An unusual cause of acute rhabdomyolysis.
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Acute Disease↗
Biomedical subjects
H Busch
Publications and source records attributed to H Busch.
Influence of spatiotemporally correlated noise on structure formation in excitable media.
We discuss the influence of additive, spatiotemporally correlated (i.e., colored) noise on pattern formation in a two-dimensional network of excitable systems. The signature of spatiotemporal stochastic resonance (STSR) is analyzed using cross-correlation and information theoretic measures. It is found that the STSR behavior is affected by both the spatial and temporal correlations of the noise due to an interplay with the length scales of the deterministic network. Increasing the spatiotemporal noise correlation shifts the occurrence of STSR to smaller values of the noise variance. Additionally, if the spatial correlation of the noise exceeds that of the network, the excitation patterns disappear in favor of cloudy structures, directly rendering the underlying spatial noise field.
Journal Article↗
Method for detecting the signature of noise-induced structures in spatiotemporal data sets.
Spatiotemporal stochastic resonance (STSR) is a phenomenon, where the stability of spatial patterns in an extended dynamical system displays a resonance-type dependence on the noise amplitude with the patterns being optimal at intermediate noise level. This dynamical behavior has been found in theoretical systems as well as in biochemical processes, where the noise level has been controlled externally. However, it is an open question how to identify the signature of a spatiotemporal stochastic resonance in a natural system, e.g., in ecology, when the noise amplitude is not known. This question is addressed in the present paper. We provide analysis tools, which allow to reconstruct the noise intensity in a spatiotemporal data set from the data alone. These tools are based on nearest-neighbor considerations inspired by cellular automata and are an appropriate method for detecting STSR, when combined with some measure of spatial order. As a test of our analysis tools, we apply them to sample data generated by four theoretical model systems. We show explicitly that without knowledge of the theoretical value of the noise amplitude for those systems displaying STSR the corresponding resonance curve can be reconstructed from the data alone. In addition, the other (nonresonant) cases are properly identified by our method with no resonance curve being found.
Journal Article↗
Effect of colored noise on networks of nonlinear oscillators.
We discuss noise-induced pattern formation in different two-dimensional networks of nonlinear oscillators, namely a sequence of biochemical reactions and the Lorenz system. The main focus of the work is on the dependence of these patterns on the correlation time (i.e., the color) of exponentially correlated Gaussian noise. It is seen that in the nonchaotic case, the homogeneity (or average cluster size) goes through a minimum with higher correlation time, while in its chaotic regime the Lorenz system shows a higher degree of synchronization when the correlation time of the noise is increased. In order to elucidate the origin of this phenomenon, the effect of colored noise on the individual oscillator is investigated. It is shown that the specific dependence of the network's homogeneity on the noise correlation time arises from an interplay of the collective behavior and the properties of the single oscillators.
Models, Biological↗
Human RNA helicase II/Gu gene: genomic organization and promoter analysis.
Human RNA helicase II/Gu (hRH II/Gu) protein unwinds double-stranded RNA, folds single-stranded RNA, and may play important roles in ribosomal RNA biogenesis, RNA editing, RNA transport, and general transcription. The genomic organization and the regulation of expression of the hRH II/Gu gene were analyzed. The hRH II/Gu gene is over 29 kb in length, and includes 15 exons. All intron-exon junctions contain consensus GT-AG sequences. An 85 bp basic promoter was identified which lacks a functional TATA box. In LNCaP cells, the hRH II/Gu promoter was up to 50 times as active as pGL2-control vector which has an SV 40 early promoter. Mutation of a putative c-Myc/USF binding site in the region between -110 and -85 caused a decrease in the promoter activity by 2- to 3-fold. A negative regulatory region was also found between -324 and -264. Studies of transcriptional regulation of hRH II/Gu under different physiological and pathophysiological conditions may provide clues to its biological function.
Base Sequence↗
Immunocytochemical localization of nucleophosmin and RH-II/Gu protein in nucleoli of HeLa cells after treatment with actinomycin D.
