bluuebunny/crossref_metadata_2025 huggingface dataset:
Glucose disposal by skeletal muscle in response to re-feeding after progressive starvation
Authors: Holness M J , Sugden M C
Published: August 2015

We investigated the extent to which increases in glucose utilization indices (GUIs) in individual skeletal muscles during chow re-feeding after 6 h, 24 h or 48 h starvation are related to the antecedent duration of starvation. Chow re-feeding after either acute or prolonged starvation led to an increase in glucose disposal by the muscle mass. Glucose intolerance after prolonged starvation was not associated with lower values of GUI in skeletal muscle. In both working and non-working muscles, the increment in GUI during the first 2 h of re-feeding was less after acute than after prolonged starvation. In non-working muscles the differential responses to re-feeding were due to higher GUI values after re-feeding rather than lower pre-prandial GUI values. Therefore the contribution of non-working muscles to glucose clearance is higher as the antecedent period of starvation is extended. Rates of glycogen deposition in non-working muscles after refeeding were similar to absolute values of GUI, and a strong relationship existed between measured GUI values and rates of glycogen deposition.
Characterization of the solubilized glibenclamide receptor in a hamster pancreatic β-cell line, HIT T15
Authors: Niki I , Welsh M , Berggren P O , Hubbard P , Ashcroft S J H
Published: August 2015

The glibenclamide receptor, a putative ATP-sensitive K+ channel in the hamster pancreatic beta-cell line HIT T15, was solubilized by using the zwitterionic detergent CHAPS. [3H]Glibenclamide binding was dependent on the incubation time and on the concentration of soluble membrane protein. Over 80% of [3H]glibenclamide bound could be displaced with 1 microM non-labelled glibenclamide. The curve relating specific binding to the concentration of [3H]glibenclamide (1-20 nM) showed saturation kinetics. Scatchard analysis suggested a single class of non-interacting binding sites with a Kd of 3.3 nM and a Bmax. of 90 fmol/mg of protein. [3H]Glibenclamide binding to solubilized membranes was inhibited by glibenclamide, tolbutamide and meglitinide. The relative potency of these agents on binding of [3H]glibenclamide to solubilized membranes was similar to that observed with microsomal preparations and paralleled their effects on K-ATP channel activity, measured as 86Rb efflux. These data show that the sulphonylurea receptor in the pancreatic beta-cell can be solubilized in an active form retaining specificity for sulphonylureas. ADP, which inhibits [3H]glibenclamide binding to microsomal preparations or intact HIT beta-cells, did not inhibit binding to the solubilized receptor. Incubation of intact HIT beta-cells with 125I-glibenclamide derivative followed by exposure to u.v. light resulted in covalent labelling of a peptide of 65 kDa on SDS/PAGE. The extent of labelling increased with 125I-glibenclamide derivative concentration (1-20 nM) and was inhibited in the presence of excess unlabelled glibenclamide.
Structural and functional studies on the human hepatic interleukin-6 receptor. Molecular cloning and overexpression in HepG2 cells
Authors: Schooltink H , Stoyan T , Lenz D , Schmitz H , Hirano T , Kishimoto T , Heinrich P C , Rose-John S
Published: August 2015

cDNAs coding for the human hepatic interleukin-6 receptor (IL-6-R) have been isolated from a library made from poly(A) RNA of dexamethasone-treated human hepatoma cells (HepG2). We found the hepatic IL-6-R to be identical to the one expressed by leucocytes. A polyclonal antiserum was raised in rabbits against the IL-6-R protein expressed in Escherichia coli. Although the entire IL-6-R protein was used for immunization, only antibodies to the cytoplasmic domain of the IL-6-R were obtained. It is demonstrated by affinity cross-linking and subsequent immunoprecipitation with antibodies against the ligand as well as against the receptor that the cloned cDNA codes for the functional IL-6-R on HepG2 cells. When the hepatic IL-6-R cDNA was overexpressed in HepG2 cells, these cells became more sensitive to low concentrations of IL-6 with respect to the induction of gamma-fibrinogen mRNA.
Activities of enzymes of glycerolipid synthesis in brown adipose tissue after treatment of rats with the adrenergic agonists BRL 26830A and phenylephrine, after exposure to cold and in streptozotocin-diabetes
Authors: Mitchell J R D , Saggerson E D
Published: August 2015

1. Measurements were made, relative to tissue DNA, of the activities of enzymes of glycerolipid synthesis in homogenates of interscapular brown adipose tissue. These were: mitochondrial and microsomal forms of glycerolphosphate acyltransferase (GPAT), Mg(2+)-dependent phosphatidate phosphohydrolase (PPH) and fatty acyl-CoA synthetase (FAS). 2. In normal animals, 3 days of cold-exposure (4 degrees C) increased all activities. The increase in mitochondrial GPAT activity was particularly pronounced (5-fold). Administration of the beta-adrenergic agonist BRL 26830A mimicked the effect of cold on microsomal GPAT activity. Mitochondrial GPAT, PPH and FAS activities were unresponsive to BRL 26830A. The alpha-adrenergic agonist phenylephrine significantly decreased activities of GPAT and PPH. 3. Streptozotocin-diabetes decreased mitochondrial GPAT activity, but did not abolish the effect of cold to increase this activity or the activity of microsomal GPAT. Diabetes abolished the effect of cold on PPH and FAS activities. 4. The findings are relevant to signals that drive early events in mitochondriogenesis and cell proliferation in brown adipose tissue on exposure to cold.
In resting conditions, the pancreatic granule membrane protein GP-2 is secreted by cleavage of its glycosylphosphatidylinositol anchor
Authors: Paul E , Leblond F A , LeBel D
Published: August 2015

