bluuebunny/crossref_metadata_2025 huggingface dataset:
Possible role for gp160 in constitutive but not insulin-stimulated GLUT4 trafficking: dissociation of gp160 and GLUT4 localization
Authors: FILIPPIS Anthony , CLARK Stella , PROIETTO Joseph
Published: August 2015

GLUT4-containing vesicles are constantly cycling in both basal and insulin-stimulated states. Our previous studies have shown that basal cycling of GLUT4 is impaired under conditions of high glucose or glucosamine and, as a consequence, GLUT4 is retained intracellularly in low-density microsomes [Filippis A., Clark, S., and Proietto, J. (1997) Biochem. J. 324, 981-985]. In addition to GLUT4 itself, a major protein component of GLUT4-containing vesicles is a glycoprotein of Mr 160000 (gp160). In all studies so far published gp160 has been co-localized with GLUT4 under all conditions. In this study, we show that retention of GLUT4 in low-density microsomes (enriched in Golgi apparatus) is associated with a decrease in gp160 levels in this compartment. A concomitant increase of gp160 in high-density microsomes (enriched in endoplasmic reticulum), demonstrates for the first time a dissociation in the localization of gp160 and GLUT4. Despite the marked decrease in gp160 levels in the GLUT4-containing compartment, insulin-stimulated translocation was normal, while little gp160 appeared in the plasma membrane in response to insulin. The retention of gp160 in the high-density microsomes is apparently not due to a change in the glycosylation state of gp160 as measured by [3H]mannose incorporation. It is concluded that, in rat adipocytes, gp160 is not required for insulin-stimulated translocation, but may be necessary for constitutive trafficking of the GLUT4-containing vesicle.
Sustained phospholipase D activation in response to angiotensin II but not carbachol in bovine adrenal glomerulosa cells
Authors: JUNG EunMi , BETANCOURT-CALLE Soraya , MANN-BLAKENEY RaShawn , FOUSHEE Tasha , ISALES M. Carlos , BOLLAG B. Wendy
Published: August 2015

We have demonstrated previously that in bovine adrenal glomerulosa cells, phospholipase D (PLD) activity can indirectly result in the generation of sn-1,2-diacylglycerol (DAG) through its production of phosphatidic acid (PA) and the subsequent action of PA phosphohydrolase. Furthermore, the PLD-generated DAG can trigger aldosterone secretion. Therefore, we characterized PLD activation by two agonists, angiotensin II (Ang II) and carbachol, to determine if the activity of the enzyme might underlie sustained aldosterone secretion. We determined that Ang II-induced PLD activation occurred via the angiotensin-1 receptor (AT1), and that a specific AT1 antagonist, losartan, inhibited this activation, whereas the same concentration of the AT2-specific antagonist, PD 123319, had no effect. Ang II activated PLD with a dose dependence similar to that observed for aldosterone secretion, with slight increases in activity induced by 0.1 nM Ang II and maximal activation at 10 nM. We also found that Ang II induced a sustained activation of PLD, but that the effect of carbachol, a stable analogue of acetylcholine, was transient; PLD activity increased within 5 min of exposure to carbachol but then ceased by 15 min. Higher carbachol concentrations were also unable to sustain PLD activation. These results suggest that the Ang II-elicited activation of PLD is associated with a sustained increase in aldosterone secretion from glomerulosa cells and further provide the first evidence, to our knowledge, of differences in the kinetics of PLD activation in response to two physiologically relevant agonists. Finally, we speculate that this disparity correlates with different functional responses induced by the two agents.
Neutralizing epitope mapping of six β1-bungarotoxin monoclonal antibodies and its application in β1-bungarotoxin peptide vaccine design
Authors: YANG C. C. , CHAN H. L.
Published: August 2015

Twenty three stable monoclonal antibodies (mAbs) against β1-bungarotoxin (β1-bgt) were prepared by the hybridoma technique. Seven of the 23 mAbs (mAbs 2, 6, 8, 11, 17, 21 and 22) could inhibit more than 70% of phospholipase A2 activity of β1-bgt and neutralize the toxin. Six of these neutralizing mAbs (mAbs 2, 6, 8, 17, 21 and 22) recognized continuous epitopes on the A chain of β1-bgt and the other one (mAb 11) recognized a conformational epitope on the toxin. The continuous epitopes of these six mAbs were mapped using synthetic peptide and proteolytic enzymes. Experimental results indicate that mAb 17 recognized the A-chain residues 31-37; mAbs 2 and 8 recognized residues 46-51; mAbs 21 and 22 recognized residues 91-98; and mAb 6 recognized residue 100-106. The competitive-antibody-binding inhibition experiments showed that the affinity of these neutralizing mAbs to the native β1-bgt is compatible with synthetic peptides. Furthermore, mice immunized with BSA-conjugated A-chain-peptide sequences A(31-37), A(46-51), A(91-98) or A(100-106) were protected from a high-dose β1-bgt challenge. Subsequently, the peptide-immunized sera were passively injected into Balb/c mice and a significantly protective effect was also observed. To our knowledge, this study is the first systematic demonstration of multiple neutralizing B-cell epitopes of β1-bgt, and this study is also the first report of the protective synthetic-peptide vaccine against β1-bgt challenge.
Chondroadherin expression changes in skeletal development
Authors: SHEN Zhenxin , GANTCHEVA Svetlana , MÅNSSON Bengt , HEINEGÅRD Dick , SOMMARIN Yngve
Published: August 2015

