bluuebunny/crossref_metadata_2025_split huggingface dataset:
Peroxisomal β‐oxidation system of Candida tropicalis
Authors: MORENO DE LA GARZA Myrthala , SCHULTZ‐BORCHARD Ursula , CRABB John W. , KUNAU Wolf‐H. 
Published: March 2005

1. A multifunctional protein from oleate‐grown cells of Candida tropicalis has been purified and partially characterized. A simple two‐step purification has been developed involving ion‐exchange chromatography followed by dye‐ligand chromatography on blue Sepharose CL‐6B. Homogeneous enzyme with a subunit Mr of 102000 is obtained in 60% yield. The native relative molecular mass, determined by three different methods, yielded values which suggest that the enzyme is dimeric. Sodium dodecyl sulfate/polyacrylamide gel electrophoresis of the purified protein revealed a single polypeptide band and reverse‐phase high‐performance liquid chromatography indicated a single component suggesting that this protein may consist either of two identical or very similar subunits.2. Three β‐oxidation activities, enoyl‐CoA hydratase, 3‐hydroxyacyl‐CoA dehydrogenase and 3‐hydroxyacyl‐CoA epimerase, co‐purified with this protein. The ratio of the three β‐oxidation enzyme activities remained constant during purification and was unchanged by additional chromatographic methods (adsorption and affinity chromatography), thus indicating the multifunctional nature of this protein. Enzymatic staining of the purified protein for 3‐hydroxyacyl‐CoA dehydrogenase and epimerase, following electrophoresis in a polyacrylamide density gradient, further supported the multifunctionality of this protein.3. After isopycnic centrifugation of a particulate fraction from oleate‐grown cells in a linear sucrose gradient the activities of all individual β‐oxidation enzymes cosedimented with catalase and with the glyoxylate bypass enzymes. This result demonstrated the peroxisomal localization of the multifunctional enzyme.4. The relationship of this multifunctional protein to the two bifunctional β‐oxidation enzymes isolated from peroxisomes of rat liver and from glyoxysomes of cucumber seeds is discussed.
Rat liver glutaminase
Authors: McGIVAN John , VADHER Mita , LACEY Jean , BRADFORD Norah 
Published: March 2005

Partially purified rat liver mitochondrial glutaminase shows a sigmoidal dependence on glutamine concentration, and an absolute requirement for inorganic phosphate as activator. Reconstitution with a mitochondrial membrane fraction changes the kinetic properties of the enzyme making the glutamine dependence more hyperbolic and reducing the concentration of phosphate required for half‐maximum activation. Glutaminase activity in isolated mitochondria is known to be increased as a result of mitochondrial swelling. In mitochondria suspended in isotonic medium, the properties of glutaminase resemble of the isolated enzyme while in swollen mitochondria the kinetic properties revert to those exhibited by the enzyme in association with the mitochondrial membrane.It is postulated that mitochondrial glutaminase is regulated in situ by reversible association with the inner mitochondrial membrane which is mediated by mitochondrial swelling. This mechanism may explain the short‐term hormonally induced activation of the enzyme observed in isolated hepatocytes.
Flavobacterium heparinum 3‐O‐sulphatase for N‐substituted glucosamine 3‐O‐sulphate
Authors: BRUCE John S. , McLEAN Maitland W. , LONG William F. , WILLIAMSON Frank B. 
Published: March 2005

