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1.
Mol Metab ; 79: 101838, 2024 Jan.
Article in English | MEDLINE | ID: mdl-37995884

ABSTRACT

OBJECTIVE: Carbohydrate Response Element Binding Protein (ChREBP) is a glucose 6-phosphate (G6P)-sensitive transcription factor that acts as a metabolic switch to maintain intracellular glucose and phosphate homeostasis. Hepatic ChREBP is well-known for its regulatory role in glycolysis, the pentose phosphate pathway, and de novo lipogenesis. The physiological role of ChREBP in hepatic glycogen metabolism and blood glucose regulation has not been assessed in detail, and ChREBP's contribution to carbohydrate flux adaptations in hepatic Glycogen Storage Disease type 1 (GSD I) requires further investigation. METHODS: The current study aimed to investigate the role of ChREBP as a regulator of glycogen metabolism in response to hepatic G6P accumulation, using a model for acute hepatic GSD type Ib. The immediate biochemical and regulatory responses to hepatic G6P accumulation were evaluated upon G6P transporter inhibition by the chlorogenic acid S4048 in mice that were either treated with a short hairpin RNA (shRNA) directed against ChREBP (shChREBP) or a scrambled shRNA (shSCR). Complementary stable isotope experiments were performed to quantify hepatic carbohydrate fluxes in vivo. RESULTS: ShChREBP treatment normalized the S4048-mediated induction of hepatic ChREBP target genes to levels observed in vehicle- and shSCR-treated controls. In parallel, hepatic shChREBP treatment in S4048-infused mice resulted in a more pronounced accumulation of hepatic glycogen and further reduction of blood glucose levels compared to shSCR treatment. Hepatic ChREBP knockdown modestly increased glucokinase (GCK) flux in S4048-treated mice while it enhanced UDP-glucose turnover as well as glycogen synthase and phosphorylase fluxes. Hepatic GCK mRNA and protein levels were induced by shChREBP treatment in both vehicle- and S4048-treated mice, while glycogen synthase 2 (GYS2) and glycogen phosphorylase (PYGL) mRNA and protein levels were reduced. Finally, knockdown of hepatic ChREBP expression reduced starch domain binding protein 1 (STBD1) mRNA and protein levels while it inhibited acid alpha-glucosidase (GAA) activity, suggesting reduced capacity for lysosomal glycogen breakdown. CONCLUSIONS: Our data show that ChREBP activation controls hepatic glycogen and blood glucose levels in acute hepatic GSD Ib through concomitant regulation of glucose phosphorylation, glycogenesis, and glycogenolysis. ChREBP-mediated control of GCK enzyme levels aligns with corresponding adaptations in GCK flux. In contrast, ChREBP activation in response to acute hepatic GSD Ib exerts opposite effects on GYS2/PYGL enzyme levels and their corresponding fluxes, indicating that GYS2/PYGL expression levels are not limiting to their respective fluxes under these conditions.


Subject(s)
Blood Glucose , Glycogen Storage Disease Type I , Animals , Mice , Carbohydrate Metabolism , Disease Models, Animal , Glucose/metabolism , Glucose-6-Phosphate/metabolism , Glycogen/metabolism , Glycogen Synthase/metabolism , Liver Glycogen/metabolism , Phosphates , RNA, Messenger/metabolism , RNA, Small Interfering/metabolism , Transcription Factors/genetics , Transcription Factors/metabolism
2.
Arch Toxicol ; 94(10): 3463-3473, 2020 10.
Article in English | MEDLINE | ID: mdl-32623605