Chondroadherin is a cartilage protein with cell binding properties. The expression of chondroadherin was studied in rat tissues and during postnatal femoral head development. For design of oligonucleotide probes and primers a 1664 bp, full length, rat chondroadherin cDNA was isolated from a rat chondrosarcoma library and sequenced. Northern blot analysis showed chondroadherin mRNA to be present in femoral head and rib cartilage, as well as in tendon. More sensitive reverse-transcriptase PCR additionally identified the mRNA in calvaria, long bone and bone marrow. Localization of chondroadherin by immunocytochemistry in the developing femoral head from postnatal day 14 to day 60 showed presence of the protein in cartilaginous regions. With increasing age a very distinct localization of chondroadherin was seen in the territorial matrix around late proliferative cells in the growth plate as well as in the developing articular cartilage in the maturing femoral head. Localization of chondroadherin mRNA by in situ hybridization was in agreement with immunocytochemistry with strong hybridization signals in late proliferative cells in the growth plate. In the articular cartilage the expression was restricted to cells in the lower regions. A three-fold increase of cartilage chondroadherin content in the growing femoral head was demonstrated by Western blot analysis. The high expression of this cell binding protein in a dynamic region of cartilage suggests an important role for chondroadherin in the regulation of chondrocyte growth and proliferation.
3-Nitrotyrosine in the proteins of human plasma determined by an ELISA method
Authors: KHAN Jamshad , BRENNAN M. David , BRADLEY Nicholas , GAO Beirong , BRUCKDORFER Richard , JACOBS Michael
Published: August 2015

The modification of tyrosine residues in proteins to 3-nitrotyrosine by peroxynitrite or other potential nitrating agents has been detected in biological systems that are subject to oxidative stress. A convenient semi-quantitative method has been developed to assay nitrated proteins in biological fluids and homogenates using a competitive ELISA developed in our laboratory. This assay selectivity detected 3-nitro-L-tyrosine residues in a variety of peroxynitrite-treated proteins (BSA, human serum albumin (HSA), α1-antiprotease inhibitor, pepsinogen and fibrinogen) and also in a nitrated peptide, but had a low affinity for free 3-nitro-L-tyrosine and 3-chloro-L-tyrosine. The IC50 values for the inhibition of antibody binding by different nitrated proteins were in the range 5-100 nM, suggesting that the antibody discriminated between nitrotyrosine residues in different environments. The presence of nitrotyrosine in plasma proteins was detected by Western blot analysis and quantified by the ELISA. A concentration of 0.12±0.01 μM nitro-BSA equivalents was measured in the proteins of normal plasma which was increased in peroxynitrite-treated plasma and was elevated in inflammatory conditions. HSA and low-density lipoprotein (LDL) isolated from plasma contained 0.085±0.04 and 0.03±0.006 nmol nitro-BSA equivalents/mg protein, respectively. Comparison of the level of nitration in peroxynitrite-treated HSA and LDL in the presence and absence of plasma indicates that nitration and presumably oxidation is inhibited by plasma antioxidants. The presence of nitrotyrosine in LDL is consistent with previous reports implicating peroxynitrite in the oxidative modification of lipoproteins and the presence of a low concentration of oxidized LDL in the blood.
Intracellular calcium signalling in rat parotid acinar cells that lack secretory vesicles
Authors: LIU Peixin , SCOTT John , SMITH Peter Matthew
Published: August 2015

Secretory vesicles from pancreatic acinar cells have recently been shown to release Ca2+ after stimulation with Ins(1,4,5)P3 [Gerasimenko, Gerasimenko, Belan and Petersen, (1996) Cell 84, 473-480]. These observations have been used in support of the hypothesis that Ca2+ release from secretory vesicles could be an important component of stimulus secretion coupling in exocrine acinar cells. In the rat, ligation of the parotid duct causes a reversible atrophy of the parotid gland. Most notably, after atrophy the acinar cells are reduced in size and no longer contain secretory vesicles [Liu, Smith, and Scott (1996) J. Dent. Res. 74, 900]. We have measured cytosolic free-Ca2+ concentration ([Ca2+]i) in single, acutely isolated, rat parotid acinar cells, and compared Ca2+ mobilization in response to acetylcholine (ACh) stimulation in cells obtained from control animals to that in cells lacking secretory vesicles obtained after atrophy of the parotid gland. Application of 50-5000 nM ACh to control cells gave rise to a typical, dose-dependent, biphasic increase in [Ca2+]i, of which the later, plateau, phase was acutely dependent on the extracellular Ca2+ concentration. An identical pattern of response was observed with cells obtained from atrophic glands. Low concentrations of ACh (10-100 nM) occasionally produced [Ca2+]i oscillations of a similar pattern in cells from both control and atrophic glands. We were able to show that Ca2+ rises first in the apical pole of the cell and the increase then spreads to the rest of the cell in cells from control glands but not in cells from atrophic glands. However, at present we are unable to determine whether this is due to the lack of secretory vesicles or whether the separation is too small to measure in the smaller acinar cells obtained from atrophic glands. We conclude therefore, that secretory vesicles make no significant contribution to overall Ca2+ mobilization in rat parotid acinar cells, nor are they required for oscillatory changes in [Ca2+]i to occur. However we are unable to eliminate completely any role for secretory vesicles in initiating Ca2+ mobilization at the apical pole of the cell.
A cDNA cloned from pregnant mouse uterus exhibits temporo-spatial expression and predicts a novel protein
Authors: KASIK W. John
Published: August 2015

