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Biomedical subjects

W Möller

Publications and source records attributed to W Möller.

At least 73 records · Page 4Linked to original sources
PubMed1989

Individuals with only one allele for a functional insulin receptor have a tendency to hyperinsulinaemia but not to hyperglycaemia.

Recently, we described a leprechaun patient with a genetically transmitted severe insulin resistance due to the absence of functional insulin receptors as inferred from the loss of insulin binding to the patients' fibroblasts and the impaired autophosphorylation of the beta-chain of the receptor. This patient was homozygous for the genetic defect which was recently found to be a leucine to proline mutation at position 233 in the alpha-chain of the insulin receptor. In the present study we have examined insulin receptor functions in relatives of this patient. Some of these individuals are heterozygous for the genetic defect and have only one allele coding for a functional insulin receptor. Insulin binding to cultured fibroblasts from the heterozygous individuals is only 20-40% of control values indicating a Mendelian mode of inheritance of the binding defect. In contrast, insulin stimulated autophosphorylation of the beta-chain of the insulin receptor shows normal values, indicating compensation mechanisms operating on this process. The stimulation of the basal level of 2-deoxyglucose uptake by insulin in fibroblasts from the homozygous patient is 1.2 fold whereas the heterozygous and control individuals show stimulation values of approximately 1.65 fold. Basal levels of 2-deoxyglucose uptake are similar in these fibroblasts. Oral glucose tolerance tests on the heterozygous individuals indicate an increased requirement for insulin of the target tissues as concluded from the tendency towards hyperinsulinaemia with no observed hyperglycaemia.(ABSTRACT TRUNCATED AT 250 WORDS)

Adult↗
PubMed1989

[Huneke test in the stomatologic practice].

It is a report about the "Huneke-test" based on a 30 year old experience of researching disorders in the jaw region. 334 teeth of 134 patients were tested by giving round-injections with local anaesthetic. 91 patients had a positive test result and 71 ill patients became free from trouble in different parts of the body with these injections or extractions. First of all dead teeth with an unsuspicious X-ray diagnosis were found out as causes of the disorders by means of the "Huneke-test". Examples of certain cases underline the recommendation of this test to dental practices.

Aged↗
PubMed1988

Phosphorylation of elongation factor 1 beta by an endogenous kinase affects its catalytic nucleotide exchange activity.

Elongation factor 1 beta (EF-1 beta) from Artemia is phosphorylated to a high percentage at serine 89 by an endogenous kinase present in EF-1 beta gamma. Protein sequencing of EF-1 beta revealed that this serine residue is located N-terminally of an acidic cluster of amino acids, (formula; see text) which is critical for casein kinase II-type substrate recognition. A number of compounds known to influence casein kinases were studied, revealing that the kinase activity as present in EF-1 beta gamma belongs to the class of casein kinase II. The rate of nucleotide exchange on EF-1 alpha as catalyzed by EF-1 beta was found to be affected reversibly by the state of phosphorylation of EF-1 beta. In the presence of dephosphorylated EF-1 beta, the exchange rate is almost twice as large compared to the rate in the presence of phosphorylated EF-1 beta. Rephosphorylation of dephosphorylated EF-1 beta diminishes the activity of the protein again. The role of casein kinase II-type enzymes in modulating the function of proteins involved in polypeptide synthesis is discussed.

Amino Acid Sequence↗
PubMed1988

Improper expression of insulin receptors on fibroblasts from a leprechaun patient.

Leprechaunism is an inherited human disorder characterized by severe insulin resistance. We have examined the properties of the insulin receptor in fibroblasts from a leprechaun patient. In vitro, severe insulin resistance is reflected by a low level of insulin binding to the patients fibroblasts and impaired insulin-mediated uptake of 2-deoxyglucose. Quantification of the receptor in detergent-solubilized total glycoprotein indicates a normal receptor number, in agreement with the observed normal level of insulin receptor mRNA on northern blots. The insulin-stimulated autophosphorylation of the patient's receptor shows a normal profile. The insulin receptor is present on the plasma membrane as indicated by cell-surface iodination experiments. No abnormalities in the molecular masses of the receptor's alpha and beta chains were observed. The results indicate that an apparently normal receptor is synthesized in sufficient amounts but functional expression of the receptor on the plasma membrane is impaired.

Cell Membrane↗
PubMed1988

Kinetic studies on the role of elongation factors 1 beta and 1 gamma in protein synthesis.