HeLa cells were studied with the use of light microscopical immunocytochemistry to obtain more information on the nucleolar localization of nucleophosmin (B23 protein) and RH-II/Gu protein. In control cells these proteins are colocalized. After structural rearrangement of nucleoli induced by actinomycin D, nucleophosmin was present in the nucleolar periphery and nucleolar peripheral caps. In contrast, RH-II/Gu protein was observed mainly in central regions of nucleoli as a compact body. However, in some nucleoli the proteins colocalized in a small portion of the peripheral nucleolar cap. The proteins were also colocalized in micronucleoli but occasionally RH-II/Gu protein was absent or both proteins were not present.
Cell Nucleolus↗
Adriamycin inhibits human RH II/Gu RNA helicase activity by binding to its substrate.
RNA helicases are enzymes important in RNA synthesis, processing, transport, and turnover. Human nucleolar RNA helicase II/Gu protein (RH II/Gu) was expressed in a baculovirus system. The purified recombinant RH II/Gu protein has RNA helicase activity on a 5' tailed ds RNA substrate in vitro. We found that Adriamycin, a widely used anticancer drug, inhibited RH II/Gu helicase activity in a dose-dependent manner with an IC(50) of 40 microM. Adriamycin bound to the RNA substrate, and the binding was disrupted by boiling or treatment with 1% SDS, suggesting that the binding of Adriamycin to RNA is reversible. Adriamycin was also found by gel electrophoresis to bind to yeast tRNA to form slow-migrating complexes. These results suggest that Adriamycin can inhibit RNA synthesis or processing by binding to RNA substrates.
Antineoplastic Agents↗
The silver reaction of nucleolar proteins in the main structural compartments of ring-shaped nucleoli in smear preparations.
The present study was undertaken to provide more information on the conditions which result in preferential silver staining of the main nucleolar structural compartments using silver stainable proteins as their markers at the light microscopic level. For this study the mostly used method in cytology and pathology in which the nucleolar silver-positive structures are "developed" with the colloidal developer (Howell and Black, 1980; Ploton et al., 1986) was selected as silver reaction. Ring-shaped nucleoli of mature human lymphocytes represent a convenient model for such a study because they consist of one large fibrillar center, adjacent nucleolar regions with dense fibrillar components and the nucleolar peripheral shell with dense granular components. All these nucleolar compartments are known to possess characteristic silver stainable proteins. The results demonstrated that proteins of the fibrillar center and possibly adjacent nucleolar regions reacted preferentially with silver after a relatively long fixation with formaldehyde or methanol in unwashed specimens before the silver reaction. In contrast, the preferential staining of proteins in the nucleolar peripheral shell with silver was achieved after the fixation with acidified methanol or ethanol as well as after short fixation with formaldehyde vapors. In addition, the commonly used fixation before the silver reaction are not necessary and may be omitted for the visualization of all silver stainable proteins present in the fibrillar center as well as in the adjacent nucleolar regions and the nucleolar peripheral shell. In addition, similar results were achieved for the simultaneous visualization of proteins in the fibrillar center and nucleolar peripheral shell after fixation with ethanol.
Cell Nucleolus↗
Cloning and characterization of GEF-H1, a microtubule-associated guanine nucleotide exchange factor for Rac and Rho GTPases.
The Rho-related small GTPases are critical elements involved in regulation of signal transduction cascades from extracellular stimuli to cell nucleus and cytoskeleton. The Dbl-like guanine nucleotide exchange factors (GEF) have been implicated in direct activation of these GTPases. Here we have identified a new member of the Dbl family, GEF-H1, by screening a human HeLa cell cDNA library. GEF-H1 encodes a 100-kDa protein containing the conserved structural array of a Dbl homology domain in tandem with a pleckstrin homology domain and is most closely related to the lfc oncogene, but additionally it contains a unique coiled-coil domain at the carboxyl terminus. Biochemical analysis reveals that GEF-H1 is capable of stimulating guanine nucleotide exchange of Rac and Rho but is inactive toward Cdc42, TC10, or Ras. Moreover, GEF-H1 binds to Rac and Rho proteins in both the GDP- and guanosine 5'-3-O-(thio)triphosphate-bound states without detectable affinity for Cdc42 or Ras. Immunofluorescence reveals that GEF-H1 colocalizes with microtubules through the carboxyl-terminal coiled-coil domain. Overexpression of GEF-H1 in COS-7 cells results in induction of membrane ruffles. These results suggest that GEF-H1 may have a direct role in activation of Rac and/or Rho and in bringing the activated GTPase to specific target sites such as microtubules.
