Structural and functional homology between the 29 kD rat liver nucleoprotein and the high mobility group 1 protein

A 29 kD soluble rat liver nucleoprotein (p29) has increased binding affinity for the hormone responsive element (RE) of the rat haptoglobin (Hp) gene during the acute-phase reaction. In this work the possibility of its structural and functional homology to the high mobility group 1 (HMG1) nonhistone protein constituent of chromatin was examined. The results of two-dimensional gel electrophoresis, Southwestern and Western immunoblot analyses, showed that p29 and HMG1 are homologous protein species. On the basis of in vitro and in vivo phosphorylation/dephosphorylation experiments, we discuss the modulatory role of phosphate groups in view of the structure and function of p29.     

Analysis of human extrachromosomal DNA elements originating from different β-satellite subfamilies

By screening total human DNA with probes derived from the small polydisperse circular (spc) DNA fraction of cultured human cells, we identified three clones that carry long stretches of -satellite DNA. Further experiments have shown that the three sequences belong to at least two different -satellite subfamilies, which are characterized by different higher order subunits. Members of one of these subfamilies are located in the cytological satellites of all acrocentric chromosomes, whereas members of another are located on the short arms of the acrocentrics on both sides of the stalk regions and also in the centromeric regions of chromosomes 1 and 9. This is the first time that -satellite sequences obtained from the spcDNA of human cells have been assigned to -satellite subfamilies that are organized as long arrays of tandemly arranged higher order monomers. This indicates that -satellite sequences can be excised from their chromosomal loci via intrastrand-recombination processes.This paper is dedicated to Prof. Dr. Ulrich Wolf on his 60th birthday, January 1993     

A competition smFRET assay to study ligand-induced conformational changes of the dengue virus protease

Ligand binding to proteins often is accompanied by conformational transitions. Here, we describe a competition assay based on single molecule Förster resonance energy transfer (smFRET) to investigate the ligand-induced conformational changes of the dengue virus (DENV) NS2B-NS3 protease, which can adopt at least two different conformations. First, a competitive ligand was used to stabilize the closed conformation of the protease. Subsequent addition of the allosteric inhibitor reduced the fraction of the closed conformation and simultaneously increased the fraction of the open conformation, demonstrating that the allosteric inhibitor stabilizes the open conformation. In addition, the proportions of open and closed conformations at different concentrations of the allosteric inhibitor were used to determine its binding affinity to the protease. The K_D value observed is in accordance with the IC₅₀ determined in the fluorometric assay. Our novel approach appears to be a valuable tool to study conformational transitions of other proteases and enzymes.     

Yeast glycogenin (Glg2p) produced in Escherichia coli is simultaneously glucosylated at two vicinal tyrosine residues but results in a reduced bacterial glycogen accumulation.

Saccharomyces cerevisiae possesses two glycogenin isoforms (designated as Glg1p and Glg2p) that both contain a conserved tyrosine residue, Tyr232. However, Glg2p possesses an additional tyrosine residue, Tyr230 and therefore two potential autoglucosylation sites. Glucosylation of Glg2p was studied using both matrix-assisted laser desorption ionization and electrospray quadrupole time of flight mass spectrometry. Glg2p, carrying a C-terminal (His6) tag, was produced in Escherichia coli and purified. By tryptic digestion and reversed phase chromatography a peptide (residues 219-246 of the complete Glg2p sequence) was isolated that contained 4-25 glucosyl residues. Following incubation of Glg2p with UDPglucose, more than 36 glucosyl residues were covalently bound to this peptide. Using a combination of cyanogen bromide cleavage of the protein backbone, enzymatic hydrolysis of glycosidic bonds and reversed phase chromatography, mono- and diglucosylated peptides having the sequence PNYGYQSSPAM were generated. MS/MS spectra revealed that glucosyl residues were attached to both Tyr232 and Tyr230 within the same peptide. The formation of the highly glucosylated eukaryotic Glg2p did not favour the bacterial glycogen accumulation. Under various experimental conditions Glg2p-producing cells accumulated approximately 30% less glycogen than a control transformed with a Glg2p lacking plasmid. The size distribution of the glycogen and extractable activities of several glycogen-related enzymes were essentially unchanged. As revealed by high performance anion exchange chromatography, the intracellular maltooligosaccharide pattern of the bacterial cells expressing the functional eukaryotic transgene was significantly altered. Thus, the eukaryotic glycogenin appears to be incompatible with the bacterial initiation of glycogen biosynthesis.     

Circadian and circannual rhythms of hormonal variables in elderly men and women

A group of fourteen men (73 ± 5 yr of age), and eighteen women (77 ± 7 yr of age) institutionalized at the Berceni Clinical Hospital, Bucharest, Romania, were studied over a 24-hr span once during each season (winter, spring, summer and fall). All subjects followed a diurnal activity pattern with rest at night and ate three meals per day with breakfast at about 0830, lunch at about 1300 and dinner at about 1830. The meals were similar, although not identical for all subjects during all seasons. On each day of sampling blood was collected at 4-hr intervals over a 24-hr span. Seventeen hormonal variables were determined by radioimmunoassay. Statistically significant circadian rhythms were detected and quantitated by population mean cosinor analysis in pooled data from all four seasons in both sexes for ACTH, aldosterone, Cortisol, C-peptide, dehydroepiandrosterone-sulfate (DHEA-S), immunoreactive insulin, prolactin, 17-OH progesterone, testosterone, total T₄ and TSH. In women, estradiol and progesterone also were determined and showed a circadian rhythm during all seasons. Total T, and FSH showed circadian rhythm detection by cosinor analysis in the men only; LH showed no consistent circadian rhythm as group phenomenon in men or women.

A circannual rhythm was detected using the circadian means of each subject at each season as input for the population mean cosinor in the women for ACTH, C-peptide, DHEA-S, FSH, LH, progesterone, 17-OH progesterone and TSH. In the men, a circannual rhythm was detected for ACTH, FSH, insulin, LH, testosterone and T₃. There were phase differences between men and women in ACTH, FSH and LH. In those functions in which both the circadian and circannual rhythms were statistically significant, a comparison of the amplitudes showed in the women a higher circannual rather than circadian amplitude for DHEA-S. In 17-OH progesterone, TSH and C-peptide, the circadian amplitude in women was larger. In men, the circannual amplitude of T₃ was larger than the circadian amplitude and in insulin the circadian amplitude was larger than the circannual amplitude. There was no statistically significant difference between the circadian and circannual amplitudes in the women in ACTH and progesterone and in the men in ACTH and testosterone.