Topographical mapping of ribosomal RNAs in situ by electron spectroscopic imaging

1984 ◽  
Vol 42 ◽  
pp. 690-691
Author(s):  
M. Boublik ◽  
G.T. Oostergetel ◽  
B. Frankland ◽  
F.P. Ottensmeyer
Keyword(s):  
Spectroscopic Techniques ◽  
Rna Molecules ◽  
Macromolecular System ◽  
Unique Distribution ◽  
Topographical Mapping ◽  
The Individual ◽  
And Function ◽  
Electron Energy Loss

Visualization of the in situ location of the individual components of any macromolecular system is important for understanding its assembly, interactions, and function. Ribosomes, which are small cellular organelles involved in protein synthesis are high molecular weight nucleoprotein complexes composed only of proteins and RNAs. This “simple” composition of ribosomes enables us topographical studies directed either towards localization of the individual ribosomal protein and RNA molecules or merely to the determination of the distribution of the protein and RNA moieties within the ribosome and its subunits. We have utilized the recent progress in the development of microanalytical electron spectroscopic techniques, electron energy loss spectroscopy (EELS) in particular, and the unique distribution of the phosphorus atoms on the ribosome (the phosphorus atoms are present only in the structural backbone of the rRNA) for the direct tracing of the RNA molecules in situ.

scholarly journals The Anomalous Influence of Polyelectrolyte Concentration on the Deposition and Nanostructure of Poly(ethyleneimine)/Poly(acrylic acid) Multilayers

Molecules ◽  
2019 ◽  
Vol 24 (11) ◽  
pp. 2141 ◽  
Author(s):  
Martin Müller
Keyword(s):  
Acrylic Acid ◽  
Attenuated Total Reflection ◽  
Silicon Substrates ◽  
Acid Base ◽  
Exponential Relationship ◽  
Individual Adsorption ◽  
The Individual ◽  
Poly Acrylic Acid

The deposition and nanostructure of polyelectrolyte (PEL) multilayers (PEMs) of branched poly(ethyleneimine)/poly(acrylic acid) (PEI/PAA) onto silicon substrates was studied in terms of the dependence of pH and the PEL concentration (cPEL) in the individual adsorption steps z. Both a commercial automatic dipping device and a homebuilt automatic stream coating device (flow cell) were used. Gravimetry, SFM, transmission (TRANS) and in situ attenuated total reflection (ATR) FTIR spectroscopy were used for the quantitative determination of the adsorbed amount, thickness, chemical composition and morphology of deposited PEMs, respectively. Firstly, the combination of pH = 10 for PEI and pH = 4 for PAA, where both PEL were predominantly in the neutral state, resulted in an extraordinarily high PEM deposition, while pH combinations, where one PEL component was charged, resulted in a significantly lower PEM deposition. This was attributed to both PEL conformation effects and acid/base interactions between basic PEI and acidic PAA. Secondly, for that pH combination an exponential relationship between PEM thickness and adsorption step z was found. Thirdly, based on the results of three independent methods, the course of the deposited amount of a PEM-10 (z = 10) versus cPEL in the range 0.001 to 0.015 M at pH = 10/4 was non-monotonous showing a pronounced maximum at cPEL = 0.005 M. Analogously, for cPEL = 0.005 M a maximum of roughness and structure size was found. Fourthly, related to that finding, in situ ATR-FTIR measurements gave evidence for the release of outermost located PEI upon PAA immersion (even step) and of outermost PAA upon PEI immersion (odd step) under formation of PEL complexes in solution. These studies help us to prepare PEL-based films with a defined thickness and morphology for interaction with biofluids in the biomedical and food fields.

scholarly journals Intracellular RNA-tracking methods

Open Biology ◽  
2018 ◽  
Vol 8 (10) ◽  
pp. 180104 ◽  
Author(s):  
Logan George ◽  
Fred E. Indig ◽  
Kotb Abdelmohsen ◽  
Myriam Gorospe
Keyword(s):  
Molecular Beacons ◽  
Live Cells ◽  
Coat Proteins ◽  
Rna Molecules ◽  
Chimeric Transcripts ◽  
Spatio Temporal ◽  
Dynamics And Function ◽  
And Function ◽  
Complementary Rnas

