bluuebunny/crossref_metadata_2025_split huggingface dataset:
Histone H1 heterogeneity in the midge, Chiromomus thummi
Authors: HOYER‐FENDER Sigrid , GROSSBACH Ulrich 
Published: March 2005

Seven subfractions of histone H1 have been isolated and purified from larvae of Chironomus thummi (Diptera). They have been denominated I‐1, II‐1, II‐2, 11‐3, III‐1, III‐2, and III‐3, according to the order of migration in two steps of preparative electrophoresis.
The amino acid compositions are similar to those of other H1 histones. Subtractions I‐1 and II‐1 were found to contain one methionine and two tyrosine residues, II‐2 contained two methionine and three tyrosine residues, and III‐1 one methionine and three tyrosine residues. The other subfractions contained one or two methionine and two or three tyrosine residues. For subfractions I‐1 and II‐1 a chain length of about 252 amino acids was estimated.
Peptide pattern analyses after chemical cleavage at the methionine and tyrosine residues, and enzymatic cleavage with thrombin and chymotrypsin, respectively, showed that all subfractions have different individual primary structures. A comparison of peptide sizes and of the positions in the peptide patterns of epitopes recognized by monoclonal antibodies was made to check whether some of the subfractions could arise by proteolytic degradation of others. This possibility can be excluded for five of the subfractions and is very improbable for the two others. Treatment of C. thummi H1 with alkaline phosphatase did not change the pattern of subfractions, while the phosphorylated subtraction of histone H2A disappeared after this treatment Most and very probably all subfractions are thus H1 sequence variants.
Inbred strains and individual larvae of C. thummi were found to comprise all seven variants. The H1 heterogeneity can therefore not be due to allelic polymorphism. Salivary gland nuclei were found to contain variant I‐1 and at least some of the other variants.
H1 from Drosophila melanogaster and from calf thymus were used as reference molecules in all cleavage experiments and yielded the peptide patterns expected from the sequence. The comparison discriminates the group of C. thummi H1 histones clearly from Drosophila and calf thymus H1. Limited trypsin digestion yielded a protected peptide of uniform size in six of the seven variants which was considerably smaller than the protected central domain of calf thymus H1.
Two other species of Chironomidae, C. pallidivittatus and Glyptotendipes barbipes were found to contain five and three H1 subfractions, respectively.
The results provide a basis for the localization of H1 variants in specific chromatin regions within the giant chromosomes by means of monoclonal antibodies and may lead to an understanding of the functional significance of H1 heterogeneity.
Cloning and expression in Escherichia coli of two additional amylase genes of a strictly anaerobic thermophile, Dictyoglomus thermophilum, and their nucleotide sequences with extremely low guanine‐plus‐cytosine contents
Authors: HORINOUCHI Sueharu , FUKUSUMI Shoji , OHSHIMA Toyoji , BEPPU Teruhiko 
Published: March 2005

An obligately anaerobic and extremely thermophilic bacterium, Dictyoglomus thermophilum, produces multiple extracellular amylases. In addition to one of the amylase genes, amyA, which we previously cloned and chgaracterized, weh have cloned two additional genes, amyB, and amyC, coding for amylases of this thermophile, into Escherichia coil and determined their nucleotide sequences. The two amylase genes were expressed under the control of E. coil promoters. Almost all activity was detected in the intracellular fraction in the E. coil cells.The molecular mass and NH2‐terminal amino acid sequence of the AmyB enzyme, which was purified from an E. coil transformant containing the amyB gene, confirmed that the reading frame of amyB consisted of 562 amino acids (Mr 67000). The molecular mass of the AmyC enzyme, estimated by activity staining of a crude extract of E. coli containing amyC, confirmed that AmyC consisted of 498 amino acids (Mr 59000). The optimal temperatures for AmyB and AmyC activities on soluble strach were 80oC and 70oC, respectively. Both AmyB and AmyC showed a pH optimum of 5.5 AmyB and AmyC showed a different pattern of strach hydrolysis when examined by thinlayer chromatography. Some homology in the amino sequences with the functional regions of Taka‐amylase A was found in both AmyB and AmyC.The condon usage in the amyA., amyB and amyC genes was highly biased, which reflects the fact that the guanine‐plus‐cytosine (G+C) content of DNA of D. thermophilum is 29 mol%. The distribution of G and C at each position of the condons was non‐random; the G+C content of the first position of condons is significantly high, whereas that of the third position is somewhat low. In addition, condons consisiting only of A and T were preferntially used in this thermophile.
Glycogenin is the priming glucosyltransferase required for the initiation of glycogen biogenesis in rabbit skeletal muscle
Authors: PITCHER Julie , SMYTHE Carl , COHEN Philip 
Published: March 2005