An equilibrium isotope exchange technique was used to measure in an Artemia system the catalytic influence of elongation factor (EF) 1 beta gamma on the dissociation of GDP from the complex of elongation factor 1 alpha.[3H] GDP in the presence of an excess of free GDP. The kinetic data demonstrate that, in analogy to procaryotes, dissociation of GDP occurs via the formation of a transient ternary complex of EF-1 alpha.GDP.EF-1 beta gamma. The rate constants for the dissociation of GDP from EF-1 alpha.GDP and from the ternary complex EF-1 alpha.GDP.EF-1 beta gamma were found to be 0.7 x 10(-3) and greater than or equal to 0.7 s-1, respectively. The equilibrium association constants of GDP to EF-1 alpha.EF-1 beta gamma and of EF-1 beta gamma to EF-1 alpha.GDP were found to be 2.3 x 10(5) and 4.2 x 10(5) M-1, respectively. Judged from the known elongation rate in vivo and kinetic constants of nucleotide exchange, it was estimated that the recycling of EF-1 alpha may be a rate-controlling step in eucaryotic translation. As a model for GTP exchange, the formation of the ternary EF-1 alpha.guanylyl (beta gamma-methylene)diphosphonate.EF-1 beta gamma complex was also studied. It was observed that both an increase of the level of aminoacyl-tRNA and of temperature favored the dissociation of this complex, thereby enabling EF-1 beta gamma to recycle as a catalyst. This behavior would explain the frequent occurrence of a heavy form of elongation factor 1 in extracts of the eucaryotic cell.

Algorithms↗
PubMed1988

Elongation factor 1 beta gamma from Artemia. Purification and properties of its subunits.

The guanine nucleotide exchange factor, elongation factor 1 beta gamma (EF-1 beta gamma) has been purified from Artemia cysts using an improved method. The protein consists of two distinct polypeptides with relative molecular masses of 26,000 (EF-1 beta) and 46,000 (EF-1 gamma). A nucleoside diphosphate phosphotransferase activity often found in EF-1 beta gamma preparations has been completely separated from the actual guanine nucleotide exchange stimulatory activity of EF-1 beta gamma, thus indicating that nucleotide diphosphate phosphotransferase is not an intrinsic property of EF-1 beta. Both EF-1 beta gamma and EF-1 beta have been shown to stimulate the following three reactions to a comparable degree: (a) exchange of GDP bound to EF-1 alpha with exogenous GDP; (b) EF-1 alpha-dependent binding of Phe-tRNA to ribosomes; (c) poly(U)-dependent poly(phenylalanine) synthesis. However, a significantly higher nucleotide exchange rate was observed in the presence of EF-1 beta gamma compared to EF-1 beta alone. Concerning elongation factor 1 gamma (EF-1 gamma) the following observations were made. In contrast to EF-1 beta, pure EF-1 gamma is rather insoluble in aqueous buffers, but the tendency to precipitate can be partially suppressed by the addition of detergents. In particular, EF-1 gamma partitions solely into the detergent phase of Triton X-114 solutions. EF-1 gamma is also more susceptible to spontaneous, specific fragmentation. It is remarkably that about 5% of the cellular pool of EF-1 beta gamma was found to be present in membrane fractions, under conditions where no EF-1 alpha was detectable in these fractions. Furthermore it was noted that EF-1 beta gamma copurified strongly with tubulin on DEAE-cellulose. Moreover, it was observed that from a mixture of EF-1 beta gamma and tubulin, EF-1 gamma coprecipitates with tubulin using a non-denaturating immunoprecipitation technique. These findings suggest that EF-1 gamma has a hydrophobic domain and interacts with membrane and cytoskeleton structures in the cell.

Animals↗
PubMed1988

Fibroblasts from a leprechaun patient have defects in insulin binding and insulin receptor autophosphorylation.

Leprechaunism is an inherited human disorder associated with an extreme resistance of the target cells towards the action of insulin. We have examined the properties of the insulin receptor in fibroblasts from a leprechaun patient (Geldermalsen, the Netherlands). In vitro, severe insulin resistance is reflected by a low level of insulin stimulated uptake of 2-deoxyglucose by these fibroblasts. This defect seems to be caused by a combination of two factors: a low level of insulin binding to intact cells and a strong decrease of insulin stimulated autophosphorylation of the receptor. The stimulation of autophosphorylation by insulin was approximately six-fold in control subjects and less than two-fold in the patient. No abnormalities were observed in the total number of insulin receptors in these cells and the molecular weights of the receptor subunits. In addition, the insulin concentration required for half maximal autophosphorylation is similar for the solubilised receptor from control and patient fibroblasts.

Cells, Cultured↗
PubMed1987

Primary structure of elongation factor 1 gamma from Artemia.

Complementary DNA corresponding to elongation factor 1 gamma, which forms a complex with EF-1 beta, has been cloned. A lambda gt11 cDNA library has been screened with an antiserum against EF-1 beta gamma. The derived amino acid sequence of EF-1 gamma corresponds to 429 amino acids excluding the initiator methionine, which is absent in the mature protein. About half of the protein was sequenced by direct protein sequence analysis. No clear homology with any other protein was found.

