Fluctuations of pol I and fibrillarin contents of the nucleoli.

Nucleoli are formed on the basis of ribosomal DNA (rDNA) clusters called Nucleolus Organizer Regions (NORs). Each NOR contains multiple genes coding for RNAs of the ribosomal particles. The prominent components of the nucleolar ultrastructure, fibrillar centers (FC) and dense fibrillar components (DFC), together compose FC/DFC units. These units are centers of rDNA transcription by RNA polymerase I (pol I), as well as the early processing events, in which an essential role belongs to fibrillarin. Each FC/DFC unit probably corresponds to a single transcriptionally active gene. In this work, we transfected human-derived cells with GFP-RPA43 (subunit of pol I) and RFP-fibrillarin. Following changes of the fluorescent signals in individual FC/DFC units, we found two kinds of kinetics: 1) the rapid fluctuations with periods of 2-3 min, when the pol I and fibrillarin signals oscillated in anti-phase manner, and the intensities of pol I in the neighboring FC/DFC units did not correlate. 2) fluctuations with periods of 10 to 60 min, in which pol I and fibrillarin signals measured in the same unit did not correlate, but pol I signals in the units belonging to different nucleoli were synchronized. Our data indicate that a complex pulsing activity of transcription as well as early processing is common for ribosomal genes.

Separation of replication and transcription domains in nucleoli.

In mammalian cells, active ribosomal genes produce the 18S, 5.8S and 28S RNAs of ribosomal particles. Transcription levels of these genes are very high throughout interphase, and the cell needs a special strategy to avoid collision of the DNA polymerase and RNA polymerase machineries. To investigate this problem, we measured the correlation of various replication and transcription signals in the nucleoli of HeLa, HT-1080 and NIH 3T3 cells using a specially devised software for analysis of confocal images. Additionally, to follow the relationship between nucleolar replication and transcription in living cells, we produced a stable cell line expressing GFP-RPA43 (subunit of RNA polymerase I, pol I) and RFP-PCNA (the sliding clamp protein) based on human fibrosarcoma HT-1080 cells. We found that replication and transcription signals are more efficiently separated in nucleoli than in the nucleoplasm. In the course of S phase, separation of PCNA and pol I signals gradually increased. During the same period, separation of pol I and incorporated Cy5-dUTP signals decreased. Analysis of single molecule localization microscopy (SMLM) images indicated that transcriptionally active FC/DFC units (i.e. fibrillar centers with adjacent dense fibrillar components) did not incorporate DNA nucleotides. Taken together, our data show that replication of the ribosomal genes is spatially separated from their transcription, and FC/DFC units may provide a structural basis for that separation.

Three-dimensional reconstruction of the S885A mutant of human mitochondrial Lon protease.

The Lon protein is a protease belonging to the superfamily of ATPases associated with diverse cellular activities (AAA+). Its main function is the control of protein quality and the maintenance of proteostasis by degradation of misfolded and damaged proteins, which occur in response to numerous stress conditions. It also participates in the regulation of levels of transcription factors that control pathogenesis, development and stress response. We focus our interest on the structure of human mitochondrial Lon (hLon) protease, whose altered expression levels are linked to some severe diseases such as epilepsy, myopathy, or lateral sclerosis. We present the first 3D structure of the ADP-bound human Lon S885A mutant obtained by electron microscopy as a result of preliminary negative staining studies. S885A appears as a hexameric ring of 120 Å diameter having 90 Å in height. Its resolution was estimated at 19 Å by the FSC = 0.5 criterion. This model is a primary step towards the understanding of the mechanism of action of the Lon protease and its involvement in the pathogenesis development.

Sub-volume averaging of repetitive structural features in angularly filtered electron tomographic reconstructions.

Electron tomographic reconstructions suffer from a number of artefacts arising from effects accompanying the processes of acquisition of a set of tilted projections of the specimen in a transmission electron microscope and from its subsequent computational handling. The most pronounced artefacts usually come from imprecise projection alignment, distