bluuebunny/crossref_metadata_2025 huggingface dataset:
Interleukin-1β prevents the stimulatory effect of transforming growth factor-β on collagen gene expression in human skin fibroblasts
Authors: Heino J , Heinonen T
Published: August 2015

Transforming growth factors beta 1 and beta 2 (TGF-beta 1 and TGF-beta 2) are well-characterized strong inducers of collagen gene expression. A 100 pM concentration of TGF-beta 1 or TGF-beta 2 increases pro alpha 1(I) collagen mRNA levels in human skin fibroblasts 6.6-fold and 7.0-fold respectively, and also increases the accumulation of procollagens in the cell culture medium. Interleukin-1 beta (IL-1 beta) is an inflammatory mediator which also regulates connective tissue metabolism. A small concentration of IL-1 beta (0.01-1.0 unit/ml) slightly increases pro alpha 1(I) collagen mRNA levels (2.2-fold). Here we provide evidence that IL-1 beta prevents the stimulatory effect of TGFs-beta on collagen synthesis in human skin fibroblasts. An IL-1 beta concentration of 1 unit/ml is enough to keep pro alpha 1(I) collagen mRNA levels at control values in cells stimulated by 100 pM-TGF-beta 1. Thus the results indicate that IL-1 beta inhibits collagen synthesis in cells activated by TGFs-beta, whereas it does not significantly change or might even stimulate collagen gene expression in non-activated cells.
Treatment of HL-60 cells with dimethyl sulphoxide inhibits the formation of β-N-acetylhexosaminidase S
Authors: Emiliani C , Falzetti F , Orlacchio A , Stirling J L
Published: August 2015

beta-N-Acetylhexosaminidase of HL-60 cells was separated into two main forms, A and S, by chromatography on DEAE-cellulose. Analysis of developmental changes in the isoenzyme pattern was complicated by the fact that the specific activity of beta-N-acetylhexosaminidase underwent a 6-fold change during the normal growth cycle. Two other lysosomal enzymes, beta-galactosidase and alpha-mannosidase, behaved similarly. Induction of differentiation of HL-60 cells with dimethyl sulphoxide at a low cell density (3 x 10(5) cells/ml) had a greater effect on the abundance of alpha-subunits of beta-N-acetylhexosaminidase, measured with 4-methylumbelliferyl-beta-N-acetylglucosaminide 6-sulphate, than of beta-subunits, measured with 4-methylumbelliferyl-beta-N-acetylglucosamine, and resulted in an isoenzyme profile in which A and B were the major forms, with the levels of form S greatly decreased.
Effect of aluminium on iron uptake and transferrin-receptor expression by human erythroleukaemia K562 cells
Authors: McGregor S J , Naves M L , Oria R , Vass J K , Brock J H
Published: August 2015

Incubation of human erythroleukaemia K562 cells with Al-transferrin inhibited iron uptake from 59Fe-transferrin by about 80%. The inhibition was greater than that produced by a similar quantity of Fe-transferrin. Preincubation of cells for 6 h with either Al-transferrin or Fe-transferrin diminished the number of surface transferrin receptors by about 40% compared with cells preincubated with apo-transferrin. Al-transferrin did not compete significantly with Fe-transferrin for transferrin receptors and, when cells were preincubated for 15 min instead of 6 h, the inhibitory effect of Al-transferrin on receptor expression was lost. Both forms of transferrin also decreased the level of transferrin receptor mRNA by about 50%, suggesting a common regulatory mechanism. Aluminium citrate had no effect on iron uptake or transferrin-receptor expression. AlCl3 also had no effect on transferrin-receptor expression, but at high concentration it caused an increase in iron uptake by an unknown, possibly non-specific, mechanism. Neither Al-transferrin nor AlCl3 caused a significant change in cell proliferation. It is proposed that aluminium, when bound to transferrin, inhibits iron uptake partly by down-regulating transferrin-receptor expression and partly by interfering with intracellular release of iron from transferrin.
Effect of lovastatin on acyl-CoA: cholesterol O-acyltransferase (ACAT) activity and the basolateral-membrane secretion of newly synthesized lipids by CaCo-2 cells
Authors: Kam N T , Albright E , Mathur S , Field F J
Published: August 2015

Lovastatin, a potent competitive inhibitor of 3-hydroxy-3-methylglutaryl-CoA (HMG-CoA) reductase activity, was used to study the regulation of cholesterol metabolism and the basolateral-membrane secretion of triacylglycerol and cholesterol in the human intestinal cell line CaCo-2. At 0.1 microgram/ml, lovastatin decreased 3H2O incorporation into cholesterol by 71%. In membranes prepared from cells incubated with lovastatin for 18 h, HMG-CoA reductase activity was induced 4-8-fold. Mevalonolactone prevented this induction. In intact cells, lovastatin (10 micrograms/ml) decreased cholesterol esterification by 50%. The reductase inhibitor decreased membrane acyl-CoA:cholesterol O-acyltransferase (ACAT) activity by 50% at 5 micrograms/ml. ACAT inhibition by lavastatin was not reversed by adding excess of cholesterol or fatty acyl-CoA to the assay. Lovastatin, in the presence or absence of mevalonolactone, decreased the basolateral secretion of newly synthesized cholesteryl esters and triacylglycerols. Lovastatin also inhibited the esterification of absorbed cholesterol and the secretion of this newly synthesized cholesteryl ester. Lovastatin is a potent inhibitor of cholesterol synthesis in CaCo-2 cells. Moreover, it is a direct inhibitor of ACAT activity, independently of its effect on HMG-CoA reductase and cholesterol synthesis.
Purification and partial characterization of the major cell-associated heparan sulphate proteoglycan of rat liver
Authors: Lyon M , Gallagher J T
Published: August 2015