A cDNA was cloned from a pregnant mouse uterus cDNA library. On conceptual translation, the cDNA has one long open reading frame that predicts a novel protein of 606 amino acids. This protein is principally composed of two CUB domains and a ZP domain; motifs found in proteins implicated in egg-sperm recognition. Probes derived from the cDNA were used to conduct Northern hybridizations. The expression of this mRNA is temporal; message first appears in the uterus 6 days prior to birth, it increases each subsequent day to attain maximal levels at 3 days prior to birth and then abruptly decreases during the last 3 days of pregnancy. The expression of this mRNA is restricted; message is abundant in the uterus during late pregnancy, but it is not found in non-pregnant uterus or in a variety of adult or fetal tissues. The temporo-spatial expression of this pregnant uterus specific mRNA and the consolidation in the predicted protein of two motifs implicated in early pregnancy events suggests that the product of the gene represented by this mRNA may play an important role in events that transpire during late pregnancy.
Protein kinase A regulates the disposition of Ca2+ which enters the cytoplasmic space through store-activated Ca2+ channels in rat hepatocytes by diverting inflowing Ca2+ to mitochondria
Authors: FERNANDO C. Kekulu , GREGORY B. Roland , BARRITT J. Greg
Published: August 2015

The roles of a trimeric GTP-binding regulatory protein, protein kinase A and mitochondria in the regulation of store-activated (thapsigargin-stimulated) Ca2+ inflow in freshly-isolated rat hepatocytes were investigated. Rates of Ca2+ inflow were estimated by measuring the increase in the fluorescence of intracellular fura-2 following the addition of extracellular Ca2+ (Ca2+o) to cells incubated in the absence of added Ca2+o. Guanosine 5ʹ-[γ-thio]-triphosphate (GTP[S]) and AlF4- inhibited the thapsigargin-stimulated Ca2+o-induced increase in cytoplasmic free Ca2+ concentration ([Ca2+]c) and this inhibition was prevented by the Rp diastereoisomer of adenosine 3ʹ,5ʹ-(cyclic)phosphoro[thioate]. cAMP, forskolin and glucagon (half-maximal effect at 10 nM) mimicked inhibition of the thapsigargin-stimulated Ca2+o-induced increase in [Ca2+]c by GTP[S], but had little effect on thapsigargin-induced release of Ca2+ from intracellular stores. Azide and carbonyl cyanide p-trifluoromethoxyphenylhydrazone inhibited the thapsigargin-stimulated Ca2+o-induced increase in [Ca2+]c in the presence of increased cAMP (induced by glucagon). In contrast, Ruthenium Red markedly enhanced the thapsigargin-stimulated Ca2+o-induced increase in [Ca2+]c in both the presence and absence of increased cAMP (induced by forskolin and dibutyryl cAMP). It is concluded that, in hepatocytes, protein kinase A regulates the disposition of Ca2+, which enters the cytoplasmic space through store-activated Ca2+ channels, by directing some of this Ca2+ to the mitochondria. The idea that caution should be exercised in using observed values of Ca2+o-induced increase in [Ca2+]c as estimates of rates of agonist-stimulated Ca2+ inflow is briefly discussed.
Cell-surface biotinylation of GLUT4 using bis-mannose photolabels
Authors: KOUMANOV Françoise , YANG Jing , JONES E. Alison , HATANAKA Yasumaru , HOLMAN D. Geoffrey
Published: August 2015

New cell-impermeant bis-mannose photolabels have been developed with biotinyl groups attached to 4-(1-azi-2,2,2-trifluoroethyl)-benzoyl-1,3-bis(d-mannos-4-yloxy)-2-propylamine (ATB-BMPA) by either a polyethoxy spacer (Bio-ATB-BMPA) or an additional hexanoic acid spacer (Bio-LC-ATB-BMPA). The half-maximal inhibition constants, Ki values, for inhibition of glucose transport activity in insulin-stimulated rat adipocytes were determined to be 359±10 and 273±28 μM for Bio-ATB-BMPA and Bio-LC-ATB-BMPA, respectively. These values are similar to those previously reported for the non-biotinylated compound ATB-BMPA. Following UV-irradiation-induced cross-linking of the biotinylated photolabels to rat adipocytes, the biotinylated glucose transporter isoform 4 (GLUT4) could be detected by non-radioactive and radioactive methods that utilized the interaction with streptavidin. Biotinylated GLUT4 from 1-2 μg of adipose cell membranes, precipitated onto magnetic streptavidin beads, could be sensitively and quantitatively detected using an electrochemiluminescent assay method. This utilized a ruthenium-tagged anti-GLUT4 antibody that on excitation at an electrode generated an electrochemiluminescent signal in an ORIGEN analyser. Alternatively, surface-biotinylated GLUT4 could be easily, but less sensitively, detected in streptavidin agarose precipitates which were analysed by conventional GLUT4 Western blotting. Data obtained using the non-radioactive methods compared favourably with those using tritiated versions of the biotinylated probes. Insulin treatment of adipocytes increased the levels of signals from surface biotinylated GLUT4 by ~ 10-fold or ~ 20-fold, respectively, when the electrochemiluminescent or the Western blot detection methods were used and these signals were blocked by cytochalasin B.
Molecular characterization of benzyl alcohol dehydrogenase and benzaldehyde dehydrogenase II of Acinetobacter calcoaceticus
Authors: GILLOOLY J. David , ROBERTSON G. S. Alan , FEWSON A. Charles
Published: August 2015