A novel bacterial sulphatase has been discovered in an extract of Flavobacterium heparinum. The enzyme hydrolyses the 3‐O‐sulphate from 2‐deoxy‐2‐sulphamido‐3‐O‐sulpho‐d‐glucose and 2‐acetamido‐2‐deoxy‐3‐O‐sulpho‐d‐glucose.The activity was purified 10800‐fold by chromatography successively on CM‐Sepharose CL‐6B, hydroxy‐apatite, taurine‐Sepharose CL‐4B and CM‐Sepharose CL‐6B. Sodium dodecylsulphate/polyacrylamide gel elec‐trophoresis showed the enzyme to be homogeneous and of relative molecular mass 56000. Two novel assays were developed using 2‐[14C]acetamido‐2‐deoxy‐3‐O‐sulpho‐d‐glucose and 2‐deoxy‐2‐sulphamido‐3‐O‐sulpho‐d‐glucose as respective substrates.The purified 3‐O‐sulphatase was shown to be free of all other known heparin‐degrading enzymes. Optimal activity was at pH 7.5 for the disulphated substrate and pH 8.0 for the N‐acetylated substrate. Enzyme activity was virtually unaffected by Na+, K+ or Mg2+ ions. A 1.2‐fold enhancement of activity was effected by 0.002 mol dm−3 Ca2+. Inorganic phosphate and sulphate inhibited 3‐O‐sulphatase activity. The Km value of the N‐acetylated substrate was determined to be 42 μmol dm−3. No activity was detected with 2‐amino‐2‐deoxy‐3‐O‐sulpho‐d‐glucose.
Uptake and processing of liposomal phospholipids by Kupffer cells in vitro
Authors: DIJKSTRA Jan , VAN GALEN Mieke , REGTS Djoeke , SCHERPHOF Gerrit 
Published: March 2005

We investigated the intracellular metabolic fate of [Me‐14C]choline‐labeled phosphatidylcholines and sphingomyelin taken up by rat Kupffer cells in maintenance culture during interaction with large unilamellar liposomes composed of cholesterol, labeled choline‐phospholipid and phosphatidylserine (molar ration 5:4:1).With both labeled compounds only small proportions of water‐soluble radioactivity were found to accumulate in the cells and in the culture medium, suggesting limited phospholipid degradation. However, after a lag period of 30 min progressively increasing proportions of cell‐associated liposomal phospholipid were found to be converted to cellular phospholipid, nearly all of which was phosphatidylcholine. This conversion as well as the limited release of watersoluble label from the cells was inhibited by the lysosomotropic agents ammonium chloride and chloroquine.With [Me‐14C]choline‐labeled lysophosphatidylcholine, label was found to become cell‐associated far in excess of an encapsulated liposomal label, [3H]inulin. Without a lag period virtually all of this was rapidly converted to phosphatidylcholine, a process which was not inhibited by the lysosomotropic agents.It is concluded that Kupffer cells, after endocytosis of liposomes, degrade the liposomal phospholipids effectively but reutilize the choline moiety for de novo synthesis of cellular phosphatidylcholine.
Isolation and characterization of peroxisomes from the liver of normal untreated rats
Authors: VÖLKL Alfred , FAHIMI H. Dariush 
Published: March 2005

The classic method of Leighton et al. [(1968) J. Cell Biol. 37, 482–513] for the isolation of peroxisomes from rat liver involves the use of Triton WR‐1339 which alters the biochemical properties of this organelle and requires the specialized type Beaufay‐rotor which is not easily available. We have employed Metrizamide as the gradient medium and a commercial type vertical rotor to obtain highly purified and structurally well‐preserved peroxisomes from normal untreated animals.The livers were homogenized in buffered 0.25 M sucrose and a slightly modified ‘light mitochondrial fraction’ was prepared by differential centrifugation. This was loaded on top of a linear Metrizamide gradient (1.12 – 1.26 g/cm3) and subjected to an integrated force of 1.252 × 106× (g× min) using a Beckman VTi 50 vertical rotor. Peroxisomes banded at the density of 1.245 g/cm3. In the isolated fraction 95% of the protein was contributed by peroxisomes, which exhibited a strong activity for cyanide‐insensitive lipid β‐oxidation. The purity of fractions was also confirmed by morphometry, which revealed that 98% of isolated particles consisted of peroxisomes. The latency for catalase was about 90% indicating a high degree of peroxisomal integrity. This corresponded to the low level of extraction of catalase in 3,3′‐diaminobenzidine‐stained filter preparations. The entire procedure took about five hours. Highly purified and structurally well preserved peroxisomes should be useful in further elucidation of the function of this organelle and especially in studies of peroxisomal enzymes with multiple intracellular localizations.
Catalytic properties of a human cytomegalovirus‐induced protein kinase
Authors: MICHELSON Susan , TARDY‐PANIT Maryse , BÂRZU Octavian 
Published: March 2005