3T3 Cells↗
The 58-kDa microspherule protein (MSP58), a nucleolar protein, interacts with nucleolar protein p120.
Protein p120 is a proliferation-related nucleolar protein which is detectable early in the G1 phase of the cell cycle and peaks early in the S phase. Most human malignant tumors contain much higher levels of protein p120 than normal resting cells. To identify p120-associated protein(s), a yeast two-hybrid screen was carried out using protein p120 as the bait. Two positive clones encoded portions of a novel protein, designated microspherule protein 58 kDa (MSP58). MSP58 mRNA is 1.9 kb and encodes an approximately 58-kDa polypeptide of 462 amino acids as shown by Western blotting of HeLa nucleolar proteins. The mouse MSP58 homolog has 97% amino acid similarity to human MSP58, but no MSP58 homolog was found in the yeast genome. The MSP58 N-terminal region contains serine-rich clusters and its C-terminal region has a coiled-coil domain. In insect Sf9 cells, recombinant p120 and MSP58 proteins associated with each other, confirming the results of the yeast two-hybrid assay. Deletion mutations revealed that the binding of MSP58 to p120 required a previously unrecognized coiled-coil domain within the N-terminal region of p120 and the C-terminal region of MSP58 protein. Immunofluorescence indicated that the MSP58 protein is localized in microspherules in the nucleolus. Anti-MSP58 Ig labeled nucleolar 'caps' when HeLa cells were treated with actinomycin D. When the MSP58 protein was overexpressed in COS-7 cells, the nucleolus became irregularly enlarged, which suggests that MSP58 may affect the size and shape of the nucleolus.
Amino Acid Sequence↗
Nucleolar protein p120 contains an arginine-rich domain that binds to ribosomal RNA.
Human proliferation-associated protein p120 has previously been shown to localize to the nucleolus, and several functional domains of p120 have been elucidated. By using a nitrocellulose filter binding assay and a Northwestern blotting procedure this study shows that recombinant p120 binds to an rRNA fragment in vitro with a dissociation constant of 4 nM. The specific RNA-binding region of p120 (residues 1-57) was identified with glutathione S-transferase-fused p120 deletion constructs and Northwestern blotting procedures. This RNA-binding region of p120, which includes the nucleolar localization signal of p120, is similar to the arginine-rich RNA-binding regions found in other RNA-binding proteins such as HIV Rev and Tat. Experiments in vivo with HeLa cell nucleolar extracts showed that p120 was associated with the 60-80S pre-ribosomal particles. This association is disrupted by treatment with either RNase A or buffer of high ionic strength. These results suggest that p120 might be involved in rRNA/ribosome maturation, consistent with the role of the yeast homologue Nop2p in rRNA biogenesis.
Amino Acid Sequence↗
CENP-G: a new centromeric protein that is associated with the alpha-1 satellite DNA subfamily.
A new constitutive centromere-specific protein (CENP) has been identified as a result of its recognition as an autoantigen by serum from a patient with gastric antral vascular ectasia disease. Conventional immunoblotting and two-dimensional double blotting with both this antiserum and a known anti-centromere antiserum showed that this antiserum predominantly recognized a Mr 95,000 protein that is different from all known CENPs. We have named this new protein CENP-G. This protein was detected at the centromeric region throughout the cell cycle. In mitosis, it was restricted to the kinetochore inner plate as shown by immunogold labeling and electron microscopy. The centromeres of some human chromosomes are known to contain two subfamilies of alpha-satellite DNA. Using immunofluorescence combined with fluorescent in situ hybridization with subfamily-specific DNA probes, we revealed that CENP-G was specifically associated with one of the subfamilies, which we have named alpha-1, but not the other. The localization and the alpha-1-specific association suggested that CENP-G may play a role in kinetochore organization and function. Like CENP-B and C, but unlike CENP-A, this protein remained with the nuclear matrix after intensive extraction. While CENP-B is absent from the human Y chromosome, the existence of CENP-G on the Y chromosome has been proven by immunofluorescence and whole chromosome painting. CENP-G was also detected in CHO, Indian muntjac and Chinese muntjac cells, suggesting that it is conserved in evolution.