ABSTRACT

Valproic acid (VPA) is a frequently prescribed anti-epileptic drug which is known to cause liver toxicity and steatosis through mitochondrial dysfunction. Nevertheless the mechanisms underlying these adverse effects are incompletely understood. In this study, we determined the effect of relatively short (3 h) or prolonged (72 h) exposure to VPA on mitochondrial function in primary human hepatocytes (PHHs). While 3 h VPA exposure did not affect oxygen consumption rates (OCRs) in PHHs, prolonged exposure (24-72 h) significantly reduced basal and maximal OCRs. Given that in particular prolonged VPA exposure is required to cause mitochondrial dysfunction, we investigated gene expression data after VPA exposure for 24, 48, 72 h and 72 h VPA followed by a 72 h washout period. We were able to reduce the comprehensive gene expression changes into a more comprehensible set of 18 TFs that were predicted to be persistently activated after 72 h of VPA exposure. Lentiviral knock-down of one of the candidate TFs, C/EBPα, partly rescued VPA-induced mitochondrial dysfunction. Furthermore, RNA-Seq analysis of shC/EBPα and shGFP control PHHs identified 24 genuine C/EBPα target genes that are regulated in response to prolonged VPA exposure in PHHs. Altogether this provides new insights on the involvement of C/EBPα in driving VPA-induced mitochondrial dysfunction in human liver cells. This hub gene, with its downstream regulators involved in this deregulation, thus represent potential new biomarkers for VPA-induced mitochondrial dysfunction.


Subject(s)
CCAAT-Enhancer-Binding Protein-alpha/genetics , Hepatocytes/drug effects , Mitochondria/drug effects , Mitochondria/metabolism , Valproic Acid/adverse effects , Anticonvulsants/adverse effects , Biomarkers , CCAAT-Enhancer-Binding Protein-alpha/metabolism , Cells, Cultured , Fatty Liver/metabolism , Gene Expression Regulation , Gene Knockdown Techniques , Humans , Liver/drug effects , Liver/metabolism , Oxygen/metabolism , RNA-Seq , Transcription Factors/genetics , Transcription Factors/metabolism
3.
BMC Bioinformatics ; 20(1): 417, 2019 Aug 13.
Article in English | MEDLINE | ID: mdl-31409281

ABSTRACT

BACKGROUND: The development of high throughput sequencing techniques provides us with the possibilities to obtain large data sets, which capture the effect of dynamic perturbations on cellular processes. However, because of the dynamic nature of these processes, the analysis of the results is challenging. Therefore, there is a great need for bioinformatics tools that address this problem. RESULTS: Here we present DynOVis, a network visualization tool that can capture dynamic dose-over-time effects in biological networks. DynOVis is an integrated work frame of R packages and JavaScript libraries and offers a force-directed graph network style, involving multiple network analysis methods such as degree threshold, but more importantly, it allows for node expression animations as well as a frame-by-frame view of the dynamic exposure. Valuable biological information can be highlighted on the nodes in the network, by the integration of various databases within DynOVis. This information includes pathway-to-gene associations from ConsensusPathDB, disease-to-gene associations from the Comparative Toxicogenomics databases, as well as Entrez gene ID, gene symbol, gene synonyms and gene type from the NCBI database. CONCLUSIONS: DynOVis could be a useful tool to analyse biological networks which have a dynamic nature. It can visualize the dynamic perturbations in biological networks and allows the user to investigate the changes over time. The integrated data from various online databases makes it easy to identify the biological relevance of nodes in the network. With DynOVis we offer a service that is easy to use and does not require any bioinformatics skills to visualize a network.


Subject(s)
Gene Regulatory Networks , User-Computer Interface , Acetaminophen/pharmacology , Computational Biology/methods , Databases, Factual , Humans , NF-kappa B/metabolism , Signal Transduction/drug effects , Signal Transduction/genetics
4.
Sci Rep ; 8(1): 4405, 2018 03 13.
Article in English | MEDLINE | ID: mdl-29535334