RNA tracking allows researchers to visualize RNA molecules in cells and tissues, providing important spatio-temporal information regarding RNA dynamics and function. Methods such as fluorescent in situ hybridization (FISH) and molecular beacons rely on complementary oligonucleotides to label and view endogenous transcripts. Other methods create artificial chimeric transcripts coupled with bacteriophage-derived coat proteins (e.g. MS2, λN) to tag molecules in live cells. In other approaches, endogenous RNAs are recognized by complementary RNAs complexed with noncatalytic Cas proteins. Each technique has its own set of strengths and limitations that must be considered when planning an experiment. Here, we discuss the mechanisms, advantages, and weaknesses of in situ hybridization, molecular beacons, MS2 tagging and Cas-derived systems, as well as how RNA tracking can be employed to study various aspects of molecular biology.

scholarly journals The Isotope Array, a New Tool That Employs Substrate-Mediated Labeling of rRNA for Determination of Microbial Community Structure and Function

2003 ◽  
Vol 69 (11) ◽  
pp. 6875-6887 ◽  
Author(s):  
Justyna Adamczyk ◽  
Martin Hesselsoe ◽  
Niels Iversen ◽  
Matthias Horn ◽  
Angelika Lehner ◽  
...  
Keyword(s):  
Community Structure ◽  
Activated Sludge ◽  
Fluorescence Labeling ◽  
Specific Substrate ◽  
Ammonia Oxidizing Bacteria ◽  
Microarray Hybridization ◽  
Independent Manner ◽  
And Function

ABSTRACT A new microarray method, the isotope array approach, for identifying microorganisms which consume a 14C-labeled substrate within complex microbial communities was developed. Experiments were performed with a small microarray consisting of oligonucleotide probes targeting the 16S rRNA of ammonia-oxidizing bacteria (AOB). Total RNA was extracted from a pure culture of Nitrosomonas eutropha grown in the presence of [14C]bicarbonate. After fluorescence labeling of the RNA and microarray hybridization, scanning of all probe spots for fluorescence and radioactivity revealed that specific signals were obtained and that the incorporation of 14C into rRNA could be detected unambiguously. Subsequently, we were able to demonstrate the suitability of the isotope array approach for monitoring community composition and CO2 fixation activity of AOB in two nitrifying activated-sludge samples which were incubated with [14C]bicarbonate for up to 26 h. AOB community structure in the activated-sludge samples, as predicted by the microarray hybridization pattern, was confirmed by quantitative fluorescence in situ hybridization (FISH) and comparative amoA sequence analyses. CO2 fixation activities of the AOB populations within the complex activated-sludge communities were detectable on the microarray by 14C incorporation and were confirmed independently by combining FISH and microautoradiography. AOB rRNA from activated sludge incubated with radioactive bicarbonate in the presence of allylthiourea as an inhibitor of AOB activity showed no incorporation of 14C and thus was not detectable on the radioactivity scans of the microarray. These results suggest that the isotope array can be used in a PCR-independent manner to exploit the high parallelism and discriminatory power of microarrays for the direct identification of microorganisms which consume a specific substrate in the environment.

Nonrandom distribution of long mono- and dinucleotide repeats in Drosophila chromosomes: correlations with dosage compensation, heterochromatin, and recombination

1989 ◽  
Vol 9 (3) ◽  
pp. 1173-1182
Author(s):  
K Lowenhaupt ◽  
A Rich ◽  
M L Pardue
Keyword(s):  
Y Chromosome ◽  
Dna Sequences ◽  
Polytene Chromosomes ◽  
Drosophila Species ◽  
Repeated Dna ◽  
Dinucleotide Repeats ◽  
Order Of Magnitude ◽  
The Individual ◽  
And Function