Heparan sulphate proteoglycans were solubilized from whole rat livers by homogenization and dissociative extraction with 4 M-guanidinium chloride containing Triton X-100 and proteinase inhibitors. The extract was subjected to trichloroacetic acid precipitation and the proteoglycan remained soluble. This was then purified to apparent homogeneity by a combination of (a) DEAE-Sephacel chromatography, (b) digestion with chondroitinase ABC followed by f.p.l.c. Mono Q ion-exchange chromatography, and (c) density-gradient centrifugation in CsCl and 4 M-guanidinium chloride. Approx. 1.5 mg of proteoglycan was obtained from 30 livers with an estimated recovery of 25%. The purified proteoglycan was eluted from Sepharose CL6B as an apparently single polydisperse population with a Kav. of 0.19 and displayed a molecular mass of greater than or equal to 200 kDa (relative to protein standards) by SDS/PAGE. Its heparan sulphate chains were eluted with a Kav. of 0.44 and have an estimated molecular mass of 25 kDa. Digestion of the proteoglycan with a combination of heparinases yielded core proteins of 77, 49 and 44 kDa. Deglycosylation using trifluoromethanesulphonic acid, though slightly decreasing the sizes, gave an identical pattern of core proteins. Electrophoretic detergent blotting demonstrated that all of the core proteins were hydrophobic and are probably integral plasma membrane molecules. The peptide maps generated by V8 proteinase digestion of the two major core proteins (77 and 49 kDa) were very similar, suggesting that these two core proteins are structurally related.
Isolation and characterization of sheep α1-proteinase inhibitor
Authors: Mistry R , Snashall P D , Totty N , Guz A , Tetley T D
Published: August 2015

Sheep plasma proteinase inhibitor, analogous to human alpha 1-proteinase inhibitor (alpha 1 PI), was isolated to homogeneity. Purification was achieved by using (NH4)2SO4 precipitation, concanavalin A-Sepharose chromatography, Mono Q ion-exchange chromatography and PAGE. Sheep alpha 1 PI had an Mr of 56,000, inhibited human leucocyte elastase, pig pancreatic elastase and bovine trypsin on a 1:1 molar basis and had a plasma concentration of 1.6 +/- 0.21 g/l (mean +/- S.D.). Amino acid/carbohydrate composition (15% glycosylated) was similar to that of human alpha 1 PI (16% glycosylated); N-terminal analysis to 31 residues revealed 48-52% identity between the human and sheep proteins. Sheep alpha 1 PI was susceptible to oxidative inactivation by chloramine-T. Re-activation with the use of methionine sulphoxide peptide reductase and dithiothreitol indicated the presence of a methionine residue at the active site. These results establish that sheep alpha 1 PI has functional and structural characteristics close to those of human alpha 1 PI.
Two types of fatty acid-binding protein in human kidney. Isolation, characterization and localization
Authors: Maatman R G H J , Van Kuppevelt T H M S M , Veerkamp J H
Published: August 2015

Two types of fatty acid-binding protein (FABP) were isolated from human kidney by gel filtration and ion-exchange chromatography. Northern-blot analysis showed the presence of two FABP transcripts in total kidney RNA, hybridizing with cDNA of human liver and muscle FABP respectively. Characterisation based on molecular mass, isoelectric point, fluorescence with dansylaminoundecanoic acid and immunological cross-reactivity showed that one, type B, was fairly similar to human heart FABP. The other, type A, showed, like human liver FABP, a high fluorescence enhancement and a wavelength shift with dansylaminoundecanoic acid as well as the binding of a variety of ligands. Antibodies raised against FABP type A and against liver FABP markedly cross-reacted in e.l.i.s.a., in Western blotting and in indirect immunoperoxidase staining on kidney and liver sections. Differences in amino acid composition and isoelectric points, however, indicate that type A is a new kidney-specific FABP type. The FABP type A is more abundant in kidney than the B type and is predominantly localized in the cortex, especially in the cells of the proximal tubules. The FABP type B is mainly present in the cells of the distal tubules. In conclusion, this study shows the presence of two types of FABP in the kidney. One type seems to be related to heart FABP, while the other type resembles, but is not identical with, liver FABP. Both types have a characteristic cellular distribution along the nephron.
Measurement of intracellular Ca2+ in single aequorin-injected and suspensions of fura-2-loaded ROS 17/2.8 cells and normal human osteoblasts. Effect of parathyroid hormone
Authors: Schöfl C , Cuthbertson K S R , Gallagher J A , Pennington S R , Cobbold P H , Brabant G , Hesch R D , von zur Mühlen A
Published: August 2015

It is known that parathyroid hormone (PTH) activates the cyclic AMP (cAMP) signalling pathway in osteoblasts. In recent years it has been suggested that an elevation of the intracellular free Ca2+ concentration ([Ca2+]i) may also be involved in the regulation of osteoblast function by PTH. However, this remains controversial. Here we investigated the effect of PTH on the [Ca2+]i of ROS 17/2.8 cells and normal human osteoblasts. The [Ca2+]i was measured in single aequorin-injected cells and in suspensions of cells loaded with fura-2. Human PTH-(1-38)-peptide (1-300 nM) had no effect on the [Ca2+]i in single aequorin-injected ROS 17/2.8 cells (n = 17) measured at various times after injection (1-20 h), or in suspensions of fura-2-loaded ROS 17/2.8 cells (n = 9). Ionomycin (1 microM) increased the [Ca2+]i in fura-2-loaded and single aequorin-injected ROS 17/2.8 cells by 285 +/- 60 nM (n = 9) and 312 +/- 99 nM (n = 6) respectively, indicating that both methods detect changes in [Ca2+]i with equal sensitivity. In contrast, human PTH-(1-38) (10-100 nM) markedly stimulated cAMP accumulation in ROS 17/2.8 cells. In single aequorin-injected normal human osteoblasts there was no change in the [Ca2+]i in response to 100 nM human PTH-(1-38) or 100 nM bovine PTH-(1-84) (n = 18). In contrast, in suspensions of normal human osteoblasts loaded with fura-2, an increase in [Ca2+]i in response to human PTH-(1-38) (100 nM) was found (60 +/- 28 nM; n = 6). Considerable variation in the magnitude of the response was observed between individual preparations and donors. These data indicate that PTH activates cAMP accumulation without affecting [Ca2+]i in ROS 17/2.8 cells and that PTH causes a rise in [Ca2+]i only in a small subset of normal human osteoblasts. We suggest that the Ca2+ response to PTH in osteoblasts is limited by the state of differentiation of the cells, and may be due either to the presence of a distinct Ca2(+)-mobilizing receptor or to a cAMP-mediated Ca2+ response.
Extracellular ATP-induced acidification leads to cytosolic calcium transient rise in single rat cardiac myocytes
Authors: Pucéat M , Clément O , Scamps F , Vassort G
Published: August 2015