ABSTRACT

Hepatic and renal energy status prior to transplantation correlates with graft survival. However, effects of brain death (BD) on organ-specific energy status are largely unknown. We studied metabolism, perfusion, oxygen consumption, and mitochondrial function in the liver and kidneys following BD. BD was induced in mechanically-ventilated rats, inflating an epidurally-placed Fogarty-catheter, with sham-operated rats as controls. A 9.4T-preclinical MRI system measured hourly oxygen availability (BOLD-related R2*) and perfusion (T1-weighted). After 4 hrs, tissue was collected, mitochondria isolated and assessed with high-resolution respirometry. Quantitative proteomics, qPCR, and biochemistry was performed on stored tissue/plasma. Following BD, the liver increased glycolytic gene expression (Pfk-1) with decreased glycogen stores, while the kidneys increased anaerobic- (Ldha) and decreased gluconeogenic-related gene expression (Pck-1). Hepatic oxygen consumption increased, while renal perfusion decreased. ATP levels dropped in both organs while mitochondrial respiration and complex I/ATP synthase activity were unaffected. In conclusion, the liver responds to increased metabolic demands during BD, enhancing aerobic metabolism with functional mitochondria. The kidneys shift towards anaerobic energy production while renal perfusion decreases. Our findings highlight the need for an organ-specific approach to assess and optimise graft quality prior to transplantation, to optimise hepatic metabolic conditions and improve renal perfusion while supporting cellular detoxification.


Subject(s)
Adaptation, Physiological , Brain Death/metabolism , Energy Metabolism , Animals , Biomarkers , Gene Expression , Kidney/metabolism , Liver/metabolism , Male , Mitochondria/metabolism , Organ Specificity , Oxidative Stress , Oxygen Consumption , Perfusion , Rats
5.
Phys Rev Lett ; 116(3): 033601, 2016 Jan 22.
Article in English | MEDLINE | ID: mdl-26849594

ABSTRACT

We probe the indistinguishability of photons emitted by a semiconductor quantum dot (QD) via time- and temperature-dependent two-photon interference (TPI) experiments. An increase in temporal separation between consecutive photon emission events reveals a decrease in TPI visibility on a nanosecond time scale, theoretically described by a non-Markovian noise process in agreement with fluctuating charge traps in the QD's vicinity. Phonon-induced pure dephasing results in a decrease in TPI visibility from (96±4)% at 10 K to a vanishing visibility at 40 K. In contrast to Michelson-type measurements, our experiments provide direct access to the time-dependent coherence of a quantum emitter on a nanosecond time scale.

6.
Metab Eng Commun ; 3: 52-63, 2016 Dec.
Article in English | MEDLINE | ID: mdl-29468113

ABSTRACT

13C labeling experiments in aerobic glucose limited cultures of Saccharomyces cerevisiae at four different growth rates (0.054; 0.101, 0.207, 0.307 h-1) are used for calculating fluxes that include intracellular cycles (e.g., storage carbohydrate cycles, exchange fluxes with amino acids), which are rearranged depending on the growth rate. At low growth rates the impact of the storage carbohydrate recycle is relatively more significant than at high growth rates due to a higher concentration of these materials in the cell (up to 560-fold) and higher fluxes relative to the glucose uptake rate (up to 16%). Experimental observations suggest that glucose can be exported to the extracellular space, and that its source is related to storage carbohydrates, most likely via the export and subsequent extracellular breakdown of trehalose. This hypothesis is strongly supported by 13C-labeling experimental data, measured extracellular trehalose, and the corresponding flux estimations.

7.
Oncogene ; 33(41): 4904-15, 2014 Oct 09.
Article in English | MEDLINE | ID: mdl-24141784

ABSTRACT

The role of the epithelial cell adhesion molecule EpCAM in cancer progression remains largely unclear. High expression of EpCAM in primary tumors is often associated with more aggressive phenotypes and EpCAM is the prime epithelial antigen in use to isolate circulating tumor cells (CTCs) and characterize disseminated tumor cells (DTCs). However, reduced expression of EpCAM was associated with epithelial-to-mesenchymal transition (EMT) and reports on a lack of EpCAM on CTCs emerged. These contradictory observations might reflect a context-dependent adaption of EpCAM expression during metastatic progression. To test this, EpCAM expression was monitored in esophageal cancer at different sites of early systemic disease. Although most of the primary esophageal tumors expressed high levels of EpCAM, the majority of DTCs in bone marrow lacked EpCAM. In vitro, downregulation of EpCAM expression at the plasma membrane was observed in migrating and invading cells, and was associated with a partial loss of the epithelial phenotype and with significantly decreased proliferation. Accordingly, induction of EMT through the action of TGFß resulted in substantial loss of EpCAM cell surface expression on esophageal cancer cells. Knock-down or natural loss of EpCAM recapitulated these effects as it reduced proliferation while enhancing migration and invasion of cancer cells. Importantly, expression of EpCAM on DTCs was significantly associated with the occurrence of lymph node metastases and with significantly decreased overall survival of esophageal cancer patients. We validated this observation by showing that high expression of EpCAM promoted tumor outgrowth after xenotransplantation of esophageal carcinoma cells. The present data disclose a dynamic expression of EpCAM throughout tumor progression, where EpCAM(high) phenotypes correlate with proliferative stages, whereas EpCAM(low/negative) phenotypes associated with migration, invasion and dissemination. Thus, differing expression levels of EpCAM must be taken into consideration for therapeutic approaches and during clinical retrieval of disseminated tumor cells.


