Efficient coherent population transfer in NO molecules using pulsed lasers.
Explore the source record for details and available documents.
Journal Article↗
Biomedical subjects
A Kuhn
Publications and source records attributed to A Kuhn.
Structural characterization of membrane insertion of M13 procoat, M13 coat, and Pf3 coat proteins.
A new, simple, and efficient purification method has been developed for the extremely hydrophobic M13 procoat, M13 coat, and Pf3 coat proteins. Homogeneous preparations were obtained in 2-propanol/0.1% TFA, where M13 coat protein is found to be dissolved in a monomeric form, and the two other proteins as dimers or trimers. The conformations of these particular proteins in different environments have been determined by circular dichroism and infrared spectroscopy. In organic solvents, the proteins adopt a conformation with an average helix content of 90%. In lipid bilayers composed of phosphatidylcholine and phosphatidylglycerol lipids, the average helix content is 50% for M13 procoat protein, 60% for M13 coat protein, and 75% for Pf3 coat protein. The orientational order parameter S alpha of the protein helices in planar lipid bilayers have been determined by polarized infrared measurements in the amide I spectral range. The helices of the three proteins are oriented preferentially parallel to the membrane normal, with S alpha = 0.63 for M13 procoat protein, S alpha = 0.58 for Pf3 coat protein, and a distinctly higher value of S alpha = 0.81 for M13 coat protein.
Amino Acid Sequence↗
Sequence-specific transactivators counteract topoisomerase II-mediated inhibition of in vitro transcription by RNA polymerases I and II.
An inhibitor of RNA polymerase II transcription in vitro has been purified from HeLa cell nuclear extracts. Partial amino acid sequences derived from the purified protein revealed that the inhibitor of transcription corresponded to human topoisomerase II. Order of addition experiments provided evidence indicating that topoisomerase II inhibited transcription by binding over the core promoter and blocking preinitiation complex formation. Topoisomerase II-mediated repression could be relieved by sequence-specific transcriptional activators, having different activating and/or DNA binding domains, but antirepression required a transcriptional activation function in addition to a DNA binding domain. Moreover, transcription by RNA polymerase I was also inhibited by topoisomerase II and this inhibition could be relieved by the RNA polymerase I transactivator UBF. These observations suggest that topoisomerase II may participate in a general repression of transcription which can be counteracted by transcriptional activators.
Amino Acid Sequence↗
The nucleolar transcription activator UBF relieves Ku antigen-mediated repression of mouse ribosomal gene transcription.
Previously we have shown that the RNA polymerase I (Pol I)-specific transcription factor UBF stimulates transcription by both facilitating transcription complex formation and by relieving repression exerted by a negative-acting factor which competes for binding of the murine factor TIF-IB to the ribosomal gene promoter (1). We have purified and functionally characterized this repressor protein from Ehrlich ascites cells. The final preparation contained two polypeptides with molecular masses of 75 and 90 kDa, respectively. Both polypeptides interact with the rDNA promoter as revealed by UV-crosslinking experiments. The specificity of binding to the ribosomal gene promoter was demonstrated in an electrophoretic mobility shift assay and by DNase footprinting. The biochemical properties of this negative-acting factor closely resemble those of the Ku antigen, a human nuclear DNA-binding heterodimer which is the target of autoantibodies in several autoimmune diseases. Anti-Ku antibodies precipitate the repressor activity and overcome transcription inhibition. The data demonstrate that regulation of Pol I gene transcription may involve an antirepression mechanism as already documented for Pol II genes and suggest that Ku protein may be causally involved in repressor-mediated down regulation of rRNA synthesis.
Animals↗
Charged residues render pro-OmpA potential dependent for initiation of membrane translocation.
We have examined the effects of positively and negatively charged residues on the translocation of outer membrane protein A precursor (pro-OmpA) across the bacterial inner membrane. Pro-OmpA does not translocate across the membrane when 2 positively charged residues are inserted immediately after the leader peptide, whereas it does insert when 2 neutral or negatively charged residues are introduced. Using a cell-free translocation system, we show that the membrane potential stimulated the rate of initial insertion of pro-OmpA with negatively charged residues, inhibited pro-OmpA with positively charged residues, and had no effect on neutral pro-OmpA. Thus, acidic residues render pro-OmpA potential-dependent for loop formation, which then initiates the translocation process.