The origin of the increase in cytosolic free Ca2+ concentration ([Ca2+]i) induced by extracellular ATP was investigated in single isolated cardiac myocytes loaded with indo-1. The nucleotide added at a concentration of 10 microM triggers a few Ca2+ spikes, followed by a cluster of Ca2+ oscillations, increasing [Ca2+]i to around 200 nM from a basal value of 70 nM. Neither caffeine nor ryanodine affects the magnitude of the Ca2+ transient, but both shorten it by preventing the Ca2+ oscillations. This indicates that the latter must be related to the release of Ca2+ from the sarcoplasmic reticulum. Since ATP also induces cell depolarization (as shown by experiments using the potential sensitive dye bis-oxonol), the initial Ca2+ spikes were attributed to the opening of voltage-dependent Ca2+ channels. A small Ca2+ transient still remains under experimental conditions designed to prevent Ca2+ influx from external medium (low-Ca2+ high-Mg2+ medium containing La3+) and after depletion of the sarcoplasmic-reticulum Ca2+ load with caffeine. Under these conditions, when this Ca2+ transient was buffered by 1,2-bis-(O-aminophenoxy)ethane-NNN′N′-tetra-acetic acid, ATP was unable to trigger the initial Ca2+ spikes. These results indicate that ATP mobilizes Ca2+ ions from an intracellular pool other than the sarcoplasmic reticulum and that this Ca2+ release is responsible for the depolarization. The effects of ATP on [Ca2+]i share the same characteristics as the acidification simultaneously induced by the nucleotide (as shown by experiments using the pH-sensitive probe snarf-1). These ionic variations are highly specific to ATP and its hydrolysis-resistant analogues. They both require the presence of Mg2+ and Cl- ions in the extracellular medium, and they are prevented by pretreatment of the cells with 4,4′-di-isothiocyanostilbene or probenecid. These results suggest that: (1) the ATP-induced acidification leads to displacement of Ca2+ ions from or close to the internal face of sarcolemma; (2) the Ca2+ ions activate a non-specific membrane conductance responsible for the depolarization of the cells; (3) the depolarization leads to a Ca2+ influx, owing to the opening of the voltage-dependent Ca2+ channels; (4) this increase in Ca2+ triggers the release of Ca2+ from the sarcoplasmic reticulum, which is facilitated by the increase in inositol trisphosphate following P2-purinergic stimulation.
Functional reconstitution of the hepatic system N amino acid transport activity
Authors: Tamarappoo B K , Kilberg M S
Published: August 2015

Hepatic System N is responsible for the active plasma-membrane transport of glutamine, histidine and asparagine. This report describes the solubilization and reconstitution of System N activity. Differential solubility resulted in an approximate enrichment of almost 600-fold compared with total cell homogenate. The results indicate that reconstitution can be utilized as a functional assay during purification of the hepatic System N carrier protein.
Succinate-driven reverse electron transport in the respiratory chain of plant mitochondria. The effects of rotenone and adenylates in relation to malate and oxaloacetate metabolism
Authors: Rustin P , Lance C
Published: August 2015

The effects of rotenone on the succinate-driven reduction of matrix nicotinamide nucleotides were investigated in Percoll-purified mitochondria from potato (Solanum tuberosum) tubers. Depending on the presence of ADP or ATP, rotenone caused an increase or a decrease in the level of reduction of the matrix nicotinamide nucleotides. The increase in the reduction induced by rotenone in the presence of ADP was linked to the oxidation of the malate resulting from the oxidation of succinate. Depending on the experimental conditions, malic enzyme (at pH 6.6 or in the presence of added CoA) or malate dehydrogenase (at pH 7.9) were involved in this oxidation. At pH 7.9, the oxaloacetate produced progressively inhibited the succinate dehydrogenase. In the presence of ATP the production of oxaloacetate was stopped, and succinate dehydrogenase was protected from inhibition by oxaloacetate. However, previously accumulated oxaloacetate transitorily decreased the level of the reduction of the NAD+ driven by succinate, by causing the reversal of the malate dehydrogenase reaction. Under these conditions (i.e. presence of ATP), rotenone strongly inhibited the reduction of NAD+ by succinate-driven reverse electron flow. No evidence for an active reverse electron transport through a rotenone-insensitive path could be obtained. The inhibitory effect of rotenone was masked if malate had previously accumulated, owing to the malate-oxidizing enzymes which reduced part or all of the matrix NAD+.
Pseudoperoxidase activity of 5-lipoxygenase stimulated by potent benzofuranol and N-hydroxyurea inhibitors of the lipoxygenase reaction
Authors: Riendeau D , Falgueyret J P , Guay J , Ueda N , Yamamoto S
Published: August 2015