Subject(s)
Antigens, Neoplasm/metabolism , Cell Adhesion Molecules/metabolism , Esophageal Neoplasms/pathology , Lymphatic Metastasis/pathology , Neoplastic Cells, Circulating/pathology , Aged , Animals , Antigens, Neoplasm/genetics , Cell Adhesion Molecules/genetics , Cell Line, Tumor , Cell Movement , Cell Proliferation , Epithelial Cell Adhesion Molecule , Epithelial-Mesenchymal Transition , Esophageal Neoplasms/metabolism , Gene Expression Regulation, Neoplastic , Heterografts , Humans , Lymphatic Metastasis/genetics , Male , Mice , Mice, Inbred NOD , Middle Aged , Neoplastic Cells, Circulating/metabolism , Phenotype , Transforming Growth Factor beta/metabolism
9.
Phys Rev Lett ; 104(1): 010502, 2010 Jan 08.
Article in English | MEDLINE | ID: mdl-20366354

ABSTRACT

We report the generation of entanglement between two individual 87Rb atoms in hyperfine ground states |F=1,M=1> and |F=2,M=2> which are held in two optical tweezers separated by 4 microm. Our scheme relies on the Rydberg blockade effect which prevents the simultaneous excitation of the two atoms to a Rydberg state. The entangled state is generated in about 200 ns using pulsed two-photon excitation. We quantify the entanglement by applying global Raman rotations on both atoms. We measure that 61% of the initial pairs of atoms are still present at the end of the entangling sequence. These pairs are in the target entangled state with a fidelity of 0.75.

10.
Cephalalgia ; 24(8): 645-56, 2004 Aug.
Article in English | MEDLINE | ID: mdl-15265053

ABSTRACT

BIBN 4096 BS ([R-(R*,S*)]-N-[2-[[5-amino-1-[[4-(4-pyridinyl)-1-piperazinyl]carbonyl]pentyl]amino]-1-[(3,5-dibromo-4-hydroxyphenyl)methyl]-2-oxoethyl]-4-(1,4-dihydro-2-oxo-3(2H)-quinazolinyl)-,1-piperidinecarboxamide) is the first selective, highly potent, small molecule, nonpeptide calcitonin gene-related peptide (CGRP) receptor antagonist, which has been developed for the treatment of acute migraine. The objective of this study was to obtain information on the safety, tolerability and pharmacokinetics of BIBN 4096 BS following single intravenous administration of rising doses (0.1, 0.25, 0.5, 1, 2.5, 5 and 10 mg) in 55 healthy male and female volunteers. The study was of single-centre, double-blind (within dose levels), placebo-controlled, randomized, single rising dose design. Blood pressure, pulse rate, respiratory rate, ECG, laboratory tests and forearm blood flow did not reveal any clinically relevant, drug-induced changes. Sixteen adverse events (AEs) were reported by eight of 41 volunteers after BIBN 4096 BS compared to five AEs reported by four of 14 volunteers after placebo. Approximately two-thirds of all AEs related to active treatment occurred at the highest dose of 10 mg. At this dose level, all AEs were confined to the three BIBN 4096 BS-treated females, and consisted mainly of transient and mild paresthesias. Paresthesias were the single most frequent AE, whereas fatigue was the AE which occurred in the highest number of subjects. Only two AEs were of moderate intensity, all remaining AEs were of mild intensity. No serious AEs were reported. The local tolerability after intravenous administration was good. In summary, intravenously administered BIBN 4096 BS revealed a very favourable safety profile over the dose range tested in both genders. Generally well tolerated at all dose levels, it was of satisfactory tolerability in female subjects at the highest dose of 10 mg. The plasma concentration-time courses of BIBN 4096 BS showed multicompartmental disposition characteristics. Mean maximum concentration (Cmax) values appeared to be dose-proportional. Based on the results from the two high dose levels (5 and 10 mg) with sufficient individual subject data, BIBN 4096 BS exhibited a total plasma clearance (CL) of approximately 12 l/h and an apparent volume of distribution at steady state (Vss) of approximately 20 l, resulting in a terminal half-life (t1/2) of approximately 2.5 h. Inter-individual variability was moderate with a coefficient of variation of approximately 45% based on the area under the plasma concentration-time curve (AUC) values. The mean renal clearance (CLR) was approximately 2 l/h, suggesting that renal excretion plays only a minor role in the elimination of unchanged BIBN 4096 BS.