Purified preparations of‐glycogen synthase are a complex of two proteins, the catalytic subunit of glycogen synthase and glycogenin, present in a 1:1 molar ratio [J. Pitcher, C. Smythe, D. G. Campbell & P. Cohen (1987) Eur. J. Biochem. 169, 497‐502]. This complex has now been found to contain a further glucosyltransferase activity that catalyses the transfer of glucose residues from UDP‐Glc to glucosylated‐glycogenin. The glucosyltransferase, which is of critical importance in forming the primer required for de novo glycogen biosynthesis, is distinct from glycogen synthase in several ways. It has an absolute requirement for divalent cations, a 1000‐fold lower Km for. UDP‐Glc and its activity is unaffected by incubation with UDP‐pyridoxal or exposure to 2 M LiBr, which inactivate glycogen synthase by 95% and 100%, respectively.The priming glucosyltransferase and glycogen synthase activities coelute on Superose 6, and the rate of glycosylation of glycogenin is independent of enzyme concentration, suggesting that the reaction is catalysed intramolecularly by a subunit of the glycogen synthase complex. This component has been identified as glycogenin, following dissociation of the subunits in 2 M LiBr and their separation on Superose 12. The glycosylation of isolated glycogenin reaches a plateau when five additional glucose residues have been added to the protein, and digestion with a‐amylase indicates that all the glycogenin molecules contain at least one glucosyl residue prior to autoglucosylation. The priming glucosyltransferase activity of glycogenin is unaffected by either glucose 6‐phos‐phate or by phosphorylation of the catalytic subunit of glycogen synthase. The mechanism of primer formation is discussed in the light of the finding that glycogenin is an enzyme that catalyses its own autoglucosylation.
Turnover of thylakoid photosystem II proteins during photoinhibition of Chlamydomonas reinhardtii
Authors: SCHUSTER Gadi , TIMBERG Rina , OHAD Itzhak 
Published: March 2005

The turnover of photosystem‐II proteins during photoinhibition was analyzed in the green alga Chlamydomonas reinhardtii. Changes in the amount of photosystem II core complex polypeptides D1, D2, 44 kDa and 51 kDa, the antennae‐CP‐29 and light‐harvesting‐complex‐II polypeptides and the water‐oxidizing complex polypeptides of 30 kDa, 23 kDa and 16 kDa were monitored by a variety of techniques. Only the D1 and D2 polypeptides were found to turnover during photoinhibition when cells were exposed to ten fold photosynthesis‐saturating light (2500 W m2 for 90 min) at 25°C. While 80% of photosystem‐II activity was lost, a reduction of only 20% was observed in the total amount of D1 and D2 proteins. However, inhibition of chloroplast translation by chloramphenicol during photoinhibition resulted in the loss of about 60% of the D1 and 40% of the D2 proteins, as demonstrated by Western blotting and dot blotting of isolated thylakoids, quantitative analysis of immunogold‐labeled whole‐cell thin sections, and chase of radioactively prelabelled proteins during photoinhibition.We propose that the light‐dependent turnover of the D1 protein is a protective mechanism against photoinhibition as far as the removal and replacement of D1 is compatible with the photoinactivation incurred by photosystem II. At light intensities at which the rate of D1 removal becomes limiting, loss of photosystem‐II activity exceeds the turnover of D1 and the stability of the D2 protein is impaired as well.
Isolation and structural characterization of low‐molecular‐mass monosialyl oligosaccharides derived from respiratory‐mucus glycoproteins of a patient suffering from bronchiectasis
Authors: VAN HALBEEK Herman , BREG Jan , VLIEGENTHART Johannes F. G. , KLEIN André , LAMBLIN Geneviéve , ROUSSEL Philippe 
Published: March 2005