Human cytomegalovirus, a DNA virus whose genome contains a fragment of transforming DNA, induces a threonine‐serine protein kinase having a molecular mass of 68 kDa (p68). p68 was extracted from cells 96–144 h after infection, and immunoprecipitated with a monoclonal antibody (F6b). Antibody‐enzyme complexes were immobilized on heat/formaldehyde‐inactivated Staphylococcus aureus.The best substrates for p68 were acidic proteins, phosvitin and casein. Glycogen synthase, phosphorylase a and histones were phosphorylated at rates not higher than 1–4% that obtained with phosvitin as substrate. ATP and GTP were equally good substrates of p68. p68 is able to autophosphorylate at the same residues (i.e. threonine and serine) as the protein substrates. Autophosphorylation does not seem to represent an intermediate in substrate phosphorylation.The protein kinase activity of p68 was not enhanced by cAMP, calcium ions, or polyamines like spermine or spermidine. Only at low Mg2+ concentration spermine enhanced by 68% the rate of casein phosphorylation. Heparin, a potent inhibitor of casein kinase II, inhibits p68 activity too, but ten‐times higher concentrations were required for the same degree of inhibition. Quercetin, a bioflavonoid, acts as a strong inhibitor of p68 protein kinase activity. The inhibitory effect of quercetin was competitive towards the nucleotide substrate (Ki= 2.8 μM), and non‐competitive towards the protein substrate (Ki= 15 μM).
Chemical modification of methionine residues of the phosphatidylcholine transfer protein from bovine liver. A spin‐label study
Authors: MEGLI Francesco M. , van LOON Douwe , BARBUTI Angela A. , QUAGLIARIELLO Ernesto , WIRTZ Karel W. A. 
Published: March 2005

The role of methionine residues in the interaction of the phosphatidylcholine transfer protein from bovine liver with phospholipid vesicles was investigated by specific modification of these residues with iodoacetamide. The modified protein was digested with cyanogen bromide in order to determine which methionine residues had become resistant to this cleavage. Automated Edman degradation on the digest indicated that after 72 h of reaction, Met‐1 was modified for 80%, Met‐73 for 50%, Met‐109 for 20%, whilst Met‐173 and Met‐203 were found to be unmodified.This distinct modification did not result in any loss of phosphatidylcholine transfer activity. The interaction of the phosphatidylcholine transfer protein with phospholipid vesicles was investigated by making use of electron spin resonance spectroscopy. The interaction of unmodified protein with vesicles composed of phos‐phatidylcholine/phosphatidic acid/spin‐labeled phosphatidylethanolamine (79:16:5, mol%) or composed of phosphatidylserine/spin‐labeled phosphatidylethanolamine (95:5, mol%), gave an increase of about 50% in the rotation correlation time.A similar increase was observed with the modified protein. This interaction was further investigated by labeling Met‐1 and Met‐73 in the transfer protein with iodoacetamidoproxyl spin‐label.Spin‐labeling did not inactivate the transfer protein. In addition, the electron spin resonance spectra of the spin‐labeled protein were not affected upon addition of vesicles composed of phosphatidylcholine/phosphatidic acid (80:20, mol%). These experiments strongly suggest that Met‐1 and Met‐73 are not part of the site that interacts with the membrane.
Diastereomers of adenosine 3′,5′‐monothionophosphate (cAMP[S] antagonize the activation of cGMP‐dependent protein kinase
Authors: HOFMANN Franz , GENSHEIMER Hans‐Peter , LANDGRAF Wolfgang , HULLIN Roger , JASTORFF Bernd 
Published: March 2005