Animals↗
Green fluorescent protein tag for studies of drug-induced translocation of nucleolar protein RH-II/Gu.
We have constructed a human osteogenic sarcoma cell line, U-2 OS/GFP-Gu, that expresses nucleolar RNA helicase RH-II/Gu tagged with green fluorescent protein (GFP). The presence of a GFP tag does not inhibit RNA helicase, RNA folding and ATPase activities of RH-II/Gu protein. The derived cell line responds to cytotoxic agents like the parental cell line U-2 OS. In the presence of either actinomycin D or toyocamycin, the GFP-RH-II/Gu fusion protein translocates from the nucleolus to the nucleoplasm in the same way as the translocation of endogenous RH-II/Gu. The drug-induced translocation of GFP-RH-II/Gu is easily monitored by direct observation of live cells in vivo. This cell line can be used to screen cytotoxic drugs and to study the mechanisms of drug-induced translocation of RH-II/Gu. The cellular localization of RH-II/Gu during the cell cycle-dependent formation of the nucleolus is readily monitored. Real-time results are obtained more quickly without the disadvantages associated with cell fixation and immunofluorescence-based staining.
Biological Transport↗
RNA-unwinding and RNA-folding activities of RNA helicase II/Gu--two activities in separate domains of the same protein.
The human RNA helicase II/Gu protein (RH-II/Gu) is a member of the D-E-A-D box protein family. It is a unique enzyme, which possesses an ATP-dependent RNA-unwinding activity and has an RNA-folding activity that introduces an intramolecular secondary structure in single-stranded RNA. This report shows that these two enzymatic activities are distinct. ATP[S], GTP and low concentrations of ATP enhance the RNA-folding activity of RH-II/Gu but not the RNA-helicase activity. High concentrations of ATP are required for the helicase activity but are inhibitory to the RNA-folding activity. Mg2+ is required for the helicase activity but not for the RNA-folding reaction. Affinity-purified anti-(RH-II/Gu) polyclonal Ig inhibit the RNA-unwinding activity but not the folding activity. Mutations of the DEVD sequence, which corresponds to the DEAD box, and the SAT motif enhanced RNA-folding activity of RH-II/Gu but completely inhibited the RNA-helicase activity. A mutant that lacks the COOH-terminal 76 amino acid residues, including the four FRGQR repeats, had unwinding activity but did not catalyze the folding of a single-stranded RNA. The two enzymatic activities of RH-II/Gu reside in distinct domains. Amino acids 1-650 are active in the RNA-unwinding reaction but lack RNA-folding activity. Amino acids 646-801 fold single-stranded RNA but lack helicase activity. This report shows distinct RNA-unwinding and RNA-folding activities residing in separate domains within the same protein.
Adenosine Triphosphatases↗
Effects of cytotoxic drugs on translocation of nucleolar RNA helicase RH-II/Gu.