The carbohydrate chains of the respiratory‐mucus glycoproteins of a patient suffering from bronchiectasis due to Kartagener's syndrome were released by alkaline borohydride treatment. Low‐molecular‐mass, monosialyl oligosaccharide‐alditols were isolated by anion‐exchange chromatography and fractionated by consecutive straight‐phase high‐performance liquid chromatography (HPLC) on a silica‐based alkylamine column, and reverse‐phase HPLC on a silica‐based octadecyl column, respectively. The structures of the oligosaccharide‐alditols were determined by 500‐MHz 1H‐NMR spectroscopy in combination with sugar composition analysis. The 24 structures established range in size from disaccharides to heptasaccharides. Novel oligosaccharides obtained from the bronchiectasis mucus glycoproteins are:

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23 of the 24 monosialyl oligosaccharides characterized can be conceived of as extensions of neutral oligosaccharides purified from the bronchial mucus of this patient [Klein, A. et al. (1988) Eur. J. Biochem. 171, 631‐642; Breg, J. et al. (1988) Eur. J Biochem. 171, 643‐654]. The sialic acid residue was found to occur either in α(2→3)‐ or α(2→6)‐linkage to a galactosyl residue or in a(2→6)‐linkage to GalNAc‐0l.
Ribosomal protein interactions in yeast
Authors: SÁENZ‐ROBLES M. Teresa , VILELLA M. Dolores , PUCCIARELLI Graciela , POLO Florentino , REMACHA Miguel , ORTÍZ Blanca L. , VIDALES Fernando J. , BALLESTA Juan P. G. 
Published: March 2005

Protein L15 from Saccharomyces cerevisiae ribosomes has been shown to interact in solution with acidic ribosomal proteins L44, L44′ and L45 by different methods. Thus, the presence of the acidic proteins changes the elution characteristics of protein L15 from CM‐cellulose and DEAE‐cellulose columns and from reverse‐phase HPLC columns. Moreover, immunoprecipitation using anti‐L15 specific monoclonal antibodies coprecipitates the acidic proteins, too. Conversely, antibodies raised against the acidic proteins immunoprecipitate protein L15. This coprecipitation seems to be specific since it does not involve other ribosomal proteins present in the sample. Similarly, plastic‐adsorbed antibodies specific for one of the components in the L15–acidic‐protein complex are able to retain the other component of the complex but cannot bind unrelated proteins. Moreover, protein L15 can be chemically cross‐linked to the acidic proteins in solution.These results indicate that protein L15 might be equivalent to bacterial ribosomal protein L10 in forming a complex with the acidic proteins. Since, on the other hand, protein L15 has been shown to be immunologically related to bacterial protein L11 [Juan Vidales et al. (1983) Eur. J. Biochem. 136, 276–281] and to interact with the same region of the large ribosomal RNA as does protein L11 [El‐Baradi et al. (1987) J. Mol. Biol. 195, 909–917], these results suggest strongly that protein L15 plays the same role in the yeast ribosome as proteins L10 and L11 do in the bacterial particles.
Expression of cytochrome P‐450 isozymes in the liver of hypophysectomized rats
Authors: LEMOINE Antoinette , MARIE Sandrine , CRESTEIL Thierry 
Published: March 2005