Amino Acid Sequence↗
Effect of various types of sodium perborate on the pH of bleaching agents.
Time-dependent changes in the pH value of various types of sodium perborate solutions used as bleaching agents were evaluated. Sodium perborate-monohydrate (MH), sodium perborate-trihydrate (TRH), and sodium perborate-tetrahydrate are available. Each perborate was mixed with 10%, 15%, or 30% fresh hydrogen peroxide or with bidistilled water in a powder to liquid ratio of 2 g:1 ml, respectively. The pH values were recorded at baseline and after 1h, 1 day, 3 days, and 7 days, respectively. At baseline the pH values of MH, TRH, and tetrahydrate in conjunction with 30% H2O2 were 8.7, 7.0, and 7.5, respectively. The pH increased significantly with decreasing concentrations of H2O2. For TRH, MH, and tetrahydrate mixed with bidistilled water more alkaline values were measured at baseline and after 1 h. Due to solidification of the samples, the pH could not be determined for MH starting day 1 and for TRH starting day 3. In conclusion, the pH of bleaching pastes depends on the content of water of crystallization in sodium perborate, H2O2 concentration, and time of measurement. The bulk of the mixtures recorded reached alkaline pH values of 10 to 11. It is recommended that the pH of the mixture being used be checked to avoid potential postbleaching root resorption.
Borates↗
[Therapeutic problem. Angiolymphoid hyperplasia with eosinophilia].
A 35-year-old female patient suffered from angiolymphoid hyperplasia with eosinophilia for 9 years, with multiple, exophytic tumours on her left pinna and the surrounding skin. Systemic treatment with gamma interferon and glucocorticoids, intralesional injections of glucocorticoids and argon laser therapy had no effect. After the tumours had been pared away by electrocautery the patient was free of symptoms for 1 year and then developed a small local recurrence. Recurrences are common in angiolymphoid hyperplasia with eosinophilia. Malignant transformation has not been observed. We therefore regard non-radical surgery as the therapy of choice.
Aged↗
Dual role of the nucleolar transcription factor UBF: trans-activator and antirepressor.
In a reconstituted system consisting of partially purified RNA polymerase I (pol I) and the initiation factors TIF-IA, TIF-IB, and TIF-IC, the nucleolar factor UBF (upstream binding factor) stimulates transcription from the rRNA-encoding DNA (rDNA) promoter at least 50-fold. This activation is not observed at high template concentrations or in the presence of highly purified pol I. Template commitment experiments suggest that UBF activates transcription by relieving inhibition exerted by a negative-acting factor(s) in the polymerase fraction that competes for TIF-IB binding to the rDNA promoter and prevents the formation of preinitiation complexes. Using purified histone H1 bound to DNA as a model for the repressed state of the rDNA promoter, we show that UBF counteracts H1-mediated repression of pol I transcription. The implications of these findings are discussed with respect to the protein-protein and protein-DNA interactions at the rDNA promoter and the possible involvement of UBF in control of ribosomal gene transcription.
Animals↗
Laser-induced population transfer in multistate systems: A comparative study.
Explore the source record for details and available documents.
Journal Article↗
Distinct domains of an oligotopic membrane protein are Sec-dependent and Sec-independent for membrane insertion.
Leader peptidase of Escherichia coli spans the plasma membrane twice with its amino terminus on the periplasmic surface of the membrane and its large carboxyl-terminal domain protruding into the periplasm. To monitor the transfer of the amino terminus of leader peptidase to the periplasm, we have constructed a fusion protein between the 18-residue amino-terminal periplasmic domain of Pf3 bacteriophage coat protein and the beginning of leader peptidase. We find that neither the SecA or SecY proteins nor a transmembrane electrochemical potential is required for insertion of the amino terminus, while the transfer of the carboxyl-terminal domain of leader peptidase has these requirements. The first 35 residues of leader peptidase, which include the first hydrophobic domain and the carboxyl-terminal positively charged cluster, are sufficient to insert the amino terminus. When positively charged residues are introduced before the first transmembrane segment, translocation of the amino terminus is abolished. These studies in protein membrane topogenesis, showing that there are different requirements for amino and carboxyl termini insertion, indicate that multiple mechanisms exist even within the same protein.