The purified 5-lipoxygenase from porcine leukocytes was found to catalyse the degradation of lipid hydroperoxides in the presence of potent inhibitors of the lipoxygenase reaction. Derivatives of diphenyl-N-hydroxyureas, 4-hydroxybenzofurans and 5-hydroxydihydrobenzofurans all stimulated the 5-lipoxygenase-mediated destruction of 13-hydroperoxyoctadecadienoic acid (13-HPOD). The reaction was dependent on inhibitor and hydroperoxide concentrations (1-10 microM) and could not be detected using heat-inactivated enzyme, when ATP and Ca2+ were omitted or when the hydroperoxide was replaced by the corresponding alcohol. The stability of the inhibitors during this pseudoperoxidase reaction was investigated by measuring the recoveries of 5-hydroxy-2-phenethyl-6-(3-phenoxypropyl)-2,3-dihydrobenzofuran and N-(4-chlorophenyl)-N-hydroxy-N'-(3-chlorophenyl)urea from the reaction mixtures using reverse-phase h.p.l.c. By using an equimolar concentration of 13-HPOD and inhibitor (10 microM) and under conditions where 50% of the 13-HPOD was consumed, the concentration of the benzofuranol decreased by 30%, whereas the N-hydroxyurea derivative could be completely recovered from the reaction mixture. A stimulation of the pseudoperoxidase reaction could be detected only with very effective inhibitors of leukotriene B4 biosynthesis by human leucocytes [IC50 (concn. causing 50% inhibition) less than 100 nM], but not with closely related structural analogues of lower potency or other inhibitors such as nordihydroguaiaretic acid, quercetin or the hydroxamate A-64077. These results demonstrate that 5-lipoxygenase possesses a pseudoperoxidase activity and indicate that potent inhibitors in both N-hydroxyurea and benzofuranol series can function as reducing agents for the enzyme.
Characterization of the enzyme activity of human plasma lipoprotein (a) using synthetic peptide substrates
Authors: Jauhiainen M , Metso J , Koskinen P , Ehnholm C
Published: August 2015

The plasma concentration of lipoprotein (a) [Lp(a)] is correlated with the risk of atherosclerosis. It is a lipoprotein particle consisting of apoprotein (a) [Lp(a)] is correlated with the risk of atherosclerosis. It is a lipoprotein particle consisting of apoprotein (a) [apo(a)], a protein showing considerable amino acid sequence identity with plasminogen. bound to low-density lipoprotein. The apo(a) portion of Lp(a) was recently shown to have serine-proteinase-type amidolytic activity and to be able to degrade the adhesive glycoprotein fibronectin. To characterize this enzyme activity further, we used chromogenic peptide substrates and inhibitors. Of the substrates tested, those with arginine at the scissile bond [N-alpha-benzoyl-L-Arg p-nitroanilide (pNA), N-alpha-benzoyl-Ile-Glu-Gly-Arg-pNA, N-alpha-benzyloxycarbonyl-Arg-Gly-Arg-pNA] gave the highest hydrolysis rates. Synthetic substrates with plasmin specificity (Val-Leu-L-Lys-pNA and Val-Phe-L-Lys-pNA) were not hydrolysed by Lp(a). Neither tissue plasminogen activator nor urokinase had any effect on the enzyme activity. The addition of antibodies to these plasminogen activators did not inhibit the enzyme activity of Lp(a). Inhibition experiments with phenylmethanesulphonyl fluoride, carbodi-imide, dichloroisocoumarin and competitive peptide inhibitors demonstrated that Lp(a) has enzyme activity that closely resembles that of serine proteinases. Whether this serine-proteinase activity of Lp(a) plays any role in the genesis of atherosclerosis remains to be established.
Lipid metabolism in the obese Zucker rat. Disposal of an oral [14C]triolein load and lipoprotein lipase activity
Authors: López-Soriano E J , Carbó N , Argilés J M
Published: August 2015

Oxidation in vivo of [14C]triolein to 14CO2 was significantly lower in obese (fa/fa) Zucker rats as compared with their lean (+/?) controls. In response to a 24 h starvation period, both lean and obese rats showed an enhanced rate of [14C]triolein oxidation. There were, however, no changes in the rate of intestinal absorption of [14C]triolein between the lean and obese animals. Conversely, the total tissular [14C]lipid accumulation was significantly higher in white adipose tissue, carcass and plasma in the obese animals, whereas that of brown adipose tissue was lower. This was associated with a marked hyperinsulinaemia and hypertriglyceridaemia in the fa/fa animals. Starvation dramatically decreased [14C]lipid accumulation in white adipose tissue of the lean Zucker rats, but had no effect in the obese rats. The lipogenic rate of the obese rats was significantly higher than that of lean rats in liver, white adipose tissue, skeletal muscle and carcass. Lipoprotein lipase activity (per g of tissue) was significantly lower in both white and brown adipose tissue of obese versus lean rats; however, total activity was higher in both tissues. Starvation significantly lowered perigenital-adipose-tissue lipoprotein lipase activity in the lean groups, and had no effect in the obese ones. These results demonstrate that the tissue capacity of exogenous lipid uptake is involved, but cannot be the only factor influencing the maintenance of obesity in these animals. Thus, in the adult fa/fa rat, the large increase in obesity is not solely dependent on a deviation of energy-producing substrate metabolism towards the storage of lipids in white fat. Other factors, such as a low rate of oxidation, a high lipogenic rate and decreased brown-adipose-tissue activity are involved in the perseverance of the obesity syndrome.
The DNA-binding properties of a rat nuclear phosphoprotein
Authors: Cowan D B , Chaban G , Liew C C
Published: August 2015