The nucleotide sequences of xylB and xylC from Acinetobacter calcoaceticus, the genes encoding benzyl alcohol dehydrogenase and benzaldehyde dehydrogenase II, were determined. The complete nucleotide sequence indicates that these two genes form part of an operon and this was supported by heterologous expression and physiological studies. Benzaldehyde dehydrogenase II is a 51654 Da protein with 484 amino acids per subunit and it is typical of other prokaryotic and eukaryotic aldehyde dehydrogenases. Benzyl alcohol dehydrogenase has a subunit Mr of 38923 consisting of 370 amino acids, it stereospecifically transfers the proR hydride of NADH, and it is a member of the family of zinc-dependent long-chain alcohol dehydrogenases. The enzyme appears to be more similar to animal and higher-plant alcohol dehydrogenases than it is to most other microbial alcohol dehydrogenases. Residue His-51 of zinc-dependent alcohol dehydrogenases is thought to be necessary as a general base for catalysis in this category of alcohol dehydrogenases. However, this residue was found to be replaced in benzyl alcohol dehydrogenase from A. calcoaceticus by an isoleucine, and the introduction of a histidine residue in this position did not alter the kinetic coefficients, pH optimum or substrate specificity of the enzyme. Other workers have shown that His-51 is also absent from the TOL-plasmid-encoded benzyl alcohol dehydrogenase of Pseudomonas putida and so these two closely related enzymes presumably have a catalytic mechanism that differs from that of the archetypal zinc-dependent alcohol dehydrogenases.
Permissive role of cAMP in the oscillatory Ca2+ response to inositol 1,4,5-trisphosphate in rat hepatocytes
Authors: CHATTON Jean-Yves , CAO Yumei , LIU Haiyan , STUCKI Jörg W.
Published: August 2015

Rat hepatocytes respond to α-adrenergic stimulation by intracellular production of myo-inositol 1,4,5-trisphosphate (IP3) which stimulates the periodic release and reuptake of intracellular store (IS) Ca2+. The generation of these Ca2+ oscillations was investigated by simultaneously monitoring Ca2+ changes in the cytosol and IS by combined fluorescence microscopy and whole-cell patch clamp. Intracellular IP3 perfusion (1-50 μM in the pipette) produced three types of Ca2+ response: understimulation, oscillations and overstimulation, i.e. with Ca2+ levels not returning to baseline. In a total of 57 experiments, only three displayed oscillations during continuous IP3 infusion, in a narrow range of IP3 concentration centred around 5-8 μM in the pipette. In oscillating cells, cytosolic Ca2+ spikes were synchronized with transient Ca2+ depletions of the IS, consistent with a direct exchange of Ca2+ between the two compartments. Application of 8-Br-cAMP to cells infused with IP3 increased the probability of eliciting Ca2+ oscillations by a factor of 4-5 for IP3 concentrations in the range 1-10 μM, whereas IP3 concentrations above 10 μM always resulted in overstimulation. IP3 photorelease experiments and measurements of IS Ca2+ content indicated that 8-Br-cAMP enhanced the affinity of the IP3 receptor and increased the pool of releasable Ca2+. We propose that cAMP has a permissive role in the generation of IP3-induced Ca2+ oscillations by extending the window of IP3 concentrations able to elicit oscillations.
3,5,3′-Tri-iodo-l-thyronine acutely regulates a protein kinase C-sensitive, Ca2+-independent, branch of the hepatic α1-adrenoreceptor signalling pathway
Authors: DAZA Francisco J. , PARRILLA Roberto , MARTÍN-REQUERO Angeles
Published: August 2015

This work aimed to investigate the acute effect of the thyroid hormone 3,5,3´-tri-iodo-l-thyronine (T3) in regulating the hepatic metabolism either directly or by controlling the responsiveness to Ca2+-mobilizing agonists. We did not detect any acute metabolic effect of T3 either in perfused liver or in isolated liver cells. However, T3 exerted a powerful inhibitory effect on the α1-adrenoreceptor-mediated responses. The promptness of this T3 effect rules out that it was the result of rate changes in gene(s) transcription. T3 inhibited the α1-adrenoreceptor-mediated sustained stimulation of respiration and release of Ca2+ and H+, but not the glycogenolytic or gluconeogenic responses, in perfused liver. In isolated liver cells, T3 enhanced the α1-agonist-induced increase in cytosolic free Ca2+ and impeded the intracellular alkalinization. Since T3 also prevented the α1-adrenoreceptor-mediated activation of protein kinase C, its effects on pH seem to be the result of a lack of activation of the Na+/H+ exchanger. The failure of T3 to prevent the α1-adrenergic stimulation of gluconeogenesis despite the inhibition of protein kinase C activation indicates that the elevation of cytosolic free Ca2+ is a sufficient signal to elicit that response. T3 also impaired some of the angiotensin-II-mediated responses, but did not alter the effects of PMA on hepatic metabolism, indicating, therefore, that some postreceptor event is the target for T3 actions. The differential effect of T3 in enhancing the α1-adrenoreceptor-mediated increase in cytosolic free Ca2+ and preventing the activation of protein kinase C, provides a unique tool for further investigating the role of each branch of the signalling pathway in controlling the hepatic functions. Moreover, the low effective concentrations of T3 (⩽ 10 nM) in perturbing the α1-adrenoreceptor-mediated response suggests its physiological significance.
Effect of replacement of His-118, His-125 and Trp-143 by alanine on the catalytic activity and subunit assembly of inorganic pyrophosphatase from thermophilic bacterium PS-3
Authors: AOKI Motoko , UCHIUMI Toshio , TSUJI Eiichi , HACHIMORI Akira
Published: August 2015

Each of two histidine residues and one tryptophan residue in thermophilic bacterium PS-3 inorganic pyrophosphatase (PPase) was replaced by alanine. The activities of the H125A and W143A variants decreased to one-fifth, whereas the activity of H118A remained unaltered. CD spectra in the near-UV region indicated that the conformations of the first two variants changed with the substitution. In contrast with wild-type PPase, which is hexameric beyond an enzyme concentration of 0.1 µM in the presence of Mg2+, the H118A and H125A variants cannot be assembled from trimers into hexamers at less than an enzyme concentration of 10 µM even at a higher concentration of Mg2+. In particular, H118A was irreversibly inactivated in a diluted state. In contrast, the enzyme concentration dependence of W143A PPase activity was almost the same as that of wild-type PPase. These results indicated that His-118 and His-125 are important for both trimer–trimer interaction and structural integrity, whereas Trp-143 is important structurally. The trimer–trimer interaction is absolutely necessary for the thermostability of the PS-3 enzyme.
A mitochondrial membrane protein is required for translocation of phosphatidylserine from mitochondria-associated membranes to mitochondria
Authors: SHIAO Young-Ji , BALCERZAK Bénédicte , VANCE Jean E.
Published: August 2015