Adenosine Triphosphatases↗
The nucleolar transcription factor mUBF is phosphorylated by casein kinase II in the C-terminal hyperacidic tail which is essential for transactivation.
UBF is a DNA binding protein which interacts with both the promoter and the enhancer of various vertebrate ribosomal RNA genes and functions as a transcription initiation factor for RNA polymerase I (pol I). We have purified murine UBF to apparent molecular homogeneity and demonstrate that its transactivating potential, but not its DNA binding activity, is modulated in response to cell growth. In vivo labelling experiments demonstrate that UBF is a phosphoprotein and that the phosphorylation state is different in growing and quiescent cells. We show that UBF is phosphorylated in vitro by a cellular protein kinase which by several criteria closely resembles casein kinase II (CKII). A major modification involves serine phosphoesterifications in the carboxy terminal hyperacidic tail of UBF. Deletions of this C-terminal domain severely decreases the UBF directed activation of transcription. The data suggest that phosphorylation of UBF by CKII may play an important role in growth dependent control of rRNA synthesis.
Amino Acid Sequence↗
Bacteriophage T4 gene 21 encodes two proteins essential for phage maturation.
The T4 prohead protease (T4 PPase) is the key enzyme in the morphopoietic pathway of the T4 phage head. It is responsible for the proteolytic processing of all head proteins allowing protein rearrangement and head expansion. To study its biochemistry and gene regulation, T4 gene 21 was cloned into an expression vector under the control of the inducible tac promoter. Two proteins of apparent molecular weights of 21.5 and 27.5 kDa were detected after induction. These proteins are synthesized using two different start codons in the same reading frame. Destruction of either start codon resulted in the loss of the respective protein. Complementation experiments with bacteriophage T4 21(-)-infected cells showed that both proteins are functional in vivo and essential for T4 phage assembly.
Base Sequence↗
[Chronic lichenoid keratosis].
We report on a 41-year-old woman with keratosis lichenoides chronica, a disorder first described by Kaposi in 1886 as "lichen moniliformis", who later also developed chronic lymphatic leukaemia. Since Kaposi's original report, 38 additional cases have been reported. Occurrence of keratosis lichenoides chronica associated with malignant disorders has not previously been described.
Adult↗
Use of site-directed mutagenesis to define the limits of sequence variation tolerated for processing of the M13 procoat protein by the Escherichia coli leader peptidase.
Leader peptidase cleaves the leader sequence from the amino terminus of newly made membrane and secreted proteins after they have translocated across the membrane. Analysis of a large number of leader sequences has shown that there is a characteristic pattern of small apolar residues at -1 and -3 (with respect to the cleavage site) and a helix-breaking residue adjacent to the central apolar core in the region -4 to -6. The conserved sequence pattern of small amino acids at -1 and -3 around the cleavage site most likely represents the substrate specificity of leader peptidase. We have tested this by generating 60 different mutations in the +1 to -6 domain of the M13 procoat protein. These mutants were analyzed for in vivo and in vitro processing, as well as for protein insertion into the cytoplasmic membrane. We find that in vivo leader peptidase was able to process procoat with an alanine, a serine, a glycine, or a proline residue at -1 and with a serine, a glycine, a threonine, a valine, or a leucine residue at -3. All other alterations at these sites were not processed, in accordance with predictions based on the conserved features of leader peptides. Except for proline and threonine at +1, all other residues at this position were processed by leader peptidase. None of the mutations at -2, -4, or -5 of procoat (apart from proline at -4) completely abolished leader peptidase cleavage in vivo although there were large effects on the kinetics of processing.(ABSTRACT TRUNCATED AT 250 WORDS)
Amino Acid Sequence↗
Palmoplantar keratoderma with tonotubular keratin.