The non-histone nuclear phosphoprotein B2 (Mr 68,000; pI 6.5-8.2) was found to bind specifically defined fragments of DNA. With the use of monoclonal IgG2A antibodies prepared against this nuclear antigen, nucleosomal DNA fragments associated with phosphoprotein B2 were isolated and cloned. Nine cloned fragments were sequenced and analysed for similarity. The clone having the most similarity to the others was chosen to serve as a model in gel shift and footprinting assays. Subsequently, the DNA binding site was found to reside within a 30 bp region. Synthetic oligonucleotides corresponding to this site confirmed the specificity of DNA binding exhibited by the nuclear antigen as demonstrated in competition assays. Moreover, a 5′-TATTAG/C-3′ motif was found to exist within the binding site and in the other sequenced clones, possibly implicating the involvement of this motif in protein binding.
Regulated expression of cytochrome P-450scc (cholesterol-side-chain cleavage enzyme) in cultured cell lines detected by antibody against bacterially expressed human protein
Authors: Hu M C , Guo I C , Lin J H , Chung B C
Published: August 2015

The first step in the synthesis of steroids is catalysed by cytochrome P-450ssc (cholesterol-side-chain cleavage enzyme). We have investigated the synthesis of this enzyme in three cultured cell lines at the protein and hormone secretion levels. Hormone levels were measured by an enzyme immunoassay using a monoclonal antibody against progesterone. The protein level was detected using polyclonal antibodies directed against a P-450scc fusion protein overproduced in Escherichia coli. Utilizing a bacteriophage T7 promoter expression system, a large amount of human P-450scc fusion protein was produced and easily purified. P-450scc was synthesized in the mouse adrenal tumour cell line Y1 and human choriocarcinoma cell line JEG-3, but not in monkey kidney cell line COS-1. The production of P-450scc in Y1 and JEG-3 cells was stimulated by 8-bromo cyclic AMP, the effect of which was not observed until 6 h after induction and was more pronounced at 24 h. Y1 and JEG-3 cells exhibited a difference in progesterone secretion after induction.
Dimeric carotenoid interaction in the light-harvesting antenna of purple phototrophic bacteria
Authors: Zurdo J , Lozano R M , Fernandez-Cabrera C , Ramirez J M
Published: August 2015

The carotenoid content of intracytoplasmic membrane vesicles isolated from purple phototrophic bacteria was reduced to a variable extent by mild extraction with light petroleum. Using preparations obtained from Rhodobacter capsulatus strains that contained the Light Harvesting System I (LHI) complex as the only major photosynthetic holochrome, it was shown that the visible circular dichroism of the carotenoids increased with the square of the membrane carotenoid content, as expected from being caused by dimeric exciton interaction. No chirality resulting from twists of the individual planar chromophore was detected. Therefore the contribution to carotenoid optical activity of non-degenerate interactions with bacteriochlorophyll or the apoprotein does not appear to be significant. The broadening of the absorption band of the bound pigment, caused by the splitting of the monomer transition, was demonstrated in membrane vesicles of both Rb, capsulatus and Rhodospirillum rubrum as a decrease of the fine structure of the band. Furthermore, the dimeric organization of the carotenoid pigments in the bacterial LHI complex accounted for the observed quantitative relationship between the fine structure of the band and the carotenoid content of the membrane.
Heterologous expression of the allelic variant mu-class glutathione transferases μ and ψ
Authors: Widersten M , Pearson W R , Engström A , Mannervik B
Published: August 2015

cDNA encoding the more acidic form, glutathione transferase (GST) psi, of the polymorphic Mu-class GSTs discovered in liver, was mutated in the 5′-end to create an NcoI site, facilitating cloning into the expression plasmid pKK233-2. The protein expressed from this construct has a point mutation Pro-2-Ala-2, but gives a catalytically functional protein. Back-mutation of the codon for amino acid residue 2 gave rise to a plasmid expressing the wild-type enzyme GST psi, or GST Mu1b-1b. A variant cDNA, differing only in specifying lysine rather than asparagine in position 173 of the coding region, was generated by site-directed mutagenesis. The variant sequence corresponds to another cDNA clone isolated from a human liver cDNA library and expresses the near-neutral GST mu, or GST Mu1a-1a. The two recombinant proteins GST Mu1a-1a and GST Mu1b-1b, by physicochemical as well as kinetic criteria, were found to be indistinguishable from GST mu and GST psi respectively, isolated from human liver. It is therefore concluded that the recombinant proteins correspond to the allelic variants observed in the human population. The two forms have different isoelectric points and correspond to the allelic variants observed in the human population. The two forms have different isoelectric points and their protein subunits can be separated by h.p.l.c. on a reverse-phase column. With standard substrates and inhibitors no differences in kinetic parameters between the two variants were detected. The mutated GST Mu1b-1b (Pro-2-Ala) was not significantly different in catalytic properties from the wild-type enzyme, even though Pro-2 is a well conserved amino acid residue in the known Mu-class GSTs.
Degradation of sucrase-isomaltase in the ileum of jejunum-bypassed rats
Authors: Shinohara H , Goda T , Takase S
Published: August 2015

At 3 weeks after jejunal-bypass operation in the rat, the proximal ileum exhibited a greater sucrase specific activity than did sham-operated controls, whereas isomaltase activity was unaffected. Electroimmunoassay of sucrase-isomaltase and a degradation product, isomaltase monomer, revealed an elevated molar ratio of sucrase subunit to isomaltase subunit in operated rats, suggesting a decrease in the extent of degradation of ileal sucrase-isomaltase after the jejunal-bypass operation.
Differential mucin gene expression in human pancreatic and colon cancer cells
Authors: Yonezawa S , Byrd J C , Dahiya R , Ho J J L , Gum J R , Griffiths B , Swallow D M , Kim Y S
Published: August 2015