Long stretches of (dC-dA)n.(dT-dG)n, abbreviated CA/TG, have a distinctive distribution on Drosophila chromosomes (M.L. Pardue, K. Lowenhaupt, A. Rich, and A. Nordheim, EMBO J. 6:1781-1789, 1987). The distribution of CA/TG suggests a correlation with the overall transcriptional activity of chromosomal regions and with the ability to undergo meiotic recombination. These correlations are conserved among Drosophila species and may indicate one or more chromosomal functions. To test the generality of these findings, we analyzed the distribution of the rest of the six possible mono- and dinucleotide repeats (A/T, C/G, AT/AT, CA/TG, CT/AG, and CG/CG). All but CG/CG were present at significant levels in the genomes of the six Drosophila species studied; however, A/T levels were an order of magnitude lower than those of the other sequences. Data base analyses suggested that the same sequences are present in other eucaryotes. Like CA/TG, both CT/AG and C/G showed increased levels on dosage-compensating chromosomes; however, the individual sites clearly differed for each sequence. In contrast, A/T and AT/AT, although present in Drosophila DNA, could not be detected in situ in polytene chromosomes. We also used in situ hybridization to analyze the neo-Y chromosome of Drosophila miranda, an ancestral autosome that has become attached to the Y chromosome and is now partially heterochromatic. The neo-Y has acquired repeated DNA sequences; we found that the added sequences are as devoid of mono- and dinucleotide repeats as other heterochromatin. The distribution and function of these sequences are likely to result from both their repetitious nature and base contents.

scholarly journals Quantum magnetic imaging of iron organelles within the pigeon cochlea

2021 ◽  
Vol 118 (47) ◽  
pp. e2112749118
Author(s):  
Robert W. de Gille ◽  
Julia M. McCoey ◽  
Liam T. Hall ◽  
Jean-Philippe Tetienne ◽  
E. Pascal Malkemper ◽  
...  
Keyword(s):  
Magnetic Susceptibility ◽  
Magnetic Particle ◽  
Imaging Techniques ◽  
Iron Storage ◽  
Magnetic Imaging ◽  
Geomagnetic Fields ◽  
The Individual ◽  
Individual Particles

The ability of pigeons to sense geomagnetic fields has been conclusively established despite a notable lack of determination of the underlying biophysical mechanisms. Quasi-spherical iron organelles previously termed “cuticulosomes” in the cochlea of pigeons have potential relevance to magnetoreception due to their location and iron composition; however, data regarding the magnetic susceptibility of these structures are currently limited. Here quantum magnetic imaging techniques are applied to characterize the magnetic properties of individual iron cuticulosomes in situ. The stray magnetic fields emanating from cuticulosomes are mapped and compared to a detailed analytical model to provide an estimate of the magnetic susceptibility of the individual particles. The images reveal the presence of superparamagnetic and ferrimagnetic domains within individual cuticulosomes and magnetic susceptibilities within the range 0.029 to 0.22. These results provide insights into the elusive physiological roles of cuticulosomes. The susceptibilities measured are not consistent with a torque-based model of magnetoreception, placing iron storage and stereocilia stabilization as the two leading putative cuticulosome functions. This work establishes quantum magnetic imaging as an important tool to complement the existing array of techniques used to screen for potential magnetic particle–based magnetoreceptor candidates.

scholarly journals Czy Konstytucja Rzeczypospolitej Polskiej wymusza na ustawodawcy ustawową regulację struktury organizacyjnej i właściwości sądów powszechnych?

2020 ◽  
pp. 119-135
Author(s):  
Bogusław Banaszak
Keyword(s):  
Public Authorities ◽  
The Supreme Court ◽  
Constitutional Tribunal ◽  
Court Proceedings ◽  
The Republic ◽  
The Individual ◽  
The Right ◽  
And Function ◽  
Statutory Regulation

The Constitution of the Republic of Poland does not define in a comprehensive manner the matter to be standardized in laws and grants the legislator considerable freedom in determining their content. In view of the subject matter of this opinion, based on views founded on the doctrine of Polish constitutional law and on the jurisprudence of the Constitutional Tribunal and the Supreme Court, it can be concluded that the legislator should regulate by law, among other matters, the following matters reserved for statutory regulation (the principle of exclusivity of the act): regulations concerning citizenship and the rights, freedoms and duties of the individual, as well as the basic competences, principles of how public authorities are organised and function. On the basis of this assumption, it was examined whether there is any indication to the legislator from the constitutional norms relevant to the judicial authority. The following were taken into account: the right to a fair trial (Article 45), the principle of the separateness and independence of the judiciary (Article 173), the principle of bi-instantiality of court proceedings and the principle of statutory determination of the system and jurisdiction of courts (Article 176), the principle of the presumption of competence being assigned to common courts (Article 177), and the principle of the non-removability and non-transferability of judges (Article 180). On the basis of an analysis of these constitutional norms, it was concluded that the Constitution of the Republic of Poland enforces statutory regulation of the organisational structure and the material, local and appeal jurisdiction of common courts, and only allows for entrusting specific matters to be regulated by executive bodies by means of a regulation. In the event of any doubts as to whether a given case should be classified into the category of specific matters, the principle of exclusivity of the Act applies.

scholarly journals Activation of protein kinase C isozymes is associated with post-mitotic events in intestinal epithelial cells in situ.