Subject(s)
Calcitonin Gene-Related Peptide Receptor Antagonists , Piperazines/administration & dosage , Piperazines/adverse effects , Piperazines/pharmacokinetics , Quinazolines/administration & dosage , Quinazolines/adverse effects , Quinazolines/pharmacokinetics , Adult , Area Under Curve , Cardiovascular Physiological Phenomena/drug effects , Female , Half-Life , Humans , Infusions, Intravenous , Male , Metabolic Clearance Rate , Middle Aged
11.
Phys Med Biol ; 45(11): 3367-72, 2000 Nov.
Article in English | MEDLINE | ID: mdl-11098910

ABSTRACT

A modified sector-integration method is presented that can predict the output factors of irregular shaped electron fields even in the case of extended source to surface distance (SSD). The model takes as input measured output factors for circular inserts of various radii. These circular fields were measured at SSDs of 100, 105 and 110 cm to determine the effective source distance as a function of radius (ESD(r)). For an arbitrary electron field at any SSD, the shape is divided into small sectors, and the contribution calculated from the radius and ESD(r). The calculated output factors were verified by direct measurements of various types of electron fields mainly based on clinical use. The energies modelled were 8, 10 and 12 MeV for applicator sizes of 10 cm x 10 cm and 14 cm x 14 cm (defined at 95 cm). The calculated values agreed with the measured data within 1% for the various rectangular cutouts including extended source to surface distance. We retrospectively modelled 97 patient inserts of irregular shape, and found agreement within 2% of measured values.


Subject(s)
Electrons , Radiotherapy, Conformal/instrumentation , Radiotherapy, Conformal/methods , Algorithms , Models, Statistical
12.
Med Phys ; 25(2): 152-60, 1998 Feb.
Article in English | MEDLINE | ID: mdl-9507474

ABSTRACT

We present a method for obtaining the line spread function (LSF) of any radiation detector from measured data. The problem of finding a LSF is essentially a discrete deconvolution from known values of the input (Monte Carlo generated data) and the output (measured data) which can be put into matrix form. We applied the total least squares (TLS) method which is particularly useful when there are errors in both the input and output data. Results from computer simulation as well as from actual data are shown. In a practical application, however, our technique is currently limited by the ability of the Monte Carlo data to simulate correctly the inherent data from the head of the linear accelerator (linac). To overcome this difficulty we have solved by deconvolution and TLS for a more realistic inherent beam profile of our linac using the information from both profile data as measured with film and the film densitometer response function. The LSF of the densitometer was estimated with a simple method of direct measurement of a slit image and a full width at half maximum (FWHM) of 0.997 mm was recorded. Additionally, using the knowledge of this realistic inherent profile of the linac, a blurring function representing the finite source size effect missing in our current Monte Carlo profile simulation was determined. Finally, with the realistic inherent beam profile we have applied the deconvolution and TLS method to find a LSF for the Markus chamber and found a resulting FWHM of 5.39 mm. The TLS approach for deconvolving can find a useful application for both finding the LSF and correcting for the detector size effect once its LSF is known. This type of correction is required when a high spatial resolution is needed (e.g., in small field off-axis measurements). Convolved and measured profiles are also presented to illustrate the effect of the blurring due to different LSFs.