The mechanism of import of phosphatidylserine (PtdSer) into mitochondria was investigated using a reconstituted system of isolated organelles in vitroin which PtdSer was translocated from donor membranes to mitochondria and was decarboxylated therein. Neither phosphatidylcholine nor phosphatidylethanolamine (PtdEtn) was translocated under the same conditions. Transfer of PtdSer from its site of synthesis on the endoplasmic reticulum and mitochondria-associated membranes [J. E. Vance (1990) J. Biol. Chem. 265, 7248–7256] to its site of decarboxylation on mitochondrial inner membranes is predicted to be mediated by membrane contact. A mitochondrial membrane protein appears to be involved in the translocation event since proteolysis of proteins exposed on the mitochondrial surface potently inhibited PtdSer transfer, whereas proteolysis of surface proteins of mitochondria-associated membranes did not impair the transfer. The nature of the membranes that donate PtdSer to mitochondria in vitrois not crucial since PtdSer of mitochondria-associated membranes, endoplasmic reticulum and microsomes was decarboxylated to PtdEtn with approximately equal efficiency. The translocation of PtdSer to mitochondria was stimulated by magnesium and calcium ions and was inhibited by incubation of mitochondria with sulphydryl group-modifying reagents. Reconstitution of PtdSer translocation/decarboxylation using digitonin-solubilized mitochondria and PtdSer-donor membranes suggested that the putative PtdSer-translocation protein is primarily localized to contract sites between mitochondrial inner and outer membranes. These studies provide evidence for the involvement of a mitochondrial membrane protein in the import of newly-synthesized PtdSer into mitochondria.
Phospholipase C-δ1 and oxytocin receptor signalling: evidence of its role as an effector
Authors: PARK Eu-Suk , WON Jong Hyun , HAN Kee Jung , SUH Pann-Ghill , RYU Sung Ho , LEE Hee Sung , YUN Hye-Young , KWON Nyoun Soo , BAEK Kwang Jin
Published: August 2015

Although the oxytocin receptor modulates intracellular Ca2+ ion levels in myometrium, the identities of signal molecules have not been clearly clarified. Our previous studies on oxytocin receptor signalling demonstrated that 80 kDa Ghα is a signal mediator [Baek, Kwon, Lee, Kim, Muralidhar and Im (1996) Biochem. J. 315, 739–744]. To elucidate the effector in the oxytocin receptor signalling pathway, we evaluated the oxytocin-mediated activation of phospholipase C (PLC) by using solubilized membranes from human myometrium and a three-component preparation containing the oxytocin receptor–Ghα–PLC-δ1 complex. PLC-δ1 activity in the three-component preparation, as well as PLC activity in solubilized membranes, was increased by oxytocin in the presence of Ca2+ and activated Ghα (GTP-bound Ghα). Furthermore the stimulated PLC-δ1 activity resulting from activation of Ghα via the oxytocin receptor was significantly attenuated by the selective oxytocin antagonist desGly-NH2d(CH2)5[Tyr(Me)2,Thr4]ornithine vasotocin or GDP. Consistent with these observations, co-immunoprecipitation and co-immunoadsorption of PLC-δ1 in the three-component preparation by anti-Gh7α antibody resulted in the PLC-δ1 being tightly coupled to activated Ghα on stimulation of the oxytocin receptor. These results indicate that PLC-δ1 is the effector for Ghα-mediated oxytocin receptor signalling.
Functional expression of TrpC1: a human homologue of the Drosophila Trp channel
Authors: SINKINS William G. , ESTACION Mark , SCHILLING William P.
Published: August 2015

TrpC1 appears to be a store-operated channel (SOC) when expressed in mammalian cells. In the present study, TrpC1 was expressed in Sf9 insect cells using the baculovirus expression system. Expression of TrpC1 caused an increase in basal cytosolic free Ca2+ concentration ([Ca2+]i) as a function of post-infection time. Basal Ba2+ influx, an index of plasmalemmal Ca2+ permeability, was also increased and was blocked by La3+. Although the thapsigargin-induced change in [Ca2+]i was greater in TrpC1-expressing cells than controls, Ba2+ influx was unaffected by thapsigargin. Whole-cell membrane currents recorded in TrpC1-expressing cells increased as a function of post-infection time and were (1) inwardly rectifying in symmetrical sodium gluconate solutions, (2) non-selective with respect to Na+, Ca2+ and Ba2+, and (3) blocked by La3+. Furthermore TrpC1 currents were unaffected by (1) thapsigargin, (2) dialysis of the cell with Ins(1,4,5)P3 or (3) dialysis of the cell with solutions containing high concentrations of the Ca2+ chelator, EGTA. These results suggest that TrpC1 forms non-selective cation channels that are constitutively active when expressed in Sf9 cells, but insensitive to depletion of the internal Ca2+ stores. Thus TrpC1 may be a subunit of a SOC which alone can form functional channels in Sf9 cells, but which requires additional subunits or cytoplasmic factors present in mammalian cells for expression of SOC activity.
The dhnA gene of Escherichia coli encodes a Class I fructose bisphosphate aldolase
Authors: THOMSON Graeme J. , HOWLETT Geoffrey J. , ASHCROFT Alison E. , BERRY Alan
Published: August 2015

The gene encoding the Escherichia coli Class I fructose-1,6-bisphosphate aldolase (FBP aldolase) has been cloned and the protein overproduced in high amounts. This gene sequence has previously been identified as encoding an E. coli dehydrin in the GenBank™ database [gene dhnA; entry code U73760; Close and Choi (1996) Submission to GenBank™]. However, the purified protein overproduced from the dhnA gene shares all its properties with those known for the E. coli Class I FBP aldolase. The protein is an 8–10-mer with a native molecular mass of approx. 340 kDa, each subunit consisting of 349 amino acids. The Class I enzyme shows low sequence identity with other known FBP aldolases, both Class I and Class II (in the order of 20%), which may be reflected by some novel properties of this FBP aldolase. The active-site peptide has been isolated and the Schiff-base-forming lysine residue (Lys236) has been identified by a combination of site-directed mutagenesis, kinetics and electrospray-ionization MS. A second lysine residue (Lys238) has been implicated in substrate binding. The cloning of this gene and the high levels of overexpression obtained will facilitate future structure–function studies.
Species-specificity in endoplasmic reticulum signal peptide utilization revealed by proteins from Trypanosoma brucei and Leishmania
Authors: AL-QAHTANI Ahmed , TEILHET Meredith , MENSA-WILMOT Kojo
Published: August 2015

N-Terminal signal peptides direct secretory and most membrane proteins into the exocytic pathway at the endoplasmic reticulum. Signal sequences can function across kingdoms. However, our attempts at translocating variant surface glycoprotein (VSG) 117, VSG MVAT7, VSG 221 and BiP from Trypanosoma brucei and gp63 from Leishmania chagasi into canine pancreas microsomes failed. On replacing the signal peptide of VSG 117 with that from yeast prepro-α-mating factor (ppαMF) the chimaeric protein was imported, indicating that the signal sequence of VSG 117 was incompatible with the protein-import machinery of mammalian microsomes. Replacement of the gp63-h-region with a hybrid composed of the N-terminal nine residues from the h-region of gp67 from Autographa californica nuclear polyhedrosis virus and the C-terminal 10 residues from the h-region of gp63 from L. major produced a functional signal peptide. Thus, the h-region of kinetoplastid signal peptides appears to be the subdomain that is non-functional at the mammalian translocon. The calculated biophysical properties and computed discriminant scores (predictive of importability of signal peptides into mammalian microsomes) of the kinetoplastid signal sequences nevertheless are similar to those of ppαMF and Escherichia coliβ-lactamase both of which were imported. These signal peptides are the first collection from one biological family that have been found to fail to function across a species barrier. They indicate that signal peptides are not as universally interchangeable as previously believed. Intriguingly, endoplasmic reticulum signal peptides from Leishmania and Crithidia fasciculata are reminiscent of signal peptides from Gram-positive bacteria.
Mevalonate-derived isopentenyl diphosphate is the biosynthetic precursor of ubiquinone prenyl side chain in tobacco BY-2 cells
Authors: DISCH Andrea , HEMMERLIN Andréa , BACH Thomas J. , ROHMER Michel
Published: August 2015

Study of the incorporation of 13C-labelled glucose or pyruvate into the isoprenoids of tobacco BY-2 cells allowed the biosynthetic origin of isopentenyl diphosphate to be determined. Sterols synthesized in the cytoplasm and the prenyl chain of ubiquinone Q10 located in mitochondria were derived from the same isopentenyl diphosphate pool, synthesized from acetyl-CoA through mevalonate, whereas the prenyl chain of plastoquinone was obtained from the mevalonate-independent glyceraldehyde 3-phosphate/pyruvate route, like all chloroplast isoprenoids from higher plants. These results are in accord with the compartmentation and complete enzymic independence of the biosynthesis of long-chain all-trans polyprenols in mitochondria and chloroplasts.
S-Nitrosoglutathione is a substrate for rat alcohol dehydrogenase class III isoenzyme
Authors: JENSEN David E. , BELKA George K. , BOIS Garrett C. Du
Published: August 2015

An enzyme isolated from rat liver cytosol (native molecular mass 78.3 kDa; polypeptide molecular mass 42.5 kDa) is capable of catalysing the NADH/NADPH-dependent degradation of S-nitrosoglutathione (GSNO). The activity utilizes 1 mol of coenzyme per mol of GSNO processed. The isolated enzyme has, as well, several characteristics that are unique to alcohol dehydrogenase (ADH) class III isoenzyme: it is capable of catalysing the NAD+-dependent oxidations of octanol (insensitive to inhibition by 4-methylpyrazole), methylcrotyl alcohol (stimulated by added pentanoate) and 12-hydroxydodecanoic acid, and also the NADH/NADPH-dependent reduction of octanal. Methanol and ethanol oxidation activity is minimal. The enzyme has formaldehyde dehydrogenase activity in that it is capable of catalysing the NAD+/NADP+-dependent oxidation of S-hydroxymethylglutathione. Treatment with the arginine-specific reagent phenylglyoxal prevents the pentanoate stimulation of methylcrotyl alcohol oxidation and markedly diminishes the enzymic activity towards octanol, 12-hydroxydodecanoic acid and S-hydroxymethylglutathione; the capacity to catalyse GSNO degradation is also checked. Additionally, limited peptide sequencing indicates 100% correspondence with known ADH class III isoenzyme sequences. Kinetic studies demonstrate that GSNO is an exceptionally active substrate for this enzyme. S-Nitroso-N-acetylpenicillamine and S-nitrosated human serum albumin are not substrates; the activity towards S-nitrosated glutathione mono- and di-ethyl esters is minimal. Product analysis suggests that glutathione sulphinamide is the major stable product of enzymic GSNO processing, with minor yields of GSSG and NH3; GSH, hydroxylamine, nitrite, nitrate and nitric oxide accumulations are minimal. Inclusion of GSH in the reaction mix decreases the yield of the supposed glutathione sulphinamide in favor of GSSG and hydroxylamine.
Effects of pH on the structure and function of neuronal nitric oxide synthase
Authors: GORREN Antonius C. F. , SCHRAMMEL Astrid , SCHMIDT Kurt , MAYER Bernd
Published: August 2015