GP-2 is the major membrane protein of the exocrine pancreatic secretory granule. It is an integral protein which is anchored by a phosphatidylinositolglycan. In addition to being present in the soluble contents of the granule, GP-2 is also actively secreted by the pancreas. Although 93% of the GP-2 in the resting secretions of anaesthetized rats could be pelleted, Triton X-114 phase extraction showed that 70% of this GP-2 had lost its hydrophobic properties. Proteases have been postulated to release GP-2 from the membrane, but phospholipases also have the capacity to release the protein from the membrane by hydrolysis of its peculiar glycosylphosphatidylinositol membrane anchor. These studies show the presence of inositol 1,2-(cyclic)monophosphate on the secreted hydrophilic GP-2, confirming the involvement of an endogenous phospholipase C in the solubilization of GP-2 by the exocrine pancreas. It is therefore concluded that most of the GP-2 secreted by the pancreas of anaesthetized rats under resting conditions is released from the membrane by a phospholipase C which hydrolyses the phosphodiester bond linking GP-2 to its diradylglycerol anchor.
Attenuated adenosine-sensitivity and decreased adenosine-receptor number in adipocyte plasma membranes in human obesity
Authors: Kaartinen J M , Hreniuk S P , Martin L F , Ranta S , LaNoue K F , Ohisalo J J
Published: August 2015

Fat-cells were isolated from patients of body-mass indices (BMIs) ranging from 17.9 to 83.9 kg/m2. Isoprenaline-stimulated cyclic AMP accumulation in cells prepared from obese subjects as compared with normal-weight subjects, was less sensitive to inhibition by the adenosine agonist N6-(phenylisopropyl)adenosine (PIA) (P = 0.047). The inhibition of 7 beta-desacetyl-7 beta-[gamma-(N-methylpiperazino) butyryl]-forskolin-stimulated adenylate cyclase by PIA in the presence of adenosine deaminase was also much attenuated in crude plasma membranes of adipocytes prepared from massively obese patients as compared with lean controls (P = 0.0143). This difference was probably not due to different cell size, because adenylate cyclase of crude plasma membranes of large adipocytes was actually more sensitive to PIA than was adenylate cyclase of membranes of smaller fat-cells co-isolated from the same individual. The stimulatory effect of PIA on glucose uptake in the presence of adenosine deaminase was depressed in adipocytes prepared from obese subjects and correlated with BMI at r = -0.626 (P = 0.007) at 100 nM-PIA. The adenosine receptors were studied by using the adenosine antagonist 1,3-[3H]dipropyl-8-cyclopentylxanthine. The binding was rapid and proportional to protein concentration. There was no difference in the affinities of receptors in membranes of obese and normal-weight subjects; Kd values of all patients averaged 3.3 nM. Bmax values were 54 and 130 fmol/mg of protein in membranes prepared from seven obese and five control patients respectively. The Bmax values calculated per mg of protein correlated with BMI at r = -0.539 (P = 0.047). The adenosine content of adipose tissue was higher in obese than in control subjects. These results demonstrate an attenuated response of cyclic AMP accumulation, adenylate cyclase and glucose uptake to adenosine in fat-cells prepared from obese subjects, and suggest that this change is at least partly due to changes in the amount of adenosine receptors, but not their affinity. The decreased receptor number could be due to higher adenosine content. A higher adenosine concentration in adipose tissue could explain why lipolysis is inhibited in situ in obesity, and the desensitization could explain the diminished response to adenosine analogues in isolated fat-cells.
Surfactant protein D (SP-D) counteracts the inhibitory effect of surfactant protein A (SP-A) on phospholipid secretion by alveolar type II cells. Interaction of native SP-D with SP-A
Authors: Kuroki Y , Shiratori M , Murata Y , Akino T
Published: August 2015

The surfactant proteins SP-A and SP-D were obtained from rats given intratracheal instillation of silica. SP-D was isolated from the 33,000 g supernatant of rat bronchoalveolar lavage fluids, and we examined whether SP-D affects surfactant secretion by alveolar type II cells. Native SP-D affected neither basal secretion nor stimulated secretion by type II cells. However, native SP-D counteracted the inhibitory effect of SP-A on surfactant secretion in a concentration-dependent manner; however, SP-D failed to counteract the inhibitory effect of concanavalin A. The activity of SP-D was unaffected by inclusion of excess methyl alpha-mannoside. Excess native SP-D competed with 125I-SP-A for high-affinity binding to type II cells. Heat treatment of SP-D and antibody against SP-D both decreased SP-D activity. Butanol extraction of native SP-D was most effective at destroying SP-D activity and attenuated the ability of the protein to compete with labelled SP-A for binding to type II cells. The butanol-soluble fraction of SP-D possessed the ability to alter the inhibitory effect of SP-A to the same extent as native SP-D. Direct binding of 125I-SP-A on nitrocellulose sheets demonstrated that SP-A could bind native SP-D, but not butanol-extracted SP-D. We conclude that native SP-D alters SP-A activity in type II cells through interaction with it via SP-D-associated lipids.
Polarized secretion of tyrosine-sulphated proteins and free tyrosine O-sulphate by filter-grown Madin-Darby canine kidney (MDCK) cells
Authors: Han J R , Liu M C
Published: August 2015

Filter-grown Madin-Darby canine kidney (MDCK) cells labelled for 24 h with [35S]sulphate were found to secrete macromolecules [35S]sulphated on their carbohydrate moieties predominantly into the basolateral medium, whereas the tyrosine-[35S]sulphated proteins synthesized were predominantly secreted into the apical medium. In contrast with the predominant apical secretin of tyrosine-[35S]sulphated proteins, the free tyrosine O-[35S]sulphate (Tyr[35S]) was released mostly into the basolateral medium. A time-lapse study using prelabelled MDCK cells incubated in fresh medium revealed that, during the 48 h time course monitored, the release of tyrosine-[35S]sulphated proteins into the apical medium was faster and quantitatively greater than that into the basolateral medium. During the same time there was a concomitant release, predominantly into the basolateral medium, of the free Tyr[35S] derived from the degradation of tyrosine-[35S]sulphated proteins. An endocytotic degradation experiment was performed to demonstrate the endocytosis of tyrosine-sulphated proteins and their degradation to generate free TyrS. It was found that free Tyr[35S] was generated and released when an apically secreted (or basolaterally secreted) tyrosine-[35S]sulphated protein preparation was added to the apical medium (or the basolateral medium) of unlabelled filter-grown MDCK cells. In both cases, the free Tyr[35S] generated was predominantly released into the basolateral medium similar to the results obtained in the time-lapse study.
The interaction of 1-fluoro-d-glucopyranosyl fluoride with glucosidases
Authors: Konstantinidis A , Sinnott M L
Published: August 2015

1. 1-Fluoro-D-glucopyranosyl fluoride undergoes pH-independent loss of F- ion at a rate of 1 x 10(-8) s-1 at 50.0 degrees C, some 10(3)-fold slower than alpha-D-glucopyranosyl fluoride and 4 x 10(4)-fold slower than beta-D-glucopyranosyl fluoride. 2. The (inverting) amyloglucosidase II of Aspergillus niger hydrolyses the difluoride according to Michaelis-Menten kinetics (Km 34 mM and kcat. 0.27 s-1), by apparently the same (simple) mechanism by which it hydrolyses alpha-D-glucopyranosyl fluoride (Km 38 mM and kcat. 730 s-1), rather than by the Hehre resynthesis-hydrolysis mechanism used to transform beta-D-glucopyranosyl fluoride. 3. The difluoride is also a substrate for the (inverting) trehalase of pig kidney [Km 17.3 mM and Vmax. 6.2 x 10(-4) relative to alpha-D-glucopyranosyl fluoride (Km 38 mM]). 4. The quantitatively similar effect of fluorine substitution on the one-step enzymic reactions and on the non-enzymic reactions suggests that they go through similar (oxocarbonium-ion-like) transition states. 5. The difluoride is a substrate for the (retaining) beta-glucosidases from Aspergillus wentii (A3 enzyme) and sweet-almond meal (B isoenzyme) and for the retaining alpha-glucosidase from rice: comparison with the appropriate monofluoride reveals a variable rate-retarding effect of the second fluorine atom on kcat./Km that correlates with other measures of oxocarbonium ion character in the transition state. 6. The difluoride is a substrate for the (retaining) alpha-glucosidase from yeast, but also gives an insidious mimicry of active-site-directed irreversible inhibition, which we tentatively attribute either to formation of the non-covalent complex or to the fluoroglucosyl-enzyme increasing the well-known tendency of this enzyme to come out of solution by adsorption on the walls of the vessel.
Regulation of the acto·myosin subfragment 1 interaction by troponin/tropomyosin. Evidence for control of a specific isomerization between two acto·myosin subfragment 1 states
Authors: McKillop D F A , Geeves M A
Published: August 2015

The co-operative binding of myosin subfragment 1 (S1) to reconstituted skeletal-muscle thin filaments has been examined by monitoring the fluorescence of a pyrene probe on Cys-374 of actin. The degree of co-operativity differs when phosphate, sulphate or ADP are bound to the S1 active site. Binding isotherms have been analysed according to the Geeves & Halsall [(1987) Biophys. J. 52, 215-220] model, which proposed that troponin and tropomyosin effected regulation of the actomyosin interaction by controlling an isomerization of the actomyosin complex. The data support the proposal that seven actin monomers associated with a single tropomyosin molecule act as a co-operative unit that can be in one of two states. In the ‘closed’ state myosin can bind to actin, but the subsequent isomerization is prevented. The isomerization is only allowed after the seven-actin unit is in the ‘open’ form. Ca2+ controls the proportion of actin filaments in the ‘closed’ and ‘open’ forms in the absence of myosin heads. The ratio of ‘closed’ to ‘open’ forms is approx. 50:1 in the absence of Ca2+ and 5:1 in its presence.
Metalloproteinase digestion of cartilage proteoglycan. Pattern of cleavage by stromelysin and susceptibility to collagenase
Authors: Hughes C , Murphy G , Hardingham T E
Published: August 2015

The action of purified rabbit bone stromelysin was investigated on proteoglycan aggregates from pig laryngeal cartilage. The enzyme caused a rapid fall in viscosity of proteoglycan aggregate solution (6 mg/ml), and the products of a partial digest (60% loss of relative viscosity) and a complete digest (95% loss of relative viscosity) were characterized. Analysis by gel chromatography on Sepharose 2B under associative conditions showed that 95% of the glycosaminoglycans in the complete digest were in small-sized fragments, whereas most of the hyaluronan-binding G1 domain and link protein remained intact and bound to hyaluronan. In contrast, there was extensive digestion of the G2 domain which resulted in 76% loss in its detection by immunoassay. Analysis of the partial digest also showed considerable loss (40%) of detection of the G2 domain, but the glycosaminoglycan-rich fragments were much larger than in the complete digest. There was also much less cleavage to create small fragments containing the G1 domain. This was evident on SDS/PAGE analysis where a 58 kDa G1 domain fragment was abundant in the complete digest, but was only present in small amounts in the partial digest. There was also only very limited conversion of link protein from a 44 kDa form to a 40 kDa form. The digestion of proteoglycan aggregate (6 mg/ml) by stromelysin was unaffected by the addition of a high concentration of extra chondroitin sulphate chains (14 mg/ml), and the digestion of proteoglycan monomer showed that the G1 domain was resistant to stromelysin digestion even when not bound to hyaluronan and link protein. The results show that stromelysin degrades the proteoglycan protein core with major cleavages close to, but not within, the G1 domain, and extensive cleavage in other regions. Experiments with purified collagenase, a metalloproteinase structurally related to stromelysin, showed that it too cleaved proteoglycan at several sites within the glycosaminoglycan-rich region of the core protein. Metalloproteinase attack on proteoglycan thus not only occurs with stromelysin but also with collagenase.
Analyses of the effect of monensin on glycosphingolipid metabolism
Authors: Gundlach B , Helland R , Radsak K , Wiegandt H
Published: August 2015

The effect of monensin on glycosphingolipid metabolism was reinvestigated. It was found that monensin increased the uptake of [3H]galactose by human fibroblasts, causing an enhanced metabolic labelling of glycosphingolipids. The preferential incorporation of radioactivity into ceramide monosaccharide in the presence of monensin was accompanied by an equally increased rate of degradation. However, using radiolabelled precursors of the ceramide moiety, a generally diminished uptake of radioactivity into all glycosphingolipids was observed under the influence of monensin, with the exception of mono- and di-hexosylceramide. The enhanced incorporation of radiolabel by these two glycolipids, as well as their increased cellular chemical content, may reflect early synthesis in a monensin-insensitive pre-Golgi compartment after disruption of the cellular Golgi transport system by monensin.
Probing of the expression of the low-density lipoprotein receptor in vivo using an anti-receptor monoclonal antibody
Authors: Gherardi E , Bowyer D E , Fitzsimmons C , Le Cras T , Hutchings A , Butcher G
Published: August 2015

MAC188 is a rat anti-[low-density lipoprotein (LDL) receptor] monoclonal antibody (McAb) which binds to the cell surface receptor with high affinity at physiological temperatures, even in the presence of high concentrations of the natural ligand, LDL. Binding of McAb MAC188 at 37 degrees C is followed by internalization and intracellular sequestering of the receptor, which results in the transient disappearance of the receptor from the cell surface. The high binding affinity and epitope specificity of McAb MAC188 suggested that this antibody could be used to quantify receptor expression in vivo. Mixtures of radiolabelled anti-receptor antibody and a control McAb (MAC221) were injected intravenously into rabbits, and the clearance from serum and uptake into tissues was determined. A fraction of the anti-receptor McAb was cleared rapidly from the circulation by a high-affinity and saturable (receptor-dependent) process. Receptor-dependent uptake of the anti-receptor McAb was measurable in liver, adrenal glands, kidneys, spleen, kidney, thoracic aorta and heart. It was highest in liver and adrenal glands and correlated well with the level of receptor protein and the rate of LDL transport in individual tissues. Anti-receptor McAbs such as MAC188, with suitable domain specificity and binding affinity at physiological temperatures, have important advantages over the natural ligand as tracers for the receptor in vivo, and may find widespread applications in studies of the receptor status (activity) in animals and man.
X.a.f.s. studies of chicken dicupric ovotransferrin
Authors: Garratt R C , Evans R W , Hasnain S S , Lindley P F , Sarra R
Published: August 2015

A comparison of Cu K-edge x.a.f.s. spectra with that of the equivalent Fe K-edge for chicken ovotransferrin (COT) indicates that the metal ions occupy essentially the same binding sites in the protein. However, in the case of the Cu2+ complex the metal appears to have reduced co-ordination. Changes are observed in the x.a.f.s. of 90%-saturated COT (Cu1.8COT) on freeze-drying. The three-dimensional X-ray structures of rabbit serum transferrin and human lactoferrin have shown that the ferric cations are co-ordinated by four protein ligands and a bidentate carbonate anion in a distorted octahedral arrangement [Anderson, Baker, Dodson, Norris, Rumball, Waters & Baker (1987) Proc. Natl. Acad. Sci. U.S.A. 84, 1768-1774; Anderson, Baker, Norris, Rice and Baker (1989) J. Mol. Biol. 209, 711-734; Bailey, Evans, Garratt, Gorinsky, Hasnain, Horsburgh, Jhoti, Lindley, Mydin, Sarra & Watson (1988) Biochemistry 27, 5804-5812]. This structural information, together with the differences in e.x.a.f.s. spectra for solution and freeze-dried samples of diferric COT [Hasnain, Evans, Garratt & Lindley (1987) Biochem. J. 247, 369-375] suggests that the synergistic carbonate anion may be capable of behaving as a unidentate linkage to the Cu2+ in the dicupric complex. Data for Cu1.8COT are consistent with only three protein ligands bound to Cu2+, monodentate binding of the synergistic anion in one lobe and its bidentate binding in the other lobe. Such flexibility in the anion co-ordination may be a requirement for the uptake and release of metals by the transferrins.
A classification of glycosyl hydrolases based on amino acid sequence similarities
Authors: Henrissat B
Published: August 2015

The amino acid sequences of 301 glycosyl hydrolases and related enzymes have been compared. A total of 291 sequences corresponding to 39 EC entries could be classified into 35 families. Only ten sequences (less than 5% of the sample) could not be assigned to any family. With the sequences available for this analysis, 18 families were found to be monospecific (containing only one EC number) and 17 were found to be polyspecific (containing at least two EC numbers). Implications on the folding characteristics and mechanism of action of these enzymes and on the evolution of carbohydrate metabolism are discussed. With the steady increase in sequence and structural data, it is suggested that the enzyme classification system should perhaps be revised.
A low-temperature method for the isolation of small-intestinal epithelium along the crypt-villus axis
Authors: Flint N , Cove F L , Evans G S
Published: August 2015

A variety of enzymic and non-enzymic methods to isolate epithelium from the small intestine have been previously published. Sequential fractionation of cells from the villus to the crypt has been reported in some of these papers, which allows the comparative study of terminally differentiated and proliferative cell phenotypes. However, these methods often involve the incubation of tissues at 37 degrees C, which may affect the structural and biochemical integrity of the cells. We have developed a rapid low-temperature (4 degrees C) method for isolating purified populations of crypt and villus cells from mouse and rat intestines. The fractionated cells have been partially characterized, and the potential value of the procedure has been indicated by the ability to analyse the comparative protein and mRNA expression along the crypt-villus axis.
Heterogeneous distribution of antithrombin-binding sites in rat brain heparan sulphate proteoglycans
Authors: Horner A A
Published: August 2015

Heparan sulphates with high binding affinity for antithrombin (HA-HS), labelled in vivo with [35S]sulphate, were extracted from rat brains and purified by chromatography on DEAE-cellulose and on antithrombin-agarose. HA-HS proteoglycans (HA-HSPG) were then separated from HA-HS chains on Sepharose CL-6B. The total HA-HSPG product was rechromatographed on antithrombin-agarose. Six HA-HSPG subfractions with differing degrees of affinity for antithrombin were recovered and treated with NaOH to release their chains. Rechromatography of these six 35S-labelled HS chain preparations on antithrombin-agarose showed that their proportions of chains with no affinity for antithrombin (NA-HS chains) ranged from 36 to 71%. There was a reciprocal relationship between the proportion of NA-HS chains in each HA-HSPG subfraction and the degree of affinity for antithrombin of the rest of its chains (assessed relative to 3H-labelled HA-heparin chains with which they were co-chromatographed). Similar characteristics of antithrombin-binding-site distribution apply to HA-heparin proteoglycans from rat skin studied previously [Horner (1987) Biochem. J. 244, 693-698]. The data suggest that the sites at which 3-O-sulphation of some glucosamine N-sulphate residues occurs in the Golgi complex of brain cells (probably endothelial cells) which synthesize HA-HSPGs (as in mast cells, which synthesize HA-heparin PGs) are distributed sparsely but not randomly.
The Enterococcus hirae R40 penicillin-binding protein 5 and the methicillin-resistant Staphylococcus aureus penicillin-binding protein 2′ are similar
Authors: el Kharroubi A , Jacques P , Piras G , Van Beeumen J , Coyette J , Ghuysen J M
Published: August 2015

The penicillin-resistant Enterococcus hirae R40 has a typical profile of membrane-bound penicillin-binding proteins (PBPs) except that the 71 kDa PBP5 of low penicillin affinity represents about 50% of all the PBPs present. Water-soluble tryptic-digest peptides were selectively produced from PBP5, their N-terminal regions were sequenced and synthetic oligonucleotides were used as primers to generate a 476 bp DNA fragment by polymerase chain reaction. On the basis of these data, the PBP5-encoding gene was cloned in Escherichia coli by using pBR322 as vector. The gene, included in a 7.1 kb insert, had the information for a 678-amino acid-residue protein. PBP5 shows similarity, in the primary structure, with the high-molecular-mass PBPs of class B. In particular, amino acid alignment of the enterococcal PBP5 and the methicillin-resistant staphylococcal PBP2′ generates scores that are 30, for the N-terminal domains, and 53, for the C-terminal domains, standard deviations above that expected for a run of 20 randomized pairs of proteins having the same amino acid compositions as the two proteins under consideration.
Morphological and functional changes in the enterocyte induced by fructose
Authors: Danielsen E M , Hansen G H , Wetterberg L L
Published: August 2015

In the presence of 10-50 mM-fructose, enterocytes of organ-cultured pig intestinal-mucosal explants fail to glycosylate correctly their newly synthesized microvillar enzymes, and instead degrade them [Danielsen (1989) J. Biol. Chem. 264, 13726-13729]. In the present work, this degradation was shown to occur extremely rapidly as the microvillar enzyme aminopeptidase N (EC 3.4.11.2) was hardly detectable after a 10 min pulse with [35S]methionine. The abnormal biosynthesis of membrane glycoproteins affected both the morphology and the function of the Golgi complex as well as the microvillar membrane. Thus the stack of Golgi cisternae was condensed and devoid of dilated rims, and the secretion of a non-glycosylated protein, apolipoprotein A-1, was almost completely blocked in the presence of fructose, showing that transport through the secretory pathway is disturbed even for proteins unaffected by the defective glycosylation. The microvilli of the brush-border membrane were markedly shortened (by about 40%) in the presence of fructose, and incorporation of newly made actin into the microvillar cytoskeleton was similarly decreased. By affecting membrane glycoprotein synthesis, the common dietary sugar fructose thus profoundly perturbs the exocytic membrane traffic in the enterocyte.
A proteolytic fragment from human link protein is taken up and processed by monocytes and B cells
Authors: Martin H , Dean M
Published: August 2015

Mild digestion of 125I-labelled human proteoglycan aggregates with trypsin or stromelysin produced specific peptides that were taken up rapidly by THP-1 monocytes. SDS/PAGE of undigested aggregate showed that the three components of molecular mass 48, 44 and 41 kDa, corresponding to isoforms of link protein originally present, had been converted into a single component of 41 kDa by trypsin treatment, and that fragments of 6-12 kDa were present in fractions containing the high-uptake peptide. Separate proteolysis of isolated proteoglycan monomer and link protein confirmed that the specific high-uptake fragment was derived from link protein. Uptake of the link fragment was rapid, reaching a maximum after 5 min, and specific, since it was blocked by metabolic or serine proteinase inhibitors and at 4 degrees C. After uptake the cleaved fragment was processed further, with 50% of the radiolabel being released as degraded peptides within 5 min. In contrast, accumulation of whole aggregate reached a maximum after 45 min and only 50% had been released after 2 h. Uptake of aggregate was less affected by inhibitors or at low temperature, suggesting that a separate mechanism existed for its turnover. The aggregate was transported to lysosomes after uptake, although the link fragment did not sediment with either lysosomes or plasma membranes, suggesting that it was present in the cytoplasm or in very labile vesicles. However, the mode of handling of the peptide by the cells remains unclear. The link fragment was taken up by several different monocytic and B cell lines, but not by mouse fibroblasts or peritoneal macrophages. These data suggest that a surface serine proteinase on monocytes and B cells enables them to process and take up a fragment of link protein derived by extracellular proteolysis.
Inhibition of interleukin 1-stimulated cartilage proteoglycan degradation by a lipophilic inactivator of cysteine endopeptidases
Authors: Buttle D J , Saklatvala J , Tamai M , Barrett A J
Published: August 2015

Inactivators of cysteine endopeptidases were tested as inhibitors of the cytokine-stimulated release of proteoglycan from cartilage. The test system consisted of bovine nasal septum cartilage maintained in organ culture, and the stimulus was provided by recombinant human interleukin 1 alpha. L-3-Carboxy-2,3-trans-epoxypropionyl-leucylamido-(4-guanidin o)butane (E64) and L-3-carboxy-2,3-trans-epoxypropionyl-leucylamido-(3-methyl)b utane (Ep475) showed no inhibition at concentrations up to 100 microM. In contrast, trans-epoxysuccinyl-leucylamido-(3-methyl)butane ethyl ester (Ep453), a ‘prodrug’ of Ep475, was an effective inhibitor. The LL-, LD- and DL-isomers gave significant inhibition at 10 microM, and the DD-isomer was inhibitory at 100 microM. None of the isomers had any detectable effect on protein synthesis or glycolysis, and their inhibitory effects were reversible. Iodoacetate inhibited proteoglycan release by a general toxic effect. Our results suggest that cysteine endopeptidase(s) play a part in cytokine-stimulated cartilage breakdown, but that effective inhibitors must pass through membranes.
Effects of insulin and guanosine 5′-[γ-thio]triphosphate on fatty acid synthesis and lipolysis within electropermeabilized fat-cells
Authors: Rutter G A , Denton R M
Published: August 2015

1. Exposure to electric fields (‘electroporation’) has been used to investigate the mechanism of the action of insulin on the regulation of fatty acid synthesis and lipolysis in isolated rat fat-cells. 2. Exposure of the cells to electric fields (six pulses at 2 or 3 kV/cm) permitted the uptake of a number of low-Mr molecules normally excluded from cells, including sucrose (Mr 342), EDTA (Mr 394) and propidium iodide (Mr 668). At least 90% of the cells were found to be permeable to these species. 3. Insulin stimulated the synthesis of fatty acids in electroporated (2 kV/cm) cells to a similar extent (0.68 +/- 0.19 to 6.7 +/- 0.7 micrograms-atoms of glucose carbon/h per g, or 3.3 +/- 0.6 to 11.2 +/- 1.6 micrograms-atoms of H2O hydrogen/h per g) to that observed in non-electroporated cells. Guanosine 5′-[gamma-thio]triphosphate (GTP[S]; 0.5 mM) also stimulated fatty acid synthesis (2-fold) and flux of glucose carbon into triacylglycerol glycerol (3-fold) in these cells, but had no effect on these parameters in non-electroporated cells. 4. Lipolysis in the electroporated cells was stimulated by isoprenaline and also by GTP[S], but only the effects of isoprenaline were inhibited by insulin. 5. Exposure to a higher field strength (3 kV/cm) gave results qualitatively similar to those described above, although the effects of insulin and isoprenaline were diminished. 6. These studies provide evidence against a role for changes in Ca2+, and probably also Na+, K+ or Mg2+, in insulin action on fat-cells, but may support a role for GTP-binding proteins.
Glucose induces the translocation and the aggregation of glycogen synthase in rat hepatocytes
Authors: Fernández-Novell J M , Ariño J , Vilaró S , Guinovart J J
Published: August 2015

Incubation of rat hepatocytes with glucose results in a decrease in the amount of glycogen synthase activity found in supernatants obtained after centrifugation of cell homogenates at 9200 g. The enzymic activity was quantitatively recovered in the sediments. This effect of translocation was dose- and time-dependent and correlated with the amount of immunoreactive enzyme determined by immunoblotting in both fractions. Hydrolysis by alpha-amylase of glycogen accumulated upon incubation with the sugar did not affect the translocation pattern. Translocation was also observed when cells were incubated with 2-deoxyglucose, which did not result in accumulation of glycogen. Immunocytochemical evidence indicates that glucose induces the aggregation of glycogen synthase molecules into clusters which are recovered in the sediments. These results indicate that glucose, in addition to activating glycogen synthase, may trigger changes in the localization of the enzyme in the cell.
Characterization of 6-phosphofructo-2-kinase from foetal-rat liver
Authors: Martín-Sanz P , Cascales M , Boscá L
Published: August 2015

Foetal and adult liver 6-phosphofructo-2-kinase (PFK-2) were purified by identical protocols. The native molecular masses of both enzymes were determined by gel filtration and were 89.1 and 100.0 kDa respectively. No differences were found in SDS/PAGE in 10%-acrylamide gel (55 kDa per subunit). The kinetic properties displayed by both enzymes were similar, except for the sensitivity to inhibition by sn-glycerol 3-phosphate. Foetal PFK-2 was a good substrate for phosphorylation by cyclic AMP-dependent protein kinase and protein kinase C, whereas the adult enzyme was phosphorylated only by cyclic AMP-dependent protein kinase. However, the phosphorylation affected only the kinetic properties of the adult enzyme, suggesting the presence in both enzymes of different sites of phosphorylation by cyclic AMP-dependent protein kinase. These differences in primary structure were consistent with the distinct chromatographic profiles of the phosphopeptides after digestion of the protein with CNBr. Western-blot analysis with antibodies specific for the N-terminal region of the liver-type PFK-2 poorly recognized the foetal enzyme, suggesting that both enzymes differ at least in the N-terminal sequence.
Role of protein kinase C in the acrosome reaction of mammalian spermatozoa
Authors: Breitbart H , Lax J , Rotem R , Naor Z
Published: August 2015