Monooxygenase activities have been examined in rat liver to determine the effects of castration and hypophysectomy on cytochrome P‐450 species. In adult males, hypophysectomy caused a decrease of total P‐450 concentration, aniline hydroxylase, benzopyrene hydroxylase, benzphetamine demethylase, testosterone hydroxylase and imipramine hydroxylase and demethylase activities. The treatment of hypophysectomized animals with human growth hormone or testosterone did not restore the full activity.
When probed with antibodies, microsomes from hypophysectomized males and females exhibited an intense reaction with a polyclonal anti‐(phenobarbital‐induced P‐450) which was not observed with a monoclonal antibody of anti‐(phenobarbital‐induced P‐450).
These microsomal preparations also reacted with an antibody raised against a developmentally regulated P‐450. No sex difference could be detected with this antibody. Furthermore, administration of human growth hormone to hypophysectomized males prevented this immunoreaction.
Total RNA has been prepared from the same liver; when probed with cDNAs, no changes occurred in the content in P‐450 b/e, PB 24 (a constitutive member of the phenobarbital subfamily) and phenobarbital‐inducible mRNA for UDP‐glucuronosyltransferase.
In contrast, P‐450 mRNA induced by pregnenolone 16α‐carbonitrile was modulated by hormonal manipulations: lower in females and castrated males than in intact males, increased in both sexes after hypophysectomy. Treatment of hypophysectomized males with human growth hormone abolished this rise in pregnenolone‐16δ‐carbonitrile‐induced P‐450 mRNA accumulation.

Data collected in this study support the assumption that hypophysectomy acts differently on the regulation of various P‐450 isozymes and that this regulation clearly does not involve the phenobarbital subfamily of P‐450s.
Alteration of the genes for glycophorin A and B in glycophorin‐A‐deficient individuals
Authors: RAHUEL Cécile , LONDON Jacqueline , VIGNAL Alain , CHERIF‐ZAHAR Baya , COLIN Yves , SIEBERT Paul , FUKUDA Minoru , CARTRON Jean‐Pierre 
Published: March 2005

Glycophorins A and B are homologous glycoproteins of the red cell membrane which carry the blood‐group MN and Ss antigens, respectively, and are encoded by two distinct genes closely linked on chromosome 4, which are probably derived from each other by duplication during evolution.The lack of glycophorin A is associated with the rare phenotype En(a–), indicating individuals who are defective for MN antigens, as well as for the Ena antigens, also located on this glycoprotein. The En(a–) condition is heterogenous and includes two categories of variants exemplified by the Finnish and the English types referred to as En(Fin) and En(UK), respectively. By Southern blot and preliminary genomic clone analyzes we have compared the status of the genes for glycophorins A and B, as well as that of the gene encoding glycophorin C, another unrelated red cell membrane glycoprotein, in the En(a –) variants and in the En(a +) control donors. Our data indicate that the En(Fin) variant is homozygous for a complete deletion of the glycophorin A gene without any detectable abnormality of the genes encoding glycophorins B or C. In the genome of the En(UK) variant, with the presumed genotype Mk/En(UK), and where the Mk condition abolishes the expression of MN and Ss antigens, we have identified several abnormalities of the glycophorin A and B genes, but the glycophorin C gene was unaffected. Our results strongly support the view that in Mk chromosome the glycophorin A and B genes are largely deleted, whereas the En(UK) chromosome probably contains a gene fusion product encoding a hybrid glycoprotein AM‐B, composed of the N‐terminal portion of a blood group M‐type glycophorin A and of the C‐terminal portion of glycophorin B. The determination of the 5′ and 3′ limits of the hybrid gene and elucidation of the mechanism involved will require sequencing of the rearranged DNA of the variant and a full knowledge of the organization of the glycophorin A and B genes.
Mycolic acids of Mycobacterium aurum
Authors: LANÉELLE Marie‐Antoinette , LACAVE Charlotte , DAFFÉ Mamadou , LANÉELLE Gilbert 
Published: March 2005