Amino Acid Sequence↗
PubMed1987

The effect of insulin, serum and dexamethasone on mRNA levels for the insulin receptor in the human lymphoblastoic cell line IM-9.

The effect of insulin, serum and dexamethasone on mRNA levels in the insulin receptor in the human lymphoblastoic cell line IM-9 was examined. To this end, mRNA levels were quantitated by Northern blot analysis using a labeled cDNA probe for the insulin receptor. The presence of 0.1 microM dexamethasone in the medium had a strong stimulatory effect on mRNA levels in insulin receptor, suggesting the presence of a glucocorticoid inducible enhancer element near the insulin receptor gene. Also, the nature of the serum had an effect on insulin receptor mRNA levels, as cells maintained in 10% fetal calf serum had insulin receptor mRNA levels that were 40-50% of those found in IM-9 cells maintained in 1% newborn serum. Variations in insulin receptor mRNA levels led in each situation to concordant variations in insulin binding. Insulin levels of up to 1 microM had no effect on hybridizable insulin receptor mRNA levels making an insulin-induced feed-back mechanism on gene expression or mRNA stability unlikely.

Blood↗
PubMed1987

A conserved amino acid sequence around Arg-68 of Artemia elongation factor 1 alpha is involved in the binding of guanine nucleotides and aminoacyl transfer RNAs.

The rate of trypsin cleavage of elongation factor 1 alpha having bound GDP is low and increases on exchange of GDP for GTP. The cleavage occurs at a unique position of the protein chain, namely at arginine-68 of Artemia EF-1 alpha. This increase in trypsin sensitivity is enhanced further in the presence of charged or uncharged transfer RNA. The local unfolding of EF-alpha at residue 68 is discussed in terms of a model in which GTP hydrolysis controls the positioning of a short 3'-terminal section of transfer RNA near the centre of peptide bond synthesis.

Adenine Nucleotides↗
PubMed1986

Localization of virginiamycin S binding site on bacterial ribosome by fluorescence energy transfer.

Virginiamycin S, a type B synergimycin inhibiting protein synthesis in bacteria, competes with erythromycin for binding to the 50S ribosomal subunits; the mechanism of action of the two antibiotics is unclear. Energy-transfer experiments between virginiamycin S (which is endowed with inherent fluorescence due to its hydroxypicolinyl moiety) and fluorescent coumarinyl derivatives of ribosomal proteins L7 and L10 have been carried out to locate the binding site of this antibiotic on the ribosome. Previous studies have indicated that two L7/L12 dimers can attach respectively to a strong binding site located on the central protuberance and to a weak binding site located on the stalk of the 50S subunits and that protein L10 is located at the base of the stalk. The distance between ribosome-bound virginiamycin S and a fluorophore located at the strong binding site of proteins L7/L12 (Lys-51 of L7) was found to be 56 (+/- 15) A. Virginiamycin S, on the other hand, was located at a distance exceeding 67 A from the weak binding site of L7/L12 dimers. A fluorophore positioned on the unique cysteine (Cys-70) of protein L10 and ribosome-bound virginiamycin S proved to be more than 60 A apart. From data available on the location of proteins L7/L12 and L10, a model is proposed, whereby the virginiamycin S binding site is placed at the base of the central protuberance of the 50S subunits, in proximity of the presumptive peptidyl transferase center. The binding sites of macrolides and lincosamides (related antibiotics of the MLS group) are expected to be very close to that of virginiamycin S.

Binding Sites↗
PubMed1986

Genes coding for the elongation factor EF-1 alpha in Artemia.

The elongation factor EF-1 alpha is one of the most abundant proteins in eukaryotic cells, where it catalyzes the binding of aminoacyl-tRNA to ribosomes. The genes coding for this protein in the brine shrimp Artemia were analyzed by gene cloning, electron microscopy and chromosomal blot hybridization. There are only a few (about four) copies of one type of gene per haploid genome. These genes contain five exons divided over 10(4) base pairs. Local rearrangements give rise to a number of gene variants. Cross-hybridizations of Artemia cDNA probes with yeast and Drosophila DNA revealed two different yeast EF-1 alpha genes and one or two different Drosophila genes, respectively. Nucleotide sequencing revealed signals for synthesis and processing of EF-1 alpha transcripts as well as the exact location of exons. One interruption in the coding sequence corresponds closely to a splice junction in the gene coding for the homologous chloroplast protein EF-Tu from Euglena gracilis, presumably of prokaryotic origin. The first exon in the chloroplast gene codes for the region of EF-Tu that is homologous to regions of the elongation factor EF-G and of the initiation factor IF2, respectively.