Mammalian spermatozoa undergo a Ca(2+)-dependent exocytotic event before fertilization which is known as the acrosome reaction. The process of exocytosis in several cell systems is mediated by a protein kinase C (PKC)-catalysed phosphorylation. Addition of phorbol 12-myristate-13-acetate or the membrane-permeant diacylglycerol analogue 1-oleoyl-2-acetylglycerol, which are potent activators of PKC, to bovine spermatozoa resulted in stimulation of the acrosome reaction. This stimulation was inhibited by low concentrations (50% inhibition at 0.7 nM) of the PKC inhibitor staurosporine. PKC specific activity in bovine spermatozoa is extremely low in comparison with other cells; however, it is comparable with the activity found in human spermatozoa. Immunohistochemical analysis using anti-PKC antibodies revealed staining in the equatorial segment, the post-acrosomal region and the upper region of the head. We propose that PKC is involved in the mammalian sperm acrosome reaction.
Characterization of the low-density-lipoprotein-receptor-independent interaction of β-very-low-density lipoprotein with rat and human parenchymal liver cells in vitro
Authors: De Water R , Kamps J A A M , Van Dijk M C M , Hessels E A M J , Kuiper J , Kruijt J K , Van Berkel T J C
Published: August 2015

beta-Migrating very-low-density lipoprotein (beta-VLDL) is a cholesteryl-ester-enriched lipoprotein which under normal conditions is rapidly cleared by parenchymal liver cells. In this study the characteristics of the interaction of beta-VLDL with rat parenchymal cells, Hep G2 cells and human parenchymal cells are evaluated. The binding of beta-VLDL to these cells follows saturation kinetics (Bmax. respectively 117, 106 and 103 ng of beta-VLDL apoliprotein/mg of cell protein), with a relatively high affinity (Kd respectively for beta-VLDL of 10.7, 5.1 and 8.4 micrograms/ml). Competition studies of unlabelled beta-VLDL, low-density lipoprotein (LDL) or acetylated LDL with the binding of radiolabelled beta-VLDL indicate that a LDL-receptor-independent, Ca(2+)-independent, specific recognition site for beta-VLDL is present on rat and human parenchymal cells, whereas with Hep G2 cells or mouse macrophages beta-VLDL recognition is performed by the LDL receptor. The binding of beta-VLDL to Hep G2 cells was down-regulated by 89% by prolonged exposure to beta-VLDL, whereas for human parenchymal and rat parenchymal cells down-regulation of 44% and 20% respectively was observed. Studies with antibodies against the LDL receptor support the presence of a LDL-receptor-independent specific beta-VLDL recognition site on rat and human parenchymal cells. It is concluded that a LDL-receptor-independent recognition site for beta-VLDL is present on rat and human parenchymal liver cells. The presence of a LDL-receptor-independent recognition site on human parenchymal cells may mediate in vivo the uptake of beta-VLDL during consumption of a cholesterol-rich diet, when LDL receptors are down-regulated, thus protecting against the extrahepatic accumulation of the atherogenic beta-VLDL constituents.
Occurrence of fatty acid epoxide hydrolases in soybean (Glycine max). Purification and characterization of the soluble form
Authors: Blée E , Schuber F
Published: August 2015

Epoxide hydrolases catalysing the hydration of cis-9,10-epoxystearate into threo-9,10-dihydroxystearate have been detected in soybean (Glycine max) seedlings. The major activity was found in the cytosol, a minor fraction being strongly associated with microsomes. The soluble enzyme, which was purified to apparent homogeneity by (NH4)2SO4 fractionation, hydrophobic, DEAE- and gel-filtration chromatographies, has a molecular mass of 64 kDa and a pI of 5.4.
Plasma-membrane-intercalated heparan sulphate proteoglycans in an osteogenic cell line (UMR 106-01 BSP)
Authors: McQuillan D J , Midura R J , Hascall V C , Yanagishita M
Published: August 2015

The heparan sulphate (HS) proteoglycans associated with the cell layer of a rat osteosarcoma cell line [UMR 106-01 (BSP)] were compared with similar cell-associated proteoglycans from other cells, and their interaction with the plasma membrane was studied. HS proteoglycans were metabolically labelled by incubation of cell cultures with [3H]glucosamine or [3H]leucine and [35S]sulphate. HS proteoglycan core protein preparation generated by heparitinase digestion of the major species from UMR 106-01 (BSP) cells co-migrated on PAGE with identical preparations from ovarian granulosa cells and parathyroid cells (at approximately 70 kDa). The hydrophobic nature of the major HS proteoglycans from these diverse cell lines, based on elution position from octyl-Sepharose, were also comparable. Linkages of the HS proteoglycan to the cell membrane were investigated by labelling plasma-membrane preparations with a lipid soluble photoactivatable reagent, 3-(trifluoromethyl)-3- (m-[125I]iodophenyl)diazirine (TID), which selectively labels plasma-membrane-spanning peptide domains. Purified HS proteoglycan from UMR 106-01 (BSP) cells was shown to be accessible to the [125I]TID, and the core protein portion of the molecule was labelled, confirming its close association with the plasma membrane. Approx. 36% of 35S-labelled HS proteoglycans were released from the cell surface by phospholipase C (Bacillus thuringiensis), which specifically cleaves phosphatidylinositol-linked proteins. In the presence of insulin, the metabolism of the phospholipase C-sensitive population was unaltered; however, release of the phospholipase C-insensitive population into the medium was increased. These data indicate that a subpopulation of HS proteoglycans are covalently bound to the plasma membrane by a glycosylphosphatidylinositol structure, with the remainder representing those species directly inserted into the plasma membrane via a hydrophobic peptide domain. These observations are similar to those reported for ovarian granulosa cells [Yanagishita & McQuillan (1989) J. Biol. Chem. 264 17551-17558], and thus may represent a general phenomenon for many cell types.
Binding of the cyclic AMP receptor protein of Escherichia coli and DNA bending at the P4 promoter of pBR322
Authors: Brierley I , Hoggett J G
Published: August 2015