The purpose of this study was to determine the quantity and nature of the mucins synthesized and secreted by four different pancreatic cancer cell lines. Well- to moderately-differentiated SW1990 and CAPAN-2 human pancreatic cancer cells were found to produce more high-Mr glycoprotein (HMG) than less-differentiated MIA PaCa-2 and PANC-1 cells. Most of the labelled HMG was secreted within 24 h. The results of chemical and enzymic degradation, ion-exchange chromatography and density-gradient centrifugation indicated that the HMG in SW1990 and CAPAN-2 cells has the properties expected for mucins, whereas much of the HMG in MIA PaCa-2 and PANC-1 cells may not be mucin, but proteoglycan. These results are consistent with immunoblots and Northern blots showing the presence of apomucin and apomucin mRNA in SW1990 and CAPAN-2 cells, but not in MIA PaCa-2 and PANC-1 cells. The Western blots and Northern blots also show that SW1990 and CAPAN-2 cells, like breast cancer cells, have the mammary-type apomucin and mRNA coded by the MUC1 gene, but lack the intestinal type apomucin and mRNA coded by the MUC2 gene. In contrast, the colon cancer cell lines tested in culture express apomucin and mRNA coded by MUC2 but not by MUC1.
Characterization of a type I collagen α 2(I) glycine-586 to valine substitution in osteogenesis imperfecta type IV. Detection of the mutation and prenatal diagnosis by a chemical cleavage method
Authors: Bateman J F , Hannagan M , Chan D , Cole W G
Published: August 2015

A chemical cleavage method for detecting mismatched bases in heteroduplexes formed between patient mRNA and control cDNA probes was employed to identify a single base mutation in a heterozygous case of osteogenesis imperfecta type IV. The parents' fibroblast mRNA did not contain the mutation. The region of the mRNA mismatch was amplified by using the polymerase chain reaction, cloned and sequenced. A point mutation of G to U at base-pair 2162 of the collagen alpha 2(I) mRNA resulted in the substitution of glycine by valine at amino acid position 586 of the helix. This substitution disrupted the critical Gly-Xaa-Yaa repeating unit of the collagen triple helix and resulted in helix destabilization, as evidenced by a decreased thermal stability. This local disturbance to helix propagation from the C-terminus to the N-terminus led to the overmodification of the collagen helix downstream towards the N-terminus. However, collagen secretion in vitro was normal, and the clinical phenotype probably resulted from the secretion into the extracellular matrix of the mutant collagen combined with a decrease in collagen production to 65% of control values. The rapid detection of the osteogenesis imperfecta mutation by using the chemical cleavage method afforded the opportunity to apply the technique to prenatal diagnosis in the next pregnancy of the mother of the osteogenesis imperfecta patient. The absence of a mismatched base in chorionic villus mRNA and control cDNA heteroduplexes indicated that the foetus did not carry the mutation, which was confirmed by the subsequent delivery of a normal baby.
The inhibition of human platelet function by ganodermic acids
Authors: Wang C N , Chen J C , Shiao M S , Wang C T
Published: August 2015

Human gel-filtered platelets aggregate at greater than 20 microM-ganodermic acid S [lanosta-7,9(11),24-triene-3 beta, 15 alpha-diacetoxy-26-oic acid] [Wang, Chen, Shiao & Wang (1989) Biochim. Biophys. Acta 986, 151-160]. This study showed that platelets at less than 20 microM-ganodermic acid S displayed both concentration- and time-dependent inhibition of function, in which the agent potency in response to inducers was ADP-fibrinogen greater than collagen greater than thrombin. The agent caused a biphasic time-dependent effect on platelet phosphoinositide metabolism. The first phase involved the decrease in the pool size of phosphoinositide by 10-20%. The second phase, in which both the resynthesis of phosphatidylinositol 4,5-bisphosphate (PIP2) and the decrease of [32P]phosphatidic acid occurred, took place after 30 min. Scanning electron microscopy also revealed a time-dependent morphological change in platelets in the presence of the agent. The cells initially became spiculate discs, then swelled to a ‘potato-like’ morphology at 60 min. Further studies on the time-dependent inhibition of thrombin response revealed that: (1) the percentage inhibition of cell aggregation was comparable with that occurring with an increase of cytosolic free Ca2+ concentration [(Ca2+]i) or the phosphorylation of marker proteins; (2) [32P]Pi-labelled platelets showed the time-dependent inhibition of thrombin-stimulated PIP2 resynthesis as indicated by first-2-min time-course studies of phosphoinositide interconversion; (3) scanning electron microscopy revealed that the aged platelet population showed an increase in the percentage of non-responding cells on prolonged incubation. The results, taken together, enabled one to discuss a possible mechanism for the time-dependent inhibition by ganodermic acid S of platelet response to thrombin.
Proteoglycans synthesized by an osteoblast-like cell line (UMR 106-01)
Authors: McQuillan D J , Findlay D M , Hocking A M , Yanagishita M , Midura R J , Hascall V C
Published: August 2015

The proteoglycans synthesized by an osteoblast-like cell line of rat origin (UMR 106-01) were defined after biosynthetic labelling with [35S]sulphate and [3H]glucosamine. Newly synthesized labelled proteoglycans were characterized by differential enzymic digestion in combination with analytical gel filtration and SDS/PAGE. UMR 106-01 cells were found to synthesize three major species of proteoglycan: a large chondroitin sulphate proteoglycan of Mr approximately 1 x 10(6), with a core protein of Mr approximately 350,000-400,000; a small chondroitin sulphate-containing species of Mr approximately 120,000 with a core protein of Mr 43,000; and a heparan sulphate proteoglycan of Mr approximately 150,000, with a core protein of Mr approximately 80,000. Over 70% of the newly synthesized intact proteoglycan species are associated with the cell layer of near-confluent cells; however, accessibility to trypsin digestion suggests an extracellular location. Chemical characteristics of the proteoglycans and preliminary mRNA hybridization indicate that the small chondroitin sulphate proteoglycan is probably PG II (decorin). The large chondroitin sulphate proteoglycan is most likely related to a hyaluronate-aggregating species from fibroblasts (versican), and the heparan sulphate proteoglycan bears striking similarities to cell-membrane-intercalated species described for a number of cell types.
Asparagine-linked glycoprotein biosynthesis in rat epididymis. Presence of a mannosidase II-like enzyme
Authors: Skudlarek M D , Orgebin-Crist M C , Tulsiani D R P
Published: August 2015

