences observed between the two transport systems is related to the distinct amino acid sequences of the enzymes.
Analysis of c-fos expression in the butyrate-induced F-98 glioma cell differentiation
Authors: Tang S J , Huang Y M , Wang F F
Published: August 2015

The functional induction of c-fos in the sodium butyrate-induced differentiation of F-98 glioma cells was studied. Fos protein level was increased by butyrate. In contrast, c-Jun protein was constitutively expressed and was not affected by butyrate. Gel-retardation assay indicates Fos as a component of the complex formed between the consensus oligonucleotide of the TPA (PMA, phorbol 12-myristate 13-acetate) response element (TRE) and nuclear extract prepared from butyrate-treated cells. Transfection studies showed that butyrate increased transcription from a multimeric TRE-driven reporter construct, and the effect was mimicked by transfecting cells with fos-expression plasmid. Furthermore, under conditions of c-fos over-expression, transactivation by butyrate was essentially abolished. These data suggest that Fos induction had a functional role in gene activation. Characterization of stable c-fos transfectants demonstrated that these cells displayed alterations in morphology, showed serum-dependent growth, had slower growth rates and grew to lower saturation densities than did untransfected F-98 cells or transfected cells that did not express c-fos. Immunofluorescent staining indicated that fos transfectants also had elevated glial fibrillary acidic protein (‘GFAP’) expression. Transfection of the c-fos promoter-chloramphenicol acetyltransferase fusion gene into F-98 cells revealed that activation of c-fos by butyrate was exerted at the promoter level, and sequences located within nucleotides -757 to -402 of the c-fos promoter were responsible for butyrate induction. Our data indicate that transcriptional activation of c-fos through its promoter by butyrate resulted in increased Fos protein expression. Transfection studies show that both c-fos and butyrate activate TRE-containing genes, and fos may be a downstream mediator of butyrate. Furthermore, expression of c-fos plays a major role in modulating the growth properties of F-98 cells.
Immunoprecipitation of opioid receptor-Go-protein complexes using specific GTP-binding-protein antisera
Authors: Georgoussi Z , Milligan G , Zioudrou C
Published: August 2015

Solubilization of opioid receptors from rat cortical membranes that retained high-affinity guanine nucleotide-sensitive agonist binding was achieved using 10 mM CHAPS. We report the nature of the interactions of mu and delta opioid receptors with the guanine nucleotide-binding protein G(o) by immunoprecipitation of CHAPS extracts with selective G(o)alpha-subunit protein antisera. Antiserum IM1 raised against amino acids 22-35 of G(o)alpha selectively co-immunoprecipitated G(o)alpha-mu and G(o)alpha-delta opioid receptor complexes detected in the immunoprecipitates by specific [3H][D-Ala2,N-Me-Phe4,Gly5-ol]enkephalin and [3H][D-Ser2,Leu5,Thr6]enkephalin binding respectively. By contrast, antisera directed against the C-terminal decapeptide (OC2) and the N-terminal hexadecapeptide (ON1) of isoforms of G(o)alpha were unable to immunoprecipitate solubilized opioid receptor-G(o) complexes, although both were able to immunoprecipitate solubilized G(o)alpha and have been shown to reduce the affinity of [D-Ala2,D-Leu5]enkephalin for opioid receptors in rat cortical membranes [Georgoussi, Carr and Milligan (1993) Mol. Pharmacol. 44, 62-69]. These findings demonstrate that CHAPS-solubilized mu and delta opioid receptors from rat cortical membranes form stable complexes with one or more variants of G(o).
Tissue- and cell-specific expression of mouse xanthine oxidoreductase gene in vivo: regulation by bacterial lipopolysaccharide
Authors: Kurosaki M , Li Calzi M , Scanziani E , Garattini E , Terao M
Published: August 2015

The expression of the xanthine oxidoreductase gene was studied in various mouse organs and tissues, under basal conditions and on treatment with bacterial lipopolysaccharide. Levels of xanthine oxidoreductase protein and mRNA were compared in order to understand the molecular mechanisms regulating the expression of this enzyme system. The highest amounts of xanthine oxidoreductase and the respective mRNA are observed in the duodenum and jejunum, where the protein is present in an unusual form because of a specific proteolytic cleavage of the primary translation product present in all locations. Under basal conditions, multiple tissue-specific mechanisms of xanthine oxidoreductase regulation are evident. Lipopolysaccharide increases enzyme activity in some, but not all tissues, mainly via modulation of the respective transcript, although translational and post-translational mechanisms are also active. In situ hybridization studies on tissue sections obtained from mice under control conditions or with lipopolysaccharide treatment demonstrate that xanthine oxidoreductase is present in hepatocytes, predominantly in the proximal tubules of the kidney, epithelial layer of the gastrointestinal mucosa, the alveolar compartment of the lung, the pulpar region of the spleen and the vascular component of the heart.
Enzymatic radiolabelling to a high specific activity of legume lipo-oligosaccharidic nodulation factors from Rhizobium meliloti
Authors: Bourdineaud J P , Bono J J , Ranjeva R , Cullimore J V
Published: August 2015

In this paper we describe the two-step coupled 35S-radiolabelling of the lipo-oligosaccharidic nodulation (Nod) factors of the bacterium Rhizobium meliloti to a specific radioactivity of 800 Ci/mmol. These radiolabelled Nod factors bind to a particulate fraction from roots of the bacterium's symbiotic host, Medicago truncatula, with an equilibrium dissociation constant (KD) of 117 nM, similar to that observed with a synthetic tritiated ligand. The first step of the 35S-labelling involves the synthesis of 3′-phosphoadenosine 5′-phospho[35S]sulphate ([35S]PAPS) from ATP and [35S]sulphate using yeast enzymes. The second step exploits the sulphotransferase activity of the R. meliloti NodH protein, which has been expressed in Escherichia coli, to transfer the labelled sulphate group from PAPS to non-sulphated Nod factors. This enzyme was found to be active in E. coli cultured at 18 degrees C but not 37 degrees C. NodH could also transfer the sulphate group from PAPS to a model substrate, tetra-N-acetyl chitotetraose, with apparent Km values of 56 and 70 microM respectively, and exhibited an apparent Km value for non-sulphated Nod factors of 28 microM. Coupling the two steps of the radiolabelling resulted in an efficiency of 35S incorporation from inorganic sulphate to the Nod factors of approximately 10%. These labelled factors will be a valuable tool in the search for high-affinity receptors for the lipo-oligosaccharidic nodulation factors.
Both nuclear and mitochondrial cytochrome c oxidase mRNA levels increase dramatically during mouse postnatal development
Authors: Kim K , Lecordier A , Bowman L H
Published: August 2015

The steady-state levels of 13 of 16 cytochrome c oxidase (COX) mRNAs and mitochondrial DNA were measured during the postnatal development of mouse skeletal muscle, ventricle, kidney and brain as well as during the differentiation of mouse myoblasts into myofibres in cell culture. These experiments indicate that large co-ordinated increases in COX mRNA levels and isoform switching are important for the elaboration of this enzyme during postnatal development and demonstrate the importance of gene-regulatory mechanisms in controlling COX activity. On a per nucleus basis, the levels of the mitochondrial- and most nuclear-encoded COX mRNAs co-ordinately increase 3-10-fold during postnatal development, with the highest levels obtained in ventricle and skeletal muscle. However, concentrations of mitochondrial and nuclear COX mRNAs remain constant during the differentiation of myoblasts into fibres in cell culture. A gradual change from the liver to the heart isoform of COX subunit VIa mRNA occurs during postnatal development of skeletal muscle and ventricle and is nearly complete 3 days after the formation of myofibres in cell culture. Mitochondrial DNA increases proportionally with COX mRNAs during mouse postnatal development but not during myoblast differentiation in cell culture, in which mitochondrial DNA levels increase 5-fold and mitochondrial mRNA levels remain constant. This suggests that mitochondrial DNA replication may control mitochondrial RNA concentrations during postnatal development but not during myoblast differentiation in cell culture.
S100β is a target protein of neurocalcin δ, an abundant isoform in glial cells
Authors: Okazaki K , Obata N H , Inoue S , Hidaka H
Published: August 2015

To clarify the function of neurocalcin delta, an isoform found abundantly in glial cells, we attempted to find its target proteins by using neurocalcin delta-affinity chromatography and the 125I-neurocalcin delta gel-overlay method. The 10, 14, 27, 36 and 50 kDa bands found on SDS/PAGE bound to 125I-neurocalcin delta, and 10, 11, 19, 24, 26, 50 and 70 kDa proteins were eluted from a neurocalcin delta-affinity column in a Ca(2+)-dependent manner. Sequence analysis of proteolytic peptides revealed the following identities: S100 beta (10 kDa), S100 alpha (11 kDa), myelin basic protein (19 kDa), glyceraldehyde-3-phosphate dehydrogenase (36 kDa) and tubulin beta-chain (50 kDa). A zero-length cross-linking study indicated that 1 mol of S100 beta bound to 1 mol of neurocalcin delta. With the gel-overlay method, purified S100 beta protein and calcyclin bound to 125I-neurocalcin delta whereas calgizarrin and calvasculin, other members of the S100 family, did not. These findings suggest that S100 beta is one of the target proteins of neurocalcin delta, and the neurocalcin delta-S100 beta complex may be involved in Ca(2+)-signalling in the glial cell.
Formation in vitro of the 3,4,6-trihydroxyphenylalanine quinone cofactor
Authors: Hanlon S P , Carpenter K , Hassan A , Cooper R A
Published: August 2015

An Escherichia coli K-12 2-phenylethylamine oxidase gene with a mutated leader sequence region produced a largely inactive form of the enzyme in the cytoplasm. This form of the enzyme was activated 30-50-fold on incubation at 30 degrees C in the absence of any added cofactors. After activation the enzyme contained a quinone which was not detected in the non-activated form. This is the first report of the formation in vitro of any quinoenzyme cofactor.
Glutamine metabolism in chick enterocytes: absence of pyrroline-5-carboxylase synthase and citrulline synthesis
Authors: Wu G , Flynn N E , Yan W , Barstow D G
Published: August 2015