1994 ◽  
Vol 126 (3) ◽  
pp. 747-763 ◽  
Author(s):  
M L Saxon ◽  
X Zhao ◽  
J D Black
Keyword(s):  
Epithelial Cells ◽  
Intestinal Epithelial Cells ◽  
Intestinal Epithelial ◽  
Kinase C ◽  
Pkc Isozymes ◽  
Pkc Alpha ◽  
The Individual ◽  
And Function ◽  
Activation Status

The mechanisms underlying control of cell growth and differentiation in epithelial tissues are poorly understood. Protein kinase C (PKC) isozymes, members of a large family of serine/threonine kinases of fundamental importance in signal transduction, have been increasingly implicated in the regulation of cell growth, differentiation, and function. Using the rat intestinal epithelium as a model system, we have examined PKC-specific activity as well as individual PKC isozyme expression and distribution (i.e., activation status) in epithelial cells in situ. Increased PKC activity was detected in differentiating and functional cells relative to immature proliferating crypt cells. Immunofluorescence and Western blot analysis using a panel of isozyme-specific antibodies revealed that PKC alpha, beta II, delta, epsilon, and zeta are expressed in rat intestinal epithelial cells and exhibit distinct subcellular distribution patterns along the crypt-villus unit. The combined morphological and biochemical approach used permitted analysis of the activation status of specific PKC isozymes at the individual cell level. These studies showed that marked changes in membrane association and level of expression for PKC alpha, beta II, delta, and zeta occur as cells cease division in the mid-crypt region and begin differentiation. Additional changes in PKC activation status are observed with acquisition of mature function on the villus. These studies clearly demonstrate naturally occurring alterations in PKC isozyme activation status at the individual cell level within the context of a developing tissue. Direct activation of PKC in an immature intestinal crypt cell line was shown to result in growth inhibition and coincident translocation of PKC alpha from the cytosolic to the particulate subcellular fraction, paralleling observations made in situ and providing further support for a role of intestinal PKC isozymes in post-mitotic events. PKC isozymes were also found to be tightly associated with cytoskeletal elements, suggesting participation in control of the structural organization of the enterocyte. Taken together, the results presented strongly suggest an involvement of PKC isoforms in cellular processes related to growth cessation, differentiation, and function of intestinal epithelial cells in situ.

Bolt assessment of wind turbine hub using nonlinear ultrasound methods

2019 ◽  
pp. 0309524X1988773
Author(s):  
Gian Piero Malfense Fierro ◽  
Michele Meo
Keyword(s):  
Large Scale ◽  
Selection Process ◽  
Detection Accuracy ◽  
Nonlinear Ultrasound ◽  
Frequency Selection ◽  
Modal Response ◽  
The Individual ◽  
Response Method

This work evaluates various nonlinear ultrasound methods for in situ structural health monitoring of the loosened state of a four-bolt structure found on large-scale wind turbines. The aim was assessment of a four bolted structure with only two piezoelectric sensors, and determination of individual bolt loosened and the extent of loosening. Nonlinear ultrasound methods have been shown to have advantages over linear methods in terms of sensitivity, although the detection accuracy and robustness of these methods can be highly dependent on correct frequency selection. Thus, a frequency selection process based on the modal response of the structure is suggested for determination of bolt-specific frequencies, which was then used to evaluate the individual bolt loosened state. Two nonlinear ultrasound techniques were used to evaluate the bolted structure: the second- and third-order nonlinearity parameters and a nonlinear acoustic moment’s method. The modal response method used for frequency selection was able to determine specific bolt frequencies based on surface and bolt velocities. Nonlinear evaluation at these frequencies showed that specific frequencies related to individual bolts, and as the bolts loosened there was a clear increase in the production of nonlinearities. Thus, the loosened status of individual bolts could be tracked using specific pre-identified frequencies.

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