The binding of the Escherichia coli cyclic AMP receptor protein (CRP) to its specific site on the P4 promoter of pBR322 has been studied by gel electrophoresis. Binding to the P4 site was about 40-50-fold weaker than to the principal CRP site on the lactose promoter at both low (0.01 M) and high (0.1 M) ionic strengths. CRP-induced bending at the P4 site was investigated from the mobilities of CRP bound to circularly permuted P4 fragments. The estimated bending angle, based on comparison with Zinkel & Crothers [(1990) Biopolymers 29, 29-38] A-tract bending standards, was found to be approximately 96 degrees, similar to that found for binding to the lac site. These observations suggest that there is not a simple relationship between strength of CRP binding and the extent of induced bending for different CRP sites. The apparent centre of bending in P4 is displaced about 6-8 bp away from the conserved TGTGA sequence and the P4 transcription start site.
Effects of clofibric acid on the activity and activity state of the hepatic branched-chain 2-oxo acid dehydrogenase complex
Authors: Zhao Y , Jaskiewicz J , Harris R A
Published: August 2015

Feeding clofibric acid to rats caused little or no change in total activity of the liver branched-chain 2-oxo acid dehydrogenase complex (BCODC). No change in mass of liver BCODC was detected by immunoblot analysis in response to dietary clofibric acid. No changes in abundance of mRNAs for the BCODC E1 alpha, E1 beta and E2 subunits were detected by Northern-blot analysis. Likewise, dietary clofibric acid had no effect on the activity state of liver BCODC (percentage of enzyme in the dephosphorylated, active, form) of rats fed on a chow diet. However, dietary clofibric acid greatly increased the activity state of liver BCODC of rats fed on a diet deficient in protein. No stable change in liver BCODC kinase activity was found in response to clofibric acid in either chow-fed or low-protein-fed rats. Clofibric acid had a biphasic effect on flux through BCODC in hepatocytes prepared from low-protein-fed rats. Stimulation of BCODC flux at low concentrations was due to clofibric acid inhibition of BCODC kinase, which in turn allowed activation of BCODC by BCODC phosphatase. Inhibition of BCODC flux at high concentrations was due to direct inhibition of BCODC by clofibric acid. The results suggest that the effects of clofibric acid in vivo on branched-chain amino acid metabolism can be explained by the inhibitory effects of this drug on BCODC kinase.
Protein kinase C enhances myosin light-chain kinase effects on force development and ATPase activity in rat single skinned cardiac cells
Authors: Clement O , Puceat M , Walsh M P , Vassort G
Published: August 2015

Many neurohormones alter the force of cardiac contraction by variations in the intracellular Ca2+ concentration. alpha 1-Adrenergic and muscarinic stimulations, rather, modify the sensitivity of contractile proteins to Ca(2+)-calmodulin-myosin light-chain kinase (MLCK) complex induces a large increase in Ca2+ sensitivity (0.14 pCa unit) of these easily accessible myofilaments. This increase is further enhanced by up to 0.19 pCa unit when protein kinase C (PKC) is added together with MLCK. Similarly, the Ca2+ ATPase activity of skinned cells in suspension is increased in the presence of MLCK and further in the presence of both kinases. 32P-labelling and SDS/PAGE show that these changes are associated with light-chain 2 (LC2) phosphorylation together with phosphorylation of troponin I and troponin T when PKC is added. Although to a smaller extent than in smooth muscle, phosphorylation of cardiac myosin LC2 may be involved in the modulation of heart contractility.
Relationship between glutathione content in liver and glutathione conjugation rate in the rat in vivo. Effect of buthionine sulphoximine pretreatment on conjugation of the two 2-bromoisovalerylurea enantiomers during intravenous infusion
Authors: Polhuijs M , Lankhaar G , Mulder G J
Published: August 2015

The relationship between hepatic glutathione content and hepatic glutathione conjugation rate in the rat in vivo was investigated. As substrate for glutathione conjugation, racemic (R,S)-2-bromoisovalerylurea (BIU) was used which gives rise to the biliary excretion of two diastereoisomeric glutathione conjugates and the urinary excretion of two diastereoisomeric mercapturates. The excretion rate of the glutathione conjugate in bile reflects hepatic conjugation exclusively. An intravenous infusion of BIU was given and the excretion rates of the metabolites in bile and urine were determined. The glutathione concentration in the liver was followed by taking biopsies every hour. Glutathione was depleted by the infused substrate; in rats that were pretreated with the inhibitor of glutathione biosynthesis, buthionine sulphoximine (BSO), the depletion of the glutathione content was more rapid. The rate of excretion of the glutathione conjugate in bile was plotted against hepatic glutathione content. These results indicate that the ‘organ Km’ for glutathione in the liver is approximately 0.5 mumol/g of liver, so that the hepatic glutathione conjugation rate is decreased only at severe glutathione depletion.