cGMP‐dependent protein kinase contains four cGMP‐binding sites which are homologeous to the four cAMP‐binding sites of cAMP‐dependent protein kinase. The interaction of the diastereomers of adenosine 3′,5′‐thionophosphate, (PS)‐cAMP[S] and (PR)‐cAMP[S], with cGMP‐dependent protein kinase has been studied.1. Autophosphorylation of cGMP‐dependent protein kinase is stimulated by cAMP and (PS)‐cAMP[S] with apparent KA values of 7 μM and 94 μM, respectively.2. cAMP‐stimulated autophosphorylation is inhibited competitively by (PR)‐cAMP[S] with a Ki value of 15 μM.3. The phosphorylation of the peptide substrate (Leu‐Arg‐Arg‐Ala‐Ser‐Leu‐Gly) is stimulated by cGMP (approx. KA 1 μM) and cAMP (approx. KA 98 μM) but neither by the (PR) nor (PS) stereomer of cAMP[S].4. (PR)‐cAMP[S] and (PS)‐cAMP[S] inhibit competitively cAMP‐ or cGMP‐stimulated phosphorylation of the peptide substrate with Ki values of 52 μM and 73 μM, respectively.5. (PS)‐cAMP[S] stimulates the phosphorylation of the peptide substrate by an autophosphorylated enzyme.6. Binding of [3H]cGMP to cGMP‐dependent protein kinase is inhibited by (PS)‐cAMP[S] and (PR)‐cAMP[S] with IC50 values of 200 μM and 15 μM, respectively.7. These results show that both diasteromers of cAMP[S] bind to cGMP‐dependent protein kinase. (PR)‐cAMP[S] has properties of a pure antagonist whereas (PS)‐cAMP[S] has properties of a partial agonist. The results provide further evidence that autophosphorylation of the enzyme affects the interaction between the cGMP‐binding sites and the catalytic center of the enzyme by facilitating the activation of the phosphotransferase reaction.
Demonstration of a bacteriophage receptor site on the Escherichia coli K12 outer‐membrane protein OmpC by the use of a protease
Authors: MORONA Renato , TOMMASSEN Jan , HENNING Ulf 
Published: March 2005

The Escherichia coli K12 outer‐membrane proteins OmpA, OmpC, OmpF, PhoE, and LamB (all of transmembrane nature) can serve as phage receptors. We have shown previously that one OmpA‐specific phage, Ox2, can give rise to the host range mutants Ox2h10 and Ox2h12, with the latter being derived from the former [Morona. R. & Henning, U. (1984) J. Bacteriol. 159, 579–582]. Unlike Ox2, both host range phages can use the OmpA and OmpC proteins as receptors and Ox2h12 is better adapted to the OmpC protein than Ox2h10. In a search for the site(s) of OmpC protein involved in phage recognition, it was found that proteinase K is able to cleave all of the proteins mentioned above. OmpC protein (Mr= 38306) could be cleaved from outside the cell by proteinase K resulting in two fragments of Mr∼ 21000 and Mr∼ 17500. The use of OmpC‐PhoE hybrid proteins allowed us to assign the ∼ 21000‐Mr fragment to the CO2H‐terminal moiety of the protein. Proteinase K treatment of intact cells abolished their activity to neutralize the OmpC‐specific phage Tulb and reduced this ability towards phage Ox2h12. The OmpA, OmpF, PhoE and LamB proteins were cleaved by the protease not in intact cells but only when acting on cell envelopes.The sizes of the OmpC protein fragments and the results obtained with the hybrid proteins very strongly suggest that the protein is cleaved from outside the cell at a region involving amino acid residues 150–178 of the 346‐residue protein, which shows homology to two regions of the OmpA protein which are involved in its phage receptor site (loc. cit.). These areas also exhibit some homology to a region of the LamB protein which is thought to be part of this protein's receptor site [Charbit et al. (1984) J. Mol. Biol. 175, 395–401]. This suggests that there is a common denominator for proteinaceous phage receptor site because the LamB‐specific phage λ and phage Tulb are of completely different nature. We conclude that the region of the OmpC protein in question is cell‐surface‐exposed and acts as a phage receptor site.
Primary structure of the two variants of a sperm‐specific histone H1 from the annelid Platynereis dumerilii
Authors: KMIECIK Daniel , SELLOS Daniel , BELAÏCHE Denise , SAUTIERE Pierre 
Published: March 2005

