bluuebunny/crossref_metadata_2025_split huggingface dataset:
Organization and complete nucleotide sequence of the core‐histone‐gene cluster of the annelid Platynereis dumerilii
Authors: SELLOS Daniel , KRAWETZ Stephen A. , DIXON Gordon H. 
Published: March 2005

The arrangement of the core‐histone genes, their transcriptional polarity and their nucleotide sequences have been determined for the polychaete annelid Platynereis dumerili.A clone containing the core‐histone genes was isolated from a annelid genomic library constructed in the EMBL‐4 phage vector, using a trout H3 genomic probe. This clone was found to contain two and a half repeats of a 6‐kbp EcoRV fragment that contained one copy of each of the core‐histone genes.The clusters are tandemly arrayed in the genome and the gene order within the core‐histone cluster does not vary. Absolutely no differences were found in the nucleotide sequences comprising the same part of two adjacent clusters (bases −225 to 2776 and bases 5821 to 8825).The number of copies of the cluster appeared to be high: approximately 660 copies/diploid cell, as also observed in sea urchins and amphibians. There are also some additional subtypes of histone gene organization: multimers of tandemly arrayed genes and isolated genes; these are present at a much lower copy number (an average of 40–50 copies/diploid genome). Two mRNAs (for H2B and H3) are transcribed from one DNA strand and the two other histone mRNAs (for H2A and H4) from the other strand as is the case for some insects and certain vertebrates.No H1‐coding sequence has been found in the completely sequenced four‐membered cluster. The organization of histone genes in P. dumerilii is similar to the clustering found in Caenorhabditis elegans but in this nematode worm several different types of organization are observed with a low copy number for each.
GAP1, the general amino acid permease gene of Saccharomyces cerevisiae
Authors: JAUNIAUX Jean‐Claude , GRENSON Marcelle 
Published: March 2005

In Saccharomyces cerevisiae, mutations at the GAP1 locus selectively abolish the activity of the general amino acid transport system. This permease catalyses active transport of apparently all biological amino acids across the plasma membrane. We have determined the nucleotide sequence of the GAP1 gene. The sequence contains an open reading frame of 601 codons corresponding to a polypeptide of Mr 65578. This polypeptide is strongly hydrophobic; it exhibits three potential glycosylation sites. Hydropathy analysis suggests 12 membrane‐spanning regions. The N‐terminal domain is charged, it does not resemble hydrophobic signal sequences found in secreted proteins. Hence the GAP1 gene encodes a protein with characteristics typical of integral membrane proteins translocating ligants across cellular membranes. The deduced amino acid sequence of GAP1 protein presents strong similarities to those of the yeast arginine, histidine and proline permeases, suggesting a common evolutionary origin for these amino acid permeases.Nitrogen‐source regulation of the GAP1 permease is believed to occur at two distinct levels, i.e. permease synthesis and permease activity [Grenson (1983) Eur. J. Biochem. 133, 135–139]. Northern analysis of GAP1‐specific transcripts in wild‐type and in mutant strains is in agreement with these views and indicates that nitrogen catabolite repression of GAP1 synthesis occurs at the RNA level.
Monoclonal antibodies against different epitopes of peptide hormones
Authors: JURZAK Mirek , BOER Rainer , FRITZSCH Günter , KOJRO Elzbieta , FAHRENHOLZ Falk 
Published: March 2005

In order to produce monoclonal antibodies directed against different epitopes of the neurohypophyseal hormone vasopressin, the hormone was coupled to carrier proteins via photoreactive groups at different positions in the vasopressin sequence: [2‐(4‐azidophenylalanine), 8‐arginine]vasopressin (peptide P1, photoreactive group at position 2) and desamino‐[8‐N6‐(4‐azidophenylamidino)lysine]vasopressin (peptide P2, photoreactive group at position 8) were conjugated to thyroglobulin by flash photolysis. Monoclonal antibodies against these conjugates bound {[3H]8‐arginine}vasopressin with dissociation constants ranging over 40–400 nM. Epitope analysis by means of competitive ELISA showed that the monoclonal antibody obtained with peptide P1 as hapten was directed against the C‐terminal acyclic tripeptide when its conformation was stabilized by interaction with the disulphide‐linked cyclic hexapeptide. In contrast, the epitope analysis of three monoclonal anti‐(peptide P2) antibodies demonstrated that they recognized antigenic determinants in the cyclic hexapeptide ring, mainly the hydrophobic surface formed by Tyr2 and Phe3.Our results suggest that monoclonal antibodies against different epitopes in small peptide hormones can be generated selectively by using photoreactive peptides in such a way that different antigenic sites are exposed in the hapten‐carrier conjugate.
Fluorescence spectroscopic analysis of ligand binding to kringle 1 + 2 + 3 and kringle 1 fragments from human plasminogen
Authors: MATSUKA Yury V. , NOVOKHATNY Valery V. , KUDINOV Stanislav A. 
Published: March 2005