Previous studies from this laboratory using p-nitrophenyl alpha-D-mannoside (p-NPM) as substrate provided no evidence for the presence of mannosidase II in the rat epididymis [Skudlarek & Orgebin-Crist (1988) J. Reprod. Fertil. 84, 611-617]. However, rat epididymal epithelial cells cultured in the presence of swainsonine, an inhibitor of mannosidase II, produce abnormally processed N-linked glycoproteins containing hybrid-type oligosaccharides instead of complex-type [Tulsiani, Skudlarek & Orgebin-Crist (1990) Biol. Reprod. 43, 130-138], a result providing indirect evidence for the presence of mannosidase II-like enzyme in rat epididymis. In the studies described here, we present evidence for the occurrence of this processing enzyme in rat epididymal Golgi membranes. This enzyme is an integral Golgi membrane component. Like liver mannosidase II, the epididymal enzyme cleaves alpha 1,3- and alpha 1,6-linked mannosyl residues from GlcNAcMan5GlcNAc. However, unlike liver mannosidase II, the epididymal enzyme shows no activity towards the synthetic substrate, p-NPM. The epididymal mannosidase cross-reacts with liver anti-(mannosidase II) antibody, a result suggesting that the two enzymes share a common antigenic site(s). Immunoblotting studies following resolution of liver and epididymal Golgi membranes on SDS/PAGE show that, whereas the liver mannosidase II was resolved as a doublet of Mr 120,000 and 122,000, only the Mr 120,000 band was observed in the epididymal Golgi membranes. Immunoblotting of the Golgi-rich fractions, resolved under non-denaturing conditions, showed different patterns of charge and/or size isomers from the two tissues. These studies demonstrate tissue-specific differences in processing enzymes with similar function.
Molecular comparisons of the β2-microglobulin-binding site in class I major-histocompatibility-complex α-chains and proteins of related sequences
Authors: Tysoe-Calnon V A , Grundy J E , Perkins S J
Published: August 2015

beta 2-Microglobulin (beta 2m) binds non-covalently to the alpha 1, alpha 2 and alpha 3 domains of the alpha-chain of Class I major-histocompatibility-complex (MHC) molecules. On the basis of the crystal structures of human leucocyte antigens HLA-A2.1 and HLA-Aw68.1, we have used molecular-graphics analyses to define 44 contact points between 19 alpha-chain residues and 18 beta 2m residues. In 88 other alpha-chain sequences from the HLA-A, HLA-B, HLA-C, HLA-D, HLA-E, HLA-F and HLA-G locus products in man and the H-2, Qa and Tla loci in mouse, 37 contact sites were conserved to 90% or more, and in beta 2m sequences from seven other species 40% of contact sites were totally conserved. Four distinct regions form the contact points between the alpha-chain and beta 2m, one on each of the alpha 1 and alpha 2 domains and two on the alpha 3 domain. We have further studied the alpha-chain sequences of three non-MHC molecules, human CD1 and rat Fc receptor (FcRn), known to bind to beta 2m, and a third molecule, the putative product of the H301 (UL18) gene of human cytomegalovirus (CMV). CMV has been shown to bind beta 2m, and it has been postulated that the H301-gene product, which has sequence similarity to Class I HLA, is the protein responsible. These sequences exhibited much lower residue conservation with the MHC-linked group, although the alpha 3 domain was the most highly conserved, and gaps and insertions were required for optimal alignments with the 90 alpha-chain sequences. Of the 44 beta 2m-alpha-chain contacts defined for Class I HLA, 24 alpha-chain contact sites were conserved in CD1, 25 in FcRn and 17 in the H301-gene product. For CD1 and FcRn, the majority of the conserved beta 2m contacts were found in the alpha 2 domain and the major contact region in the alpha 3 domain. Together with the use of secondary-structure predictions, it was concluded that the binding of beta 2m in CD1 and FcRn was MHC-like at the alpha 3 domain, and probably also at the alpha 2 domain for FcRn, but non-MHC-like for the alpha 1 domain of both molecules and the alpha 2 domain of CD1. In the H301-gene product sequence, only the beta 2m contacts with the main region of the alpha 3 domain were noticeably conserved.(ABSTRACT TRUNCATED AT 400 WORDS)
Kinetics of the specific binding of a second RNA polymerase to the standard bacterial-transposon-Tn3 bla promoter complex
Authors: Schmitt B , Reiss C
Published: August 2015