Mycobacterium aurum (type strain) was analyzed for its mycolate content. Three types of mycolates were identified: di‐unsaturated, oxo and dicarboxy mycolates, each type being constituted by two subtypes. The acid released by pyrolysis was identified as docosanoic acid. By use of mass spectrometry and oxidation techniques, the structures of these six subtypes of mycolates were elucidated. They contain Z and E double bonds, the latter having an adjacent methyl branch. No cyclopropane ring was observed. All the methyl branches occurring in these mycolates derived from methionine, the methyl‐branched chiral center adjacent to the double bond having an R configuration.The structures of the major di‐unsaturated mycolates suggest that they cannot be the precursors of oxo mycolates.The amount of long‐chain secondary alcohol (2‐octadecanol) obtained from the whole cells was found to be much greater than that expected from hydrolysis of wax ester mycolate (ester of 2‐octadecanol and dicarboxymycolic acid). Further investigations showed that 2‐octadecanol was also present in triacylglycerols, esterifying the ω‐carboxyl group of long‐chain fatty acids structurally related to dicarboxy‐mycolates.
Characterization of proteolytic fragments of the laminin‐nidogen complex and their activity in ligand‐binding assays
Authors: MANN Karlheinz , DEUTZMANN Rainer , TIMPL Rupert 
Published: March 2005

Some 12 new nidogen and laminin fragments were purified from elastase, thrombin and trypsin digests and characterized by their sizes (22 kDa to > 300 kDa), subunit patterns on electrophoresis, partial amino acid sequences, content of specific epitopes and their binding to laminin or nidogen structures in radioligand assays. This permitted the various fragments to be ordered along the dumbbell‐shaped structure of nidogen and to compare them with previously described nidogen fragments arising by endogenous proteolysis. Two nidogen fragments (E‐50, E‐90; 50 kDa and 90 kDa) remain associated with a large laminin fragment in elastase digests of the complex and could be dissociated with 2 M guanidine · HCl. Recombination studies demonstrated Kd= 10–20 nM for this interaction. Nidogen fragments devoid of binding activity included the tryptic peptide T‐40 (40 kDa) corresponding to the rod‐like domain and several larger fragments extending more to the N‐terminus of nidogen. An N‐terminal thrombin fragment of about 50 kDa was also inactive. Together the data show a lack of laminin binding to the N‐terminal globule and rod of nidogen and provide indirect evidence that this activity is located within or close to its C‐terminal globular domain.Nidogen‐binding structures of laminin were obtained as two large fragments (> 300 kDa), P1X and E1X. They correspond to the short arm structure of laminin with one (E1X) or two (P1X) arms decreased in size to the inner rod‐like segment. Shortening in E1X is mainly due to the B1 chain segment including the central globular domain which was identified as a new laminin fragment E10. Binding of E1X and P1X to nidogen was comparable to that of laminin while much lower activity was found for other laminin fragments. A 10‐fold lower binding potential was also observed for the laminin‐nidogen complex whose structure can now be defined in more precise molecular terms.
Inhibition of uridine phosphorylase by pyrimidine nucleoside analogs and consideration of substrate binding to the enzyme based on solution conformation as seen by NMR spectroscopy
Authors: VERES Zsuzsa , NESZMÉLYI András , SZABOLCS Anna , DÉNES Géza 
Published: March 2005