Animals↗
PubMed1986

The primary structure of the alpha subunit of human elongation factor 1. Structural aspects of guanine-nucleotide-binding sites.

The primary structure of the alpha subunit of elongation factor 1 (EF-1 alpha) from human MOLT 4 cells was determined by cDNA sequencing. The data show that the conservation of the amino acid sequence is more than 80% when compared with yeast and Artemia EF-1 alpha. An inventory of amino acid sequences around the guanine-nucleotide-binding site in elongation factor Tu from Escherichia coli and homologous amino acid sequences in G proteins, initiation and elongation factors and proteins from the RAS family shows two regions containing conserved sequence elements. Region I has the sequence apolar-Xaa-Xaa-Xaa-Gly-Xaa-Xaa-Yaa-Xaa-Gly-LYs-Thr(Ser)- -Xaa-Xaa-Xaa-Xaa-X-apolar. Except for RAS proteins, Yaa is always an acidic amino acid residue. Region II is characterized by the invariant sequence apolar-apolar-Xaa-Xaa-Asn-Lys-Xaa-Asp. In order to facilitate sequence comparison we have used a graphic display, which is based on the hydrophilicity values of individual amino acids in a sequence.

Amino Acid Sequence↗
PubMed1985

Phosphate-binding sequences in nucleotide-binding proteins.

In the three-dimensional model of adenylate kinase, the phosphate-binding site for AMP and ATP has been identified [Pai, E.F. et al. (1977) J. Mol. Biol. 114, 37--45]. In this region one can distinguish a sequence glycine XXXX glycinelysine. The same sequence is found in many other mononucleotide-binding proteins including elongation factors and oncogenic P21 proteins. Dinucleotide-binding proteins display a pyrophosphate-binding unit with a glycine pattern different from that of mononucleotide-binding proteins. It has been found that P21 ras protein possesses a strand motif typical for (pyro)phosphate binding of a mononucleotide. A single mutation at position 12 can confer oncogenic activity on the protein. Based on the assumption that amino acid residues which are critical for function are preferentially conserved, we predict from the sequence that glycine residue 15 rather than residue 12 is important for (pyro)phosphate binding.

Adenine Nucleotides↗
PubMed1985

Molecular cloning and analysis of cDNA sequences for two ribosomal proteins from Artemia. The coordinate expression of genes for ribosomal proteins and elongation factor 1 during embryogenesis of Artemia.

The large subunit of eukaryotic ribosomes contains acidic phosphoproteins which are related to L7/L12 from Escherichia coli. In the brine shrimp Artemia these proteins are designated eL12 and eL12'. We have isolated cDNA clones for these proteins from a cDNA bank that was constructed by the use of size-fractionated poly(A)-rich RNA (8-10S fraction) from Artemia and a synthetic oligonucleotide as primer. Clones containing DNA sequences coding for eL12 and eL12 were characterized by hybrid-selected translation and DNA sequencing. The proteins eL12 and eL12' share an identical peptide of 22 amino acids at their carboxy termini whereas the remaining part of the protein shows little sequence homology. The nucleotide sequences show a different codon use for the amino acids in the common carboxy terminus, thereby excluding a common exon coding for this part of both proteins. Despite the differences in amino acid sequence in the major part of eL12 and eL12' the proteins have a considerable degree of homology on the basis of the distribution of hydrophobic and hydrophilic amino acids over the polypeptide chains, in agreement with a related folding and function of both proteins. Relative levels of mRNA coding for eL12, eL12' and elongation factor 1 alpha were determined during the development of Artemia from a dormant cyst to a nauplius. The data show a coordinate expression of the genes for EF-1 alpha and both ribosomal proteins, excluding a differential expression of the genes for these related ribosomal proteins during embryogenesis. Analysis of the gene copy number for eL12 and eL12' indicates the presence of a few genes for each protein.

Animals↗
PubMed1984

Localization of Lys-51 of L7/L12 on 50S ribosomes from Escherichia coli.

Ribosomal protein L7/L12 from Escherichia coli was modified specifically at Lys-51 with 4-(6-formyl-3-azido-phenoxy)butyrimidate. Reconstitution of ribosomal cores, lacking L7/L12, with imidate-modified L7/L12 resulted in back formation of 50S particles which were fully active in elongation-factor-dependent processes. By use of the formylazidophenoxy moiety as hapten, the position of Lys-51 of L7/L12 on the 50S ribosome was determined by immune electron microscopy. The results show that an L7/L12 dimer is present in the L7/L12 stalk in such a way that Lys-51 is located at the far cytoplasmic end of the stalk. The experimental data are discussed in relation to a proposed model for the L7/L12 dimer.

Bacterial Proteins↗