It was shown previously, that at moderate excess of RNA polymerase (RNAP) relative to DNA, the complex of the bla promoter from bacterial transposon Tn3 with RNAP is locked in an inactive, heparin-resistant, isomeric state which is not that of the ‘open’ complex. This 1:1 isomer can accommodate a second RNAP, which becomes tightly and specifically bound just upstream of the first RNAP [Duval-Valentin & Reiss (1990) Mol. Microbiol. 4, 1465-1475]. Both the resulting 2:1 complex and its antecedent 1:1 complex formed at excess of RNAP are immediately and permanently inhibited for transcription initiation. Using the gel-retardation technique, we investigate here the kinetics of formation and decay of the 2:1 complex under various experimental conditions. The data are consistent with pseudo-first-order kinetics at moderate excess of RNAP. The salt-dependence of rate and equilibrium constants has been analysed within the framework of the theoretical model described by Lohman, Dehaseth & Record [(1978) Biophys. Chem. 8, 281-294]. It was found that the salt-dependence is consistent with the existence of a transient intermediate during formation of the 2:1 complex, which forms rapidly on the time scale of its isomerization to the final 2:1 complex. The intermediate is characterized by the release of about seven cations from the 1:1 complex, one additional cation being released upon its final isomerization. Formation of the 2:1 complex at high excess of RNAP becomes inhibited, probably as a result of a ‘bumping’ effect of the complex by the enzyme, also observed with several other promoters. We conclude that formation of the 2:1 complex closely mimics that of the standard 1:1 complex, except that the final isomerization step to an ‘open’ complex is lacking. A mechanism of the formation of the 2:1 complex and of its role in transcription regulation of constitutive promoter by RNAP is proposed.
An improved assay for pyruvate dehydrogenase in liver and heart
Authors: Paxton R , Sievert L M
Published: August 2015

A radiochemical assay was developed to measure pyruvate dehydrogenase complex (PDC) activity in liver and heart without interference by branched-chain 2-oxo acid dehydrogenase (BCODH). Decarboxylation of pyruvate by BCODH was eliminated by using low pyruvate concentration (0.5 mM), a preferred substrate for BCODH (3-methyl-2-oxopentanoate) that is not used by PDC, and a competitive inhibitor of BCODH, dichloroacetate. This method was validated by assaying a combination of both purified enzymes and tissue homogenates with known amounts of added BCODH. The actual percentage of active PDC decreased after 48 h starvation from 13.6 to 3.1 in liver and from 77.1 to 9.0 in heart. Total PDC activity (munits of PDC/units of citrate synthase) in starved rats was increased by 34% in liver and decreased by 23% in heart. Total PDC activity (munits/g wet wt.) in fed- and starved-rat liver was 0.8 and 1.3, and in heart was 6.6 and 5.8, respectively.
Raf-1 is a potential substrate for mitogen-activated protein kinase in vivo
Authors: Anderson N G , Li P , Marsden L A , Williams N , Roberts T M , Sturgill T W
Published: August 2015

MAP (mitogen-activated protein) kinase is shown to phosphorylate baculovirally expressed Raf-1 in vitro, generating one major tryptic phosphopeptide which co-migrated with a peptide from Raf-1 32P-labelled in situ. This peptide also undergoes an insulin-dependent increase in labelling. Thus the serine/threonine kinase Raf-1 may be a substrate for MAP kinase in vivo.
Modulation of maximal glycogenolysis in perfused rat liver by adenosine and ATP
Authors: Vanstapel F , Waebens M , Van Hecke P , Decanniere C , Stalmans W
Published: August 2015

Rat livers perfused at constant flow via the portal vein with dibutyryl cyclic AMP produced glucose equivalents at a steady maximal rate (6 mumol/min per g of liver). Addition of adenosine (150 microM) caused a biphasic effect. (i) First, the glycogenolytic rate rose transiently, to a mean peak of 150% of control levels after 2 min. This glycogenolytic burst was reproduced by two P1-receptor agonists, but not by ATP, and was blocked by a P1-antagonist (8-phenyltheophylline), as well as by inhibitors of eicosanoid synthesis (indomethacin, ibuprofen or aspirin). It did not occur in phosphorylase-kinase-deficient livers. The adenosine-induced glycogenolytic burst coincided with moderate and transient changes in portal pressure (+6 cmH2O) and O2 consumption (-20%), but it could not be explained by an increase in cytosolic Pi, since the n.m.r. signal fell precipitously. (ii) Subsequently, the rate of glycogenolysis decreased to one-third of the preadenosine value, in spite of persistent maximal activation of phosphorylase. The decrease could be linked to the decline in cytosolic Pi: both changes were prevented by the adenosine kinase inhibitor 5-iodotubercidin, whereas they were not affected by ibuprofen or 8-phenyltheophylline, and were not reproduced by non-metabolized adenosine analogues. In comparison with adenosine, ATP caused a slower decrease of Pi and of glycogenolysis. The fate of the cytosolic Pi was unclear, especially with administered ATP, which did not increase the n.m.r.-detectable intracellular ATP.
Structure-activity relationships of retinoids as inhibitors of calmodulin-dependent human erythrocyte Ca2+-ATPase activity and calmodulin binding to membranes
Authors: Davis F B , Smith T J , Davis P J , Blas S D
Published: August 2015

All-trans retinoic acid displaces the binding of radiolabelled calmodulin to human erythrocyte membranes, and inhibits the activity of plasma membrane Ca(2+)-stimulated, Mg(2+)-dependent ATPase (Ca(2+)-ATPase; EC 3.6.1.3). This enzyme is dependent upon the action of calmodulin. In this study we explored the structural attributes of the retinoids which confer this ability to inhibit enzyme activity and calmodulin binding. With respect to the fatty acid side-chain, a clear requirement for inhibition is a trans-configuration of the polar end-group. The importance of the ring structure is indicated by the ineffectiveness of polyprenoic acid and a benzene ring retinoid analogue as inhibitors of enzyme activity and calmodulin binding. There was good correlation between the relative potencies of the analogues as enzyme inhibitors and as inhibitors of calmodulin binding. The ability of selected retinoid analogues, at physiological concentrations with respect to all-trans retinoic acid, to inhibit erythrocyte Ca(2+)-ATPase activity and membrane binding of calmodulin underscores the structurally specific effects of these compounds on the interaction of calmodulin with the membrane-bound enzyme.