We investigated how pH affects rat brain neuronal nitric oxide synthase (nNOS) with regard to spin-state equilibrium and the thiolate ligand bond of the haem group, catalytic activity, and monomer ↔ dimer equilibrium. At neutral pH, nNOS containing 1 equiv. of (6R)-5,6,7,8-tetrahydro-l-biopterin (BH4) per dimer was mostly high-spin (λmax at 398 nm), whereas the BH4-free enzyme consisted of a mixture of the high-spin and two low-spin forms (λmax at 418 nm, and at 376 and 456 nm respectively). With BH4-free nNOS, an appreciable high-spin fraction was only observed between pH 7 and 8; at pH 6 and 9, the 418 and 376/456 nm low-spin forms predominated respectively. With nNOS containing 1 equiv. of BH4 per dimer, similar observations were made, but these involved only half of the enzyme; the other half, presumably the BH4-containing subunits, remained high-spin. Since the spin state in the BH4-free subunit appeared little affected by the state of the other subunit, we conclude that, in dimeric nNOS, the two haem groups function independently. Low pH destabilized thiolate binding and the interaction between NOS subunits, as indicated by CO-binding studies and gel electrophoresis respectively. Formation of l-citrulline was optimal between pH 7.0 and 7.5; the decrease in NOS activity at lower pH proved to be due to uncoupling of NADPH oxidation, resulting in increased formation of H2O2. At high pH strict coupling of l-arginine and NADPH oxidation was maintained, even in the absence of exogenous BH4. The possible pathophysiological implications of the uncoupling at low pH are discussed.
Alanine-261 in intracellular loop III of the human gonadotropin-releasing hormone receptor is crucial for G-protein coupling and receptor internalization
Authors: MYBURGH David B. , MILLAR Robert P. , HAPGOOD Janet P.
Published: August 2015

Gonadotropin-releasing hormone (GnRH) is a decapeptide that regulates reproductive function via binding to the GnRH receptor, which is a G-protein-coupled receptor (GPCR). For several members of this family, the C-terminal domain of intracellular loop III is important in ligand-mediated coupling to G-proteins; mutations in that region can lead to constitutive activity. A specific alanine residue is involved in certain GPCRs, the equivalent of which is Ala-261 in the GnRH receptor. Mutation of this residue to Leu, Ile, Lys, Glu or Phe in the human GnRH receptor did not result in constitutive activity and instead led to complete uncoupling of the receptor (failure to support GnRH-stimulated inositol phosphate production). When this residue was mutated to Gly, Pro, Ser or Val, inositol phosphate production was still supported. All the mutants retained the ability to bind ligand, and the affinity for ligand, where measured, was unchanged. These results show that Ala-261 cannot be involved in ligand binding but is critical for coupling of the receptor to its cognate G-protein. Coupling is also dependent on the size of the residue in position 261. When the amino acid side chain has a molecular mass of less than 40 Da efficient coupling is still possible, but when its molecular mass exceeds 50 Da the receptor is uncoupled. Internalization studies on the Ala261 → Lys mutant showed a marked decrease in receptor internalization compared with the wild type, indicating that coupling is necessary for effective receptor internalization in the GnRH receptor system. Activation of protein kinase C (with PMA), but not protein kinase A (with forskolin) markedly increased the internalization of the mutant receptor while having a small effect on the wild-type receptor.
Rac1, and not Rac2, is involved in the regulation of the intracellular hydrogen peroxide level in HepG2 cells
Authors: COOL Robbert H. , MERTEN Evelyne , THEISS Christiane , ACKER Helmut
Published: August 2015

In order to elucidate the components of the oxygen sensory complex in HepG2 cells which regulates the production of erythropoietin, we have microinjected recombinant variants of the human small GTP-binding protein hRac1 and measured their effects on the production of reactive oxygen species (ROS) by the dihydrorhodamine-123 technique. The dominant-negative mutant hRac1(T17N) inhibits the NADH-stimulated production of ROS in HepG2 cells, whereas the constitutively activated hRac1(G12V) leads to an increase in intracellular ROS concentration. Reverse transcriptase PCR analysis showed that the hRac1, but not the hRac2, gene is expressed in HepG2 cells. These results demonstrate that hRac1, and not hRac2, is involved in the regulation of ROS production in HepG2 cells and suggest that hRac1 specifically functions in the non-phagocytic NAD(P)H oxidase complex.
An esterase from Escherichia coli with a sequence similarity to hormone-sensitive lipase
Authors: KANAYA Shigenori , KOYANAGI Tomoyoshi , KANAYA Eiko
Published: August 2015