Subject(s)
Radiometry/methods , Biophysical Phenomena , Biophysics , Computer Simulation , Humans , Least-Squares Analysis , Monte Carlo Method , Photons , Radiometry/statistics & numerical data , Radiosurgery , Radiotherapy, High-Energy
13.
Int J Syst Bacteriol ; 43(2): 204-9, 1993 Apr.
Article in English | MEDLINE | ID: mdl-8494735

ABSTRACT

Strains of a new type of slowly growing mycobacterium were repeatedly isolated from sputum from a patient with pulmonary disease. This photochromogenic organism grew at 22, 31, 37, and 41 degrees C, possessed catalase, acid phosphatase, esterase, beta-galactosidase, and arylsulfatase activities, and hydrolyzed Tween. It did not produce nicotinic acid or have nitrate reductase, acetamidase, benzamidase, isonicotinamidase, nicotinamidase, pyrazinamidase, succinidamidase, and acid phosphatase activities. Urease activity was variable. The organism is susceptible to ethambutol and resistant to isoniazid and streptomycin. A mycolic acid analysis revealed the presence of alpha-mycolates, alpha'-mycolates, and keto-mycolates. The results of comparative 16S rRNA sequencing placed this organism at an intermediate position between the rapidly and slowly growing mycobacteria. On the basis of the pattern of enzymatic activities and metabolic properties, the results of fatty acid analyses, and the unique 16S rRNA sequence, we propose that this organism represents a new species, for which we propose the name Mycobacterium intermedium. The type strain is strain 1669/91; a culture of this strain has been deposited in the Deutsche Sammlung von Mikroorganismen und Zellkulturen as strain DSM 44049.


Subject(s)
Mycobacterium/classification , Mycobacterium/isolation & purification , Bacterial Typing Techniques , Base Sequence , Fatty Acids/analysis , Humans , Lung Diseases/microbiology , Molecular Sequence Data , Mycolic Acids/analysis , Phylogeny , Polymerase Chain Reaction , RNA, Ribosomal/genetics , RNA, Ribosomal, 16S/genetics , Sequence Analysis, RNA , Sputum/microbiology
14.
J Clin Microbiol ; 30(11): 2772-5, 1992 Nov.
Article in English | MEDLINE | ID: mdl-1280641

ABSTRACT

A 1.5-kb segment of the DNA that encodes 16S rRNA was amplified by polymerase chain reaction, and 880 nucleotide positions were determined from each of the described biovariants of Mycobacterium fortuitum. Signature sequences which allow rapid identification of M. fortuitum strains at the biovariant level are described. Our data demonstrate a close phylogenetic relationship between Mycobacterium senegalense and M. fortuitum and indicate that the described biovariants of M. fortuitum represent genetically distinct taxa.


Subject(s)
Nontuberculous Mycobacteria/genetics , Base Sequence , DNA, Bacterial/genetics , Genes, Bacterial , Genetic Variation , Genotype , Humans , Molecular Sequence Data , Nontuberculous Mycobacteria/classification , Nontuberculous Mycobacteria/metabolism , RNA, Bacterial/genetics , RNA, Ribosomal, 16S/genetics , Species Specificity
16.
Int J Syst Bacteriol ; 42(3): 337-43, 1992 Jul.
Article in English | MEDLINE | ID: mdl-1380284