The amino acid sequences of the two variants (H1a 121 residues and H1b 119 residues) of the sperm‐specific histone H1 from the polychaete annelid Platynereis dumerilii have been completely established.Comparison of the sequences of these two variants shows one deletion of two residues in histone H1b and 22 substitents, of which most occur in the globular domain. The two variants differ highly in a sequence of nine residues adjacent to the conservative phenylalanine residue of histone H1 (64–72 in H1a, 62–70 in H1b) which makes H1a less hydrophobic than H1b.The small molecular size of Platynereis H1a and H1b is a unique feature among the histones H1 of which the size ranges between 189 residues (chicken erythrocyte H5) and 248 residues (sea urchin sperm H1).H1a and H1b have short N‐ and C‐terminal basic domains but the size of the globular domain (∼ 80 residues) is similar to that of other H1s.In the globular region the variant H1a exhibits a close relationship with somatic or sperm H1s whereas the variant H1b is more related to H5 histones.
Regulation of the human‐erythrocyte hexose‐monophosphate shunt under conditions of oxidative stress
Authors: THORBURN David R. , KUCHEL Philip W. 
Published: March 2005

The regulation of the hexose monophosphate shunt of human erythrocytes under conditions of oxidative stress has been investigated by monitoring the reduction of oxidised glutathione (GSSG) to reduced glutathione (GSH) in erythrocytes containing high levels of GSSG; 1H NMR and a biochemical assay were used to measure the changes.A reconstituted metabolic system prepared with the purified erythrocyte enzymes was used in conjunction with studies of intact cells and haemolysates to determine the dependence of the rate of GSH production on the activities of hexokinase and glucose‐6‐phosphate dehydrogenase. Both of these enzymes have previously been claimed to be the rate‐limiting step of oxidatively stimulated flux through the hexose monophosphate shunt.The absence of a kinetic isotope effect on the rate of GSH production in these systems, when [1‐2H]glucose replaced glucose as the source of reducing equivalents, showed that glucose‐6‐phosphate dehydrogenase activity was not a strong determinant of the flux. The dependence of the rate of GSH production on the concentration of the hexokinase inhibitors glucose 1,6‐bisphosphate and glycerate 2,3‐bisphosphate showed that, under conditions of oxidative stress, hexokinase was the principal determinant of flux through the shunt. Glucose 1,6‐bisphosphate at the concentration present in vivo appears to be more important in limiting hexokinase activity, and thus the rate of glucose utilisation, than was previously assumed.A detailed computer model of the system was developed based on the reported kinetic parameters of the enzymes involved. A sensitivity analysis of this model predicted that the hexokinase reaction would have a sensitivity coefficient of 0.995 with respect to the maximal rate of GSH production.
Modifications of the expression of liver‐specific and non‐specific messenger RNAs during azo‐dye hepatocarcinogenesis
Authors: LAMAS Eugénia , SCHWEIGHOFFER Fabien , VAN ELS Cécile , BACHNER Lucien , MÅIE Joëlle , KAHN Axel 
Published: March 2005