Some 3′‐ and/or 5′‐substituted pyrimidine nucleosides, as well as anhydropyrimidine nucleosides, which have no flexibility about the N‐glycosidic bond were studied as inhibitors of thymidine phosphorylase and uridine phosphorylase. The conformation of some analogs was also investigated in order to obtain information on substrate binding to the enzyme.The above compounds, including the potential anti‐(human immunodeficiency virus) agent, 3′‐azido‐2′,3′‐dideoxy‐5‐methyluridine were not substrates for either thymidine phosphorylase or uridine phosphorylase. (The only exception was arabinofuranosyl‐5‐ethyluracil, which proved to be a poor substrate for uridine phosphorylase).The phosphorolysis of thymidine by thymidine phosphorylase was slightly or not at all altered by these pyrimidine nucloside analogs. The lowest Ki was obtained in the case of 3′‐azido‐2′,3′‐dideoxy‐5‐methyluridine and the highest in the case of 2′‐deoxylyxofuranosyl‐5‐ethyluracil, when studying the analogs with flexible structure as inhibitors of uridine phosphorylase. The Ki for 2,3′‐ and 2,5′‐anhydro‐2′‐deoxy‐5‐ethyluridine was 5–6 orders of magnitude higher than that for 2,2′‐anhydro‐5‐ethyluridine. Competitive inhibition was observed in all cases.For these three molecules computer‐aided molecular modelling predicts the following glycosidic torsion angles X(O4,‐C1,‐N1‐C2): 109° for 2,2′‐anhydro‐5‐ethyluridine, and 78° and 71° for 2,3′‐ and 2,5′‐anhydro‐2′‐deoxy‐5‐ethyluridine respectively. These values are corroborated by high‐resolution 13C‐ and 1H‐NMR studies.2′‐Deoxy‐5‐ethyluridine is predicted to have a syn conformation with X= 46° and ΔE about 2.5 kJ/mol over the minimum energy (in anti position, X=−147°). 1H and 13C data including homonuclear Overhauser enhancements complete the information about the solution conformation.Considering the Ki values obtained, it is likely that substrates of uridine phosphorylase will bind to the enzyme in the same conformation as 2,2′‐anhydro‐5‐ethyluridine. The >30° deviation from the N‐glycosidic torsion angle of 2,2′‐anhydro‐5‐ethyluridine results in much higher Ki values.
Accumulation of carbamoylphosphate‐synthetase and phosphoenolpyruvate‐carboxykinase mRNA in embryonic rat hepatocytes
Authors: van ROON Marian A. , ZONNEVELD Duco , CHARLES Robert , LAMERS Wouter H. 
Published: March 2005

The aim of this study was to establish whether the initial accumulation of hepatocyte‐specific proteins after hormone induction is regulated at the pretranslational and/or the translational level. To this end, mRNA molar concentrations were determined and compared with rates of protein synthesis from previous studies [van Roon, M. A., Charles, R. & Lamers, W. H. (1987Eur. J. Biochem. 165, 229–234]. In vivo, carbamoylphosphate‐synthetase mRNA starts to accumulate at day 17 of pregnancy. Phosphoenol‐pyruvate‐carboxykinase mRNA starts to accumulate only just prior to birth. Embryonic day 14 (i.e. 8 days before the expected day of birth), livers were chosen to study the regulation of the initiation of hepatocyte‐specific mRNA accumulation in vitro. Accumulation of carbamoylphosphate‐synthetase and phosphoenolpyruvate‐carboxykinase mRNA is regulated by the same hormones as accumulation of the respective proteins. The rate at which carbamoylphosphate‐synthetase and phosphoenolpyruvate‐carboxykinase mRNA molecules accumulate in cultured embryonic, hepatocytes is relatively low, compared to that of postnatal hepatocytes. However, the increase of the rate of synthesis of carbamoylphosphate‐synthetase and phosphoenolpyruvate‐carboxykinase protein is even 3‐6‐fold slower than that of mRNA. This shows that initially mRNAs accumulate intracellularly to a relatively high concentration without being efficiently translated or translatable. Only after the mRNA concentration reaches a plateau at 72 h and 48 h respectively, the cellular capacity to synthesize the respective proteins increases. Therefore, the translational efficiency is certainly one of the major rate‐limiting factors of the initial phases of expression of the hepatocyte‐specific genes for carbamoylphosphate synthetase and phosphoenolpyruvate carboxykinase.