ABSTRACT

The 16S rRNAs from nine rapidly growing Mycobacterium species were partially sequenced by using the dideoxynucleotide-terminated, primer extension method with cDNA generated by reverse transcriptase. The sequences were aligned with 47 16S rRNA or DNA sequences that represented 30 previously described and 5 undescribed species of the genus Mycobacterium, and a dendrogram was constructed by using equally weighted distance values. Our results confirmed the phylogenetic separation of the rapidly and slowly growing mycobacteria and showed that the majority of the slowly growing members of the genus represent the most recently evolved organisms. The 24 strains which represented 21 rapidly growing species constituted several sublines, which were defined by the following taxa: (i) Mycobacterium neoaurum and M. diernhoferi, (ii) M. gadium, (iii) the M. chubuense cluster, (iv) the M. fortuitum cluster, (v) M. kommossense, (vi) M. sphagni, (vii) M. fallax and M. chitae, (viii) M. aurum and M. vaccae, (ix) the M. flavescens cluster, and (x) M. chelonae subsp. abscessus. Our phylogenetic analysis confirmed the validity of the phenotypically defined species mentioned above, but our conclusions disagree with most of the conclusions about intrageneric relationships derived from numerical phenetic analyses.


Subject(s)
Mycobacterium/genetics , Nontuberculous Mycobacteria/genetics , Phylogeny , Base Sequence , Molecular Sequence Data , Mycobacterium/classification , Nontuberculous Mycobacteria/classification , RNA, Bacterial/genetics , RNA, Ribosomal, 16S/genetics , Sequence Homology, Nucleic Acid
17.
Nucleic Acids Res ; 20(8): 1843-50, 1992 Apr 25.
Article in English | MEDLINE | ID: mdl-1374559

ABSTRACT

Variable length hairpins in 16S-like rRNA show a predominance for tetra-loops, its degree correlates with the protein content of the ribosome. The number of base-pairs adjacent to the loop (the tip size) and the nearest neighbor composition contribute to the stability of hairpin structures. The average tip size in length variable hairpins correlates with the thermophilicity of the organism, i.e. in temperate environments less stable stem structures are tolerated or even necessary. The most abundant loop families UUCG, GCAA, and CUUG occur most frequently at loop sizes 3, 2, and 7, respectively. Short tips of size less than or equal to 4 generally prefer nearest-neighbor combinations that result in CCC-GGG. Loop-specific tipmost nearest neighbors are revealed at longer tips: CUC(UUCG)GAG, GUA(GCAA)UAC with a maximum at tip sizes 5-6, and GWG(CUUG)CWC. Conserved hairpins, however, prefer variants of the UUCG and GCAA motifs with additional purines. Minor loop families and single motifs such as UUUA, UUUU, CUUGU, UUCGG, and UUU are investigated for preferable tip sizes and nearest-neighbor composition. Specific features are revealed for prominent hexa-loops.


Subject(s)
Nucleic Acid Conformation , RNA, Ribosomal, 16S/chemistry , Bacteria/genetics , Base Sequence , Eukaryotic Cells , Molecular Sequence Data , RNA, Bacterial/chemistry , RNA, Bacterial/genetics , RNA, Ribosomal, 16S/genetics , Ribonucleoproteins/chemistry , Temperature
18.
Int J Syst Bacteriol ; 42(2): 257-62, 1992 Apr.
Article in English | MEDLINE | ID: mdl-1374623

ABSTRACT

A new rapidly growing mycobacterium was isolated from human sputum. This organism grew at 22, 31, 37, and 41 degrees C and possessed catalase, acid phosphatase, acetamidase, urease, nicotinamidase, pyrazinamidase, and nitrate reductase activities. It did not produce nicotinic acid, hydrolyze Tween, or have benzamidase, isonicotinamidase, succinidamidase, and arylsulfatase activities. A mycolic acid analysis revealed a simple, unique pattern. The organism is susceptible to antituberculotic drugs. A comparative 16S rRNA sequence analysis placed this organism within the confines of the genus Mycobacterium, most closely related to the thermotolerant rapidly growing species. On the basis of the pattern of enzymatic activities and metabolic properties, as well as the unique 16S rRNA sequence, we propose that our single strain represents a new species, for which we propose the name Mycobacterium confluentis. The type strain is strain 1389/90; a culture of this strain has been deposited in the German Collection of Microorganisms and Cell Cultures as strain DSM 44017.