The expression of specific and non‐specific rat liver messenger RNAs has been studied during 3′ ‐methyl‐4‐(dimethylamino)azobenzene (3′‐MeDAB) carcinogenesis, using cDNA probes complementary to mRNAs encoding aldolase A and B, L‐type pyruvate kinase, albumin, α‐foetoprotein, transferrin and an unidentified 2.7 × 103‐base mRNA. mRNAs specific for undifferentiated cells, such as those encoding aldolase A and the unidentified 2.7 × 103‐base species were re‐expressed very early, being easily detectable at the 1st week of 3′‐MeDAB treatment. They reached a maximum of expression at the 4th week. Simultaneously the levels of aldolase B and L‐type pyruvate kinase mRNAs dramatically decreased as compared to controls, but remained responsive to induction by a high‐carbohydrate diet.Albumin and transferrin mRNA levels were only slightly modified in the course of the carcinogenic diet. At the terminal stage of hepatocarcinogenesis, i.e. in malignant hepatoma cells, expression and inducibility of aldolase B and L‐type pyruvate kinase mRNAs were similar to those in normal adult rats while mRNAs specific for undifferentiated or foetal stages were also synthesized.The very early changes in gene expression for aldolases A and B, L‐type pyruvate kinase and the 2.7 ×103 ‐base mRNA species could indicate that carcinogenic diet modifies gene control mechanisms long before inducing hepatoma.
The subcellular location in rat kidney of the peripheral benzodiazepine acceptor
Authors: O'BEIRNE Gerard B. , WILLIAMS D. Clive 
Published: March 2005

In rat kidney high‐affinity binding sites for [3H]Ro‐5‐4864 and [3H]PK‐11195 with the properties of the peripheral‐type acceptor were found enriched in mitochondrial (M) and light‐mitochondrial‐lysosomal (L) fractions on differential centrifugation. When the combined M and L fractions were subjected to sucrose density gradient centrifugation, these binding sites were found enriched at a density of 1.155 g/ml coincident with a population of light mitochondria, whereas a population of heavier mitochondria (ϱ= 1.175 g/ml) had few or no binding sites. Transmission electron microscopy showed that whereas the heavier mitochondria appeared highly pure and intact, the lighter mitochondria appeared less intact and to be contaminated with vesicular structures. After fractionation of the light mitochondria and vesicles by centrifugation, both fractions showed the same ratio of [3H]Ro‐4864 binding sites to monoamine oxidase activity consistent with the vesicles being of mitochondrial outer‐membrane origin.Digitonin pre‐treatment had no effect on the density of acceptor‐rich fractions on sucrose density gradient centrifugation. However, pretreatment with succinate/iodophenylnitrophenylphenyltetrazolium (INT) perturbed equally the density of acceptor‐rich fractions and mitochondrial marker enzymes.When mitochondrial fractions were subjected to sonication prior to density gradient centrifugation the binding sites were now found highly enriched in a much lighter fraction coincident with the monoamine oxidase activity and thus consistent with being outer‐membrane vesicles.When a mitochondrial fraction was subjected to hypotonic treatment before assay no evidence for activation/ unmasking of binding sites was found. The hypotonic treatment did not release any inhibitor of the binding sites.These results are consistent with the peripheral benzodiazepine acceptor having an outer‐membrane location on a sub‐population of rat kidney mitochondria. Those mitochondria showing high levels of the acceptor are either light mitochondria or appear more susceptible to osmotic damage than those mitochondria in which the acceptor is absent or at low levels.
What Can PISA Tell Us About Teacher Shortages?
Authors: WHITE PATRICK , SMITH EMMA 
Published: March 2005

AbstractThe OECD's recent research initiative, Attracting, Developing and Retaining Effective Teachers (OECD 2002), has highlighted teacher supply and demand as an area of international concern. Using data collected in this area as part of a wider OECD initiative (the PISA 2000 study) an individual‐level analysis was conducted on the views on teacher recruitment and retention of 5416 headteachers, principals and administrators working in secondary schools in 25 countries. The findings showed considerable variation between countries in terms of perceptions of the impact of teacher shortages, inadequacy and turnover. Surprisingly, however, differences in the reports of headteachers working in the same countries were only rarely related to the size, location, or other characteristics of the schools in which they worked.Although there has been considerable previous research into headteachers’ views on this issue, much of this research has been, by necessity, small scale and restricted to a single nation. This article provides a much larger scale perspective and allows international comparisons to be made.