The ligand binding of kringle 1 + 2 + 3 and kringle 1 from human plasminogen has been investigated by fluorescence spectroscopy. Analysis of fluorescence titration of kringle 1 + 2 + 3 with 6‐aminohexanoic acid shows that this fragment, besides the high‐affinity lysine‐binding site with Kd= 2.9 μM, contains two additional lysine‐binding sites which differ in binding strength (Kd= 28 μM and Kd= 220 μM). This strongly suggests the existence of a lysine‐binding site in each of the first three kringles.6‐Aminohexanoic acid, pentylamine, pentanoic acid and arginine were used for investigation of the ligand specificity of isolated kringle 1 prepared by pepsin hydrolysis of kringle 1 + 2 + 3. It has been established that kringle 1 has high affinity to 6‐aminohexanoic acid, pentylamine and arginine (Kd values are 3.2 μM, 4.8 μM and 4.3 μM, respectively). At the same time pentanoic acid did not bind with kringle 1. These facts indicate, firstly, a broad ligand specificity of kringle 1 and, secondly, the paramount importance of the positively charged group of the ligand for its interaction with lysine‐binding site of this kringle.
Phospholipid specificity of bovine heart bc1 complex
Authors: SCHÄGGER Hermann , HAGEN Thomas , ROTH Brigitte , BRANDT Ulrich , LINK Thomas Andreas , von JAGOW Gebhard 
Published: March 2005

Bovine heart bc1 complex was reversibly inactivated by a new simple and effective chromatographic delipidation method. Upon phospholipid replenishment, catalytic activity increased from values near zero to values 2–6‐times higher than those of the original preparation. Compared to original preparations maximally activated by additional phospholipid, the degree of reactivation was up to 100%. By this delipidation method, the 6.4‐kDa protein subunit was removed with the phospholipid. The loss of this protein neither diminished electron transport activity nor abolished proton translocation.Two requirements were necessary to obtain quantitative data: (a) only bc1 complexes, homogeneously dissolved before and after relipidation had to be used and (b) the phospholipid bound to the complex had to be determined. The correlation of catalytic activity to bound phospholipid was studied in the range of low phospholipid/protein ratios, which had previously been insufficiently resolved. Catalytic activity increased linearly with added phospholipid up to a molar ratio of 80–100 lipid molecules/dimeric complex. This corresponds to the number of phospholipid molecules that complete a single bilayer annulus.The activating effect of phospholipid is not merely due to a hydrophobic phase effect, since it strongly depends on the nature of the polar head group of the added phospholipid. Of the three major phospholipids bound to the bc1 complex, only phosphatidylethanolamine and phosphatidylcholine activated when added as sole phospholipid. Tightly bound diphosphatidylglycerol was needed for preservation of the native complex structure.
A critical evaluation of the hydropathy profile of membrane proteins
Authors: DEGLI ESPOSTI Mauro , CRIMI Massimo , VENTUROLI Giovanni 
Published: March 2005

New membrane‐preference scales are introduced for categories of membrane proteins with different functions. A statistical analysis is carried out with several scales to verify the relative accuracy in the prediction of the transmembrane segments of polytopic membrane proteins. The correlation between some of the scales most used and those calculated here provides criteria for selecting the most appropriate methods for a given type of protein. The parameters used in the evaluation of the hydropathy profiles have been carefully ascertained in order to develop a reliable methodology for hydropathy analysis. Finally, an integrated hydropathy analysis using different methods has been applied to several sequences of related proteins.The above analysis indicates that (a) microsomal cytochrome P450 contains only one hydrophobic region at the N‐terminus that is consistently predicted to transverse the membrane; (b) only four of the six or seven putative transmembrane helices of cytochrome oxidase subunit III are predicted and correspond to helices I, III, V and VI of the previous nomenclature; (c) the product of the mitochondrial ATPase‐6 gene (or the chloroplast ATPase‐IV gene) of F0‐F1‐ATPase shows that helix IV is not consistently predicted to traverse the membrane, suggesting a four‐helix model for this family of proteins.
Coupling of insulin‐responsive glucose transport to receptors for insulin‐like growth factor 1 in primary human fibroblasts
Authors: MAASSEN J. Antonie , van der ZON Gerard C. M. 
Published: March 2005