Some cytotoxic drugs cause translocation of nucleophosmin/B23 and other nucleolar proteins to the nucleoplasm. The present study shows that these drugs caused a similar translocation of RH-II/Gu, a nucleolar RNA helicase. Other nucleolar proteins including p120, UBF, RNA polymerase I large subunit, fibrillarin, p40, and Ren-1 did not translocate. A 2-h treatment of MCF-7 breast cancer cells with 0.008 or 0.16 microM actinomycin D resulted in translocation of RH-II/Gu to the nucleoplasm; these effects were not reversed by 100 microM guanosine. The effects of 0.008 microM actinomycin D, but not 0.16 microM actinomycin D, on the translocation of RH-II/Gu were reversed when the drug was removed. However, the effects of 0.008 or 0.16 microM actinomycin D on the translocation of nucleophosmin/B23 were not reversible. The translocation effects of 50 microM toyocamycin on RH-II/Gu were reversed when the drug was replaced with fresh medium. RH-II/Gu mostly relocalized to the nucleoli within 15 min after toyocamycin was withdrawn; only partial relocalization of nucleophosmin/B23 occurred 40 h after removal of the drug. The effects of toyocamycin were not blocked by 100 microM guanosine. Mycophenolic acid (50 microM, 2-h treatment) caused partial translocation of RH-II/Gu; this effect was slowly reversed upon drug removal and was inhibited by 100 microM guanosine, in a manner similar to the effects of mycophenolic acid on the localization of nucleophosmin/B23. This study shows similarities and differences in the drug-induced translocation and relocalization of RH-II/Gu and nucleophosmin/B23. Analysis of translocation of specific nucleolar proteins may offer a quantitative approach to assessment of potency and duration of effects of cytotoxic agents.
Adenocarcinoma↗
Ion channels in the vacuoles of the seagrass Posidonia oceanica.
Voltage-dependent ionic channels were investigated by the patch-clamp technique in the vacuolar membrane from the leaves of the seagrass Posidonia oceanica. Vacuoles extruded from the meristematic white part of the leaves displayed rectifying slow currents which activated in several seconds at positive potentials and deactivated at negative voltages within a few hundreds of ms. Like the Slow Vacuolar (SV) channel already identified in the tonoplast of terrestrial plants, the SV voltage-dependent channel of Posidonia leaves was activated by micromolar concentrations of Ca2+ and was equally permeable to K+ and Na+. The single-channel conductance of the Posidonia SV-type channel was 106 +/- 12 pS (in symmetric 400 mM K+). In the same ionic solutions, another channel, occasionally observed in vacuoles from the green part of the leaves, displayed a single-channel conductance of 47 +/- 4 pS. To our knowledge, this is the first electrophysiological characterization of ion transport pathways in Posidonia, a marine plant of crucial importance for the ecology of the Mediterranean sea.
Calcium↗
Cloning and characterization of Gu/RH-II binding protein.
Gu/RNA helicase II (Gu/RH-II) is the first reported mammalian nucleolar RNA helicase that is a member of the D-E-A-D (Asp-Glu-Ala-Asp) box family of proteins. It has an ATP-dependent RNA unwinding (helicase) activity and a separate RNA folding activity (introduction of intramolecular secondary structure into single-stranded RNA). To determine which proteins may bind to Gu/RH-II, a yeast two-hybrid system was used. A cDNA which encoded a protein, called Gu/RH-II binding protein or GBP, was isolated and sequenced. The GBP protein is localized to the nucleus in speckled or diffuse nucleoplasmic patterns. The GBP mRNA level is highest in testis, 9- to 49-fold greater than other tissues. When GBP interacts with Gu/RH-II, proteolytic cleavage of Gu/RH-II occurs; the amino-terminal portion of Gu/RH-II is critical for this proteolysis.
Amino Acid Sequence↗
Human proliferation-related protein p120 interacts with HSRP1.
Protein p120 is a proliferation-related nucleolar protein, which is detectable early in the G1-phase of the cell cycle and peaks early in the S-phase. Most human malignant tumor cells contain much higher levels of protein p120 than do normal resting cells. To learn more about p120-associated proteins, a yeast two-hybrid screen was carried out using protein p120 as the bait. Five positive clones were identified from 2 million clones for further analysis. Three of them encoded portions of the same protein, which had identity to human SRP1. The recombinant p120 and HSRP1 proteins produced in Sf9 cells are associated with each other, confirming the results of the yeast genetic assay. Protein SRP1 was originally characterized as a suppressor of RNA polymerase I mutations, and recently human SRP1 was identified as a receptor for nuclear localization sequences. In the present study, use of deletion mutations revealed that the binding of human SRP1 to p120 required the p120 nuclear localization signal (amino acids 96-119) and the C-terminus of human SRP1 (amino acids 453-491).
Animals↗