This study was designed to determine whether pyrroline-5-carboxylate (P-5-C) synthase is deficient in chick enterocytes therefore resulting in the lack of synthesis of ornithine and citrulline from glutamine. Post-weaning pig enterocytes, which are known to contain P-5-C synthase and to synthesize both ornithine and citrulline from glutamine, were used as positive controls. Enterocytes were incubated at 37 degrees C for 0-30 min in the presence of 2 mM [U-14C]glutamine or 2 mM ornithine plus 2 mM NH4Cl. In chick enterocytes, glutamine was metabolized to NH3, CO2, glutamate, alanine and aspartate, but not to ornithine, citrulline, arginine or proline. Likewise, there was no formation of citrulline, arginine, alanine or aspartate from ornithine in chick enterocytes. Furthermore, the rate of conversion of ornithine into proline in chick enterocytes was only about 4% of that in cells from pigs. To elucidate the reason for the inability of chick enterocytes to synthesize ornithine and citrulline from glutamine, the activities of the enzymes involved were measured. No activity of P-5-C synthase or ornithine carbamoyltransferase was found in chick enterocytes, in contrast with cells from post-weaning pigs. It was also demonstrated that the activity of ornithine aminotransferase in chick enterocytes was only 3% of that in cells from pigs. Thus the present findings elucidate the biochemical reason for the lack of endogenous synthesis of ornithine and citrulline in chicks. Our results also explain previous observations that ornithine cannot replace arginine or proline in the diet of chicks. We suggest that the absence of P-5-C synthase and ornithine carbamoyltransferase in enterocytes is the metabolic basis for the nutritional requirement of arginine in the chick.
Detection of acyl-CoA-binding protein in human red blood cells and investigation of its role in membrane phospholipid renewal
Authors: Fyrst H , Knudsen J , Schott M A , Lubin B H , Kuypers F A
Published: August 2015

Acyl-CoA-binding protein (ACBP) has been identified in a number of tissues and shown to affect the intracellular distribution and utilization of acyl-CoA. We have detected ACBP in the cytosol but not the membrane of human red blood cells and, using an e.l.i.s.a. with antibodies prepared against human liver ACBP, found that its concentration was 0.5 microM. To investigate the role of ACBP in human red blood cells, we added purified human liver ACBP and radiolabelled acyl-CoA to isolated membranes from these cells. ACBP prevented high concentrations of acyl-CoA from binding to the membrane but could not keep the acyl-CoA in the aqueous phase at low concentrations. This suggested the presence of a pool in the membrane with a binding affinity for acyl-CoA that was greater than that of ACBP for acyl-CoA. In the presence of lysophospholipid, this membrane-bound pool of acyl-CoA was rapidly used as a substrate by acyl-CoA:lysophospholipid acyltransferase (LAT) to generate phospholipid from lysophospholipid. We also found that ACBP-bound acyl-CoA was preferred over free acyl-CoA as a substrate by LAT. These results are the first documentation that human red blood cells contain ACBP and that this protein can affect the utilization of acyl-CoA in plasma membranes of these cells. The interactions between acyl-CoA, ACBP and the membrane suggest that there are several pools of acyl-CoA in the human red blood cell and that ACBP may have a role in regulating their distribution and fate.
Structure of heparin fragments with high affinity for lipoprotein lipase and inhibition of lipoprotein lipase binding to α2-macroglobulin-receptor/low-density-lipoprotein-receptor-related protein by heparin fragments
Authors: LarnkjÆr A , NykjÆr A , Ølivecrona G , Thøgersen H , Ostergaard P B
Published: August 2015

Heparin-derived deca- and octa-saccharides were subjected to affinity chromatography on lipoprotein lipase-Sepharose and the fractions eluted at high salt concentration were analysed by strong-anion-exchange chromatography. Two high-affinity decasaccharides were isolated and the structure determined by one- and two-dimensional 1H-n.m.r. spectroscopy. The affinities of 3H-labelled low-molecular-mass heparin and size-fractionated deca-, octa-, and hexa-saccharides for lipoprotein lipase immobilized on microtitre plates were determined from saturation curves. From competition experiments the affinities of unlabelled heparins and pure deca- and hexa-saccharide fragments were determined. The binding was size- and charge-dependent, but structural dependency was also indicated. Thus substitution of a 2-O-sulphated L-iduronic acid with D-glucuronic acid was less important than the sulphation pattern of the D-glucosamine residue for affinity for lipoprotein lipase. Heparin inhibits binding of lipoprotein lipase to alpha 2-macroglobulin-receptor/low-density-lipoprotein receptor-related protein. The effects of size, charge and structure for this inhibition were studied. The ability of the heparin fragments to inhibit binding correlated with their affinity for lipoprotein lipase. This indicates that the inhibition of the binding of lipoprotein lipase to alpha 2-macroglobulin-receptor/low-density-lipoprotein receptor-related protein by heparin is exclusively mediated by binding of heparin to lipoprotein lipase.