An esterase from Escherichia colithat is a member of the hormone-sensitive lipase (HSL) family was overproduced, purified and characterized. It is encoded by the ybaCgene and composed of 319 amino acid residues with an Mr of 36038. The enzymic activity was determined by using various p-nitrophenyl esters of fatty acids as a substrate at 25 °C and pH 7.1. The enzyme showed hydrolytic activity towards substrates with an acyl chain length of less than 8, whereas it showed little hydrolytic activity towards those with an acyl chain length of more than 10. In addition, it showed little hydrolytic activity towards trioleoylglycerol and cholesterol oleate. Determination of the kinetic parameters for the hydrolyses of the substrates from C2 to C8 indicates that C4 and C5 substrates are the most preferred. Close agreement between the Mr determined by SDS/PAGE (37000) and column chromatography (38000) suggests that the enzyme exists in a monomeric form. It is an acidic protein with a pI value of 4.1. The far-UV CD spectrum suggests that its helical content is 26.1%. Comparison of the amino acid sequence of this enzyme with those involved in the HSL family allows us to propose that Ser165, Asp262 and His292 constitute the catalytic triad of E. coliesterase.
Spectrin self-association site: characterization and study of β-spectrin mutations associated with hereditary elliptocytosis
Authors: NICOLAS Gaël , PEDRONI Sophie , FOURNIER Catherine , GAUTERO Huguette , CRAESCU Constantin , DHERMY Didier , LECOMTE M. Christine
Published: August 2015

Most of hereditary elliptocytosis (HE) cases are related to a spectrin dimer (SpD) self-association defect. The severity of haemolysis is correlated with the extent of the SpD self-association defect, which itself depends on the location of the mutation regarding the tetramerization site. This site is presumed to involve the first C helix of the α chain and the last two helices, A and B, of the β chain to reconstitute a triple helical structure (A, B and C), as observed along spectrin. Using recombinant peptides, we demonstrated that the first C helix of the α chain and the last two helices of the β chain alone are not sufficient to establish interactions, which only occurred when a complete triple-helical repeat was added to each partner. One adjacent repeat is necessary to stabilize the conformation of both N- and C-terminal structures directly involved in the interaction site and is sufficient to generate a binding affinity similar to that observed in the native molecule. Producing peptides carrying a βHE mutation, we reproduced the tetramerization defect as observed in patients. Therefore, the βW2024R and βW2061R mutations, which replace the invariant tryptophan and a residue located in the hydrophobic core, respectively, affect α–β interactions considerably. In contrast, the βA2013V mutation, which modifies a residue located outside any presumed interacting regions, has a minor effect on the interaction.
Reactivation of triosephosphate isomerase from three trypanosomatids and human: effect of Suramin
Authors: GAO Xiu-Gong , GARZA-RAMOS Georgina , SAAVEDRA-LIRA Emma , CABRERA Nallely , de GÓMEZ-PUYOU Marietta T. , PEREZ-MONTFORT Ruy , GÓMEZ-PUYOU Armando
Published: August 2015

The reactivation of the homodimeric triosephosphate isomerases (TIMs) from Trypanosoma brucei, T. cruzi, Leishmania mexicana and humans was determined after their denaturation with guanidine hydrochloride. In the range of 2–32 µg of T. brucei TIM per ml and 0.2–5 µg of the other enzymes per ml, the rate and extent of TIM reactivation depended on protein concentration, indicating that at these protein concentrations, the rate-limiting step of reactivation is monomer association and not monomer folding. The rate of monomer association was more than one order of magnitude lower in the T. brucei enzyme than in the other three enzymes. Suramin is a drug of choice in the treatment of sleeping sickness, but its mechanism of action is not known. At micromolar concentrations, Suramin inhibited the reactivation of the four enzymes, but the extent of inhibition by Suramin decreased with increasing protein concentration as consequence of a diminution of the life time of the folded monomer. Since the life time of the monomer of T. brucei TIM is longer than that of the other enzymes, Suramin is a more effective inhibitor of the reactivation of TIM from T. brucei, particularly at monomer concentrations above 1 µg of protein per ml (monomer concentration approx. 37 nM). Compounds that are structurally related to Suramin also inhibit TIM reactivation; their effect was about five times more pronounced in the enzyme from T. brucei than in human TIM.
Nitroarginine and tetrahydrobiopterin binding to the haem domain of neuronal nitric oxide synthase using a scintillation proximity assay
Authors: ALDERTON Wendy K. , BOYHAN Angela , LOWE Peter N.
Published: August 2015

Nitric oxide synthases (NOS) have a bidomain structure comprised of an N-terminal oxygenase domain and a C-terminal reductase domain. The oxygenase domain binds haem, (6R)-5,6,7,8-tetrahydro-l-biopterin (tetrahydrobiopterin) and arginine, is the site where nitric oxide synthesis takes place and contains determinants for dimeric interactions. A novel scintillation proximity assay has been established for equilibrium and kinetic measurements of substrate, inhibitor and cofactor binding to a recombinant N-terminal haem-binding domain of rat neuronal NOS (nNOS). Apparent Kd values for nNOS haem-domain-binding of arginine and Nω-nitro-l-arginine (nitroarginine) were measured as 1.6 µM and 25 nM respectively. The kinetics of [3H]nitroarginine binding and dissociation yielded an association rate constant of 1.3×104 s-1·M-1 and a dissociation rate constant of 1.2×10-4 s-1. These values are comparable to literature values obtained for full-length nNOS, suggesting that many characteristics of the arginine binding site of NOS are conserved in the haem-binding domain. Additionally, apparent Kd values were compared and were found to be similar for the inhibitors, l-NG-monomethylarginine, S-ethylisothiourea, N-iminoethyl-l-ornithine, imidazole, 7-nitroindazole and 1400W (N-[3-(aminomethyl) benzyl] acetamidine). [3H]Tetrahydrobiopterin bound to the nNOS haem domain with an apparent Kd of 20 nM. Binding was inhibited by 7-nitroindazole and stimulated by S-ethylisothiourea. The kinetics of interaction with tetrahydrobiopterin were complex, showing a triphasic binding process and a single off rate. An alternating catalytic site mechanism for NOS is proposed.