We have recently described an insulin‐resistant patient with leprechaunism (leprechaun G.) having a homozygous leucine → proline mutation at amino acid position 233 in the α‐chain of the insulin receptor. The mutation results in a loss of insulin binding to cultured fibroblasts. Fibroblasts from the patient and control individuals were used to quantify the stimulation of 2‐deoxyglucose uptake by insulin and insulin‐like growth factor 1 (IGF‐1). Insulin hardly stimulates basal 2‐deoxyglucose uptake in the patient's fibroblasts whereas in control fibroblasts the uptake of 2‐deoxyglucose is stimulated by insulin approximately 1.7 times. In contrast, IGF‐1 stimulates hexose uptake in the patient's fibroblasts 1.8 times, a similar value to that obtained by stimulation of control fibroblasts with insulin or IGF‐1. With both types of fibroblasts, maximal IGF‐1 response is reached at about 10 nM IGF‐1, the ED50 being approximately 4 nM.The results indicate that the insulin responsive glucose transport in primary fibroblasts is functionally linked to the receptor for IGF‐1. Insulin binds with an approximately 200‐fold lower affinity to IGF‐1 receptors, compared to homologous IGF‐1 binding. As an insulin concentration of 10 μM is unable to give maximal stimulation of glucose uptake in the patient's fibroblasts, which is already seen with 10 nM IGF‐1, it seems that occupation of IGF‐1 receptors by insulin on the patient's cells is less efficient at stimulating hexose uptake compared to homologous activation.
Thermodynamics of information transfer between subunits in oligomeric enzymes and kinetic cooperativity
Authors: GIUDICI‐ORTICONI Marie‐Thérèse , BUC Jean , BIDAUD Michel , RICARD Jacques 
Published: March 2005

The theory and the methods that have been described in the two preceding papers in this journal have been used to analyze the kinetic properties of chloroplast fructose bisphosphatase. The enzyme is a tetramer made up of apparently identical subunits and displays a sigmoidal kinetics with respect to its substrate, fructose bis‐phosphate. The free ionic species, magnesium and fructose bisphosphate bind to the enzyme and the chelate fructosé‐bisphosphate‐magnesium does not affect the sigmoidicity of the rate curves.The Hill coefficient with respect to free fructose bisphosphate is equal to 2.3, which is indeed incompatible with the view that the enzyme behaves as a dimer of dimers. This conclusion is confirmed by direct analysis of the rate curve. On the basis of the sum of the residuals, their sum of squares, the standard error of the kinetic parameters of the equation, the kinetic scheme associated with a dimer of dimers may be ruled out. On the basis of the same criteria, the fit of an Adair equation to the rate data cannot be retained as satisfactory. This is a direct proof that neither the Monod nor the Koshland model can correctly fit these kinetic data. In fact the model that fits these data best is a structural kinetic scheme where information transfer occurs between each subunit and its three neighbors (‘tetrahedral’ mode of information transfer).The fit of these models to a large number of kinetic data allows one to compute the free energy profile during the successive binding processes of the four substrate molecules to the enzyme. Whereas the first two steps are associated with an increase of free energy, all the other subsequent steps are associated with a decrease of free energy.
Purification and fractionation of lipopolysaccharide from Gram‐negative bacteria by hydrophobic interaction chromatography
Authors: FISCHER Werner 
Published: March 2005

By hydrophobic interaction chromatography on octyl‐Sepharose, lipopolysaccharide (LPS) of Escherichia coli Re mutant and of wild‐type smooth‐form (S‐form) Salmonella typhimurium and Salmonella abortus equi is fractionated according to increasing amount of fatty acids. Thereby a fractionation of S‐form LPS according to the length of the O‐polysaccharide chain also occurs, because with increasing of fatty acids there is a decrease in the mean length of the O‐polysaccharide chain from approximately 30 to 4 repeating units. Molecular species of Re‐mutant LPS contain four 3‐hydroxytetradecanoyl residues in addition to which dodecanoic, tetradecanoic and possibly hexadecanoic acid, appear in this sequence. Among the molecular species of S‐form LPS, dodecanoic, tetradecanoic and hexadecanoic acids appear in the same order, but in contrast to Re‐mutant LPS a significant fraction of S‐form LPS contains less than four 3‐hydroxytetradecanoyl residues.Hydrophobic interaction chromatography also proved an effective one‐step purification procedure of LPS as was shown with a crude preparation from S‐form S. typhimurium.