A 61-year-old man with palmoplantar keratoderma with an unusual tonotubular keratin is reported. The histologic findings, genetic transmission, and clinical course were similar to epidermolytic palmoplantar keratoderma (Voerner type), but keratinocytes ultrastructurally displayed a tonotubular cytoskeleton, which has not been previously described, instead of a tonofilamentous one. Electrophoretically, we found no difference in the keratin pattern of normal plantar skin, skin in palmoplantar keratoderma of Voerner, and that of our patient's skin. Therefore the tubular keratin most likely formed as a result of a posttranslational change of keratin polymerization.
Humans↗
The function of a leader peptide in translocating charged amino acyl residues across a membrane.
Insertion of bacteriophage coat proteins into the membrane of infected bacterial cells can be studied as a model system of protein translocation across membranes. The coat protein of the filamentous bacteriophage Pf3--which infects Pseudomonas aeruginosa--is 44 amino acids in length and has the same basic structure as the coat protein of bacteriophage M13, which infects Escherichia coli. However, unlike the Pf3 coat protein, the M13 coat protein is synthesized as a precursor (procoat) with a typical leader (signal) sequence, which is cleaved after membrane insertion. Nevertheless, when the gene encoding the Pf3 coat protein is expressed in E. coli, the protein is translocated across the membrane. Hybrid M13 and Pf3 coat proteins were constructed in an attempt to understand how the Pf3 coat protein is translocated without a leader sequence. These studies demonstrated that the extracellular regions of the proteins determined their cellular location. When three charged residues in this region were neutralized, the leader-free M13 coat protein was also inserted into the membrane. Differences in the water shell surrounding these residues may account for efficient membrane insertion of the protein without a leader sequence.
Amino Acid Sequence↗
Specific interaction of the murine transcription termination factor TTF I with class-I RNA polymerases.
The 18-base-pair sequence element AGGTCGACCAGTACTCCG (the Sal box) signals termination of mouse ribosomal gene transcription. This sequence is recognized by a sequence-specific DNA-binding protein, TTF I, which mediates the termination of transcription by RNA polymerase I (pol I). Subsequently, the ends of the primary transcripts are trimmed by 10 nucleotides in a sequence-dependent 3'-terminal processing reaction. We have now investigated whether TTF I bound to its target sequence will block elongation by any RNA polymerase by steric hindrance, or whether it is specific for elongation by pol I. The results demonstrate that TTF I directs transcription termination with RNA polymerase I from species as divergent as mouse and yeast, but fails to affect elongation by heterologous polymerases (eukaryotic RNA polymerases II and III, Escherichia coli or bacteriophage T3 RNA polymerase). By contrast, purified lac repressor bound to its operator sequence stops elongation by both RNA polymerase I and II.
Animals↗
Efficient translocation of positively charged residues of M13 procoat protein across the membrane excludes electrophoresis as the primary force for membrane insertion.
The coat protein of bacteriophage M13 is inserted into the Escherichia coli plasma membrane as a precursor protein, termed procoat, with a typical leader peptide of 23 amino acid residues. Its membrane insertion requires the electrochemical potential but not the cellular components SecA and SecY. Since the electrochemical gradients result in the periplasmic side of the membrane being positively charged, the membrane potential could contribute to the transfer of the negatively charged central region of procoat across the membrane. Here we demonstrate that the central domain following the leader peptide can be translocated across the membrane even when the net charge of the region is changed from -3 to +3. This rules out an electrophoresis-like insertion mechanism for procoat. We also show that the sec independence of procoat insertion is linked to the presence of the second apolar domain. The deletion of most of the second apolar domain from a procoat fusion protein results in sec dependent membrane insertion of the hybrid protein. Moreover, like other proteins that require the sec genes, translocation of this sec dependent procoat protein is inhibited when positively charged residues are introduced after the leader peptide. Loop models involving one or two hydrophobic regions are presented that account for the differences in tolerance of positively charged residues.
Amino Acid Sequence↗