Subject(s)
Mycobacterium/classification , Base Sequence , Chromatography, Thin Layer , DNA, Bacterial , Molecular Sequence Data , Mycobacterium/genetics , Phylogeny , RNA, Bacterial/genetics , RNA, Ribosomal, 16S/genetics
19.
Mol Gen Genet ; 231(3): 460-8, 1992 Feb.
Article in English | MEDLINE | ID: mdl-1371580

ABSTRACT

Leaf thionins of several barley cultivars and wild barley species were analysed. We found large differences in the numbers of leaf thionin genes in different Hordeum species. While, for instance, cultivars of Hordeum vulgare (Section Hordeum) contain more than 50 copies of thionin genes per haploid genome, the numbers are much lower in Hordeum species belonging to the sections Critesion and Stenostachys. The apparent number of genes correlates with the concentration of leaf thionin and its mRNA, which differs more than 100-fold among various Hordeum species. Leaf thionins are synthesized as high molecular weight precursor proteins that contain a signal peptide domain, a thionin domain and an acidic polypeptide domain. Analysis of cDNA clones of leaf thionins revealed a family of related transcripts. When the predicted amino acid sequences of the precursor molecules of wild barley species were compared, differences in the sequence variability of the three domains became apparent. The frequency of amino acid exchanges is much higher within the thionin domain than in the signal peptide and acidic polypeptide domains. The amino acid exchanges within the thionin domain do not occur at random but are confined to variable regions that alternate with highly conserved areas. Conserved regions comprise mostly cysteine residues and adjacent amino acids and may be important for the correct formation of the specific disulphide configuration of thionins.


Subject(s)
Hordeum/genetics , Multigene Family , Phylogeny , Plant Proteins/genetics , Amino Acid Sequence , Antimicrobial Cationic Peptides , DNA/genetics , DNA/isolation & purification , Hordeum/physiology , Light , Molecular Sequence Data , Plant Proteins/isolation & purification , Poly A/genetics , Poly A/isolation & purification , RNA/genetics , RNA/isolation & purification , RNA, Messenger , Sequence Homology, Nucleic Acid , Transcription, Genetic/drug effects
20.
Curr Genet ; 21(1): 73-81, 1992 Jan.
Article in English | MEDLINE | ID: mdl-1735127

ABSTRACT

Cryptomonads are thought to have arisen from a symbiotic association between a eukaryotic flagellated host and a eukaryotic algal symbiont, presumably related to red algae. As organellar DNAs have proven to be useful tools in elucidating phylogenetic relationships, the plastid (pt) DNA of the cryptomonad alga Pyrenomonas salina has been characterized in some detail. A restriction map of the circular 127 kb ptDNA from Pyrenomonas salina was established. An inverted repeat (IR) region of about 5 kb separates two single-copy regions of 15 and 102 kb, respectively. It contains the genes for the small and large subunit of rRNA. Ten protein genes, coding for the large subunit of ribulose-1,5-bisphosphate carboxylase, the 47 kDa, 43 kDa and 32 kDa proteins of photosystem II, the ribosomal proteins L2, S7 and S11, the elongation factor Tu, as well as the alpha- and beta-subunits of ATP synthase, have been localized on the restriction map either by hybridization of heterologous gene probes or by sequence homologies. The gene for the plastidal small subunit (SSUr) RNA has been sequenced and compared to homologous SSU regions from the cyanobacterium Anacystis nidulans and plastids from rhodophytes, chromophytes, euglenoids, chlorophytes, and land plants. A phylogenetic tree constructed with the neighborliness method and indicating a relationship of cryptomonad plastids with those of red algae is presented.


Subject(s)
DNA, Circular/genetics , Eukaryota/genetics , Organelles/chemistry , Phylogeny , Base Sequence , Biological Evolution , Cloning, Molecular , DNA, Circular/chemistry , DNA, Circular/isolation & purification , Eukaryota/classification , Genes , Molecular Sequence Data , Operon , RNA, Ribosomal/genetics , Restriction Mapping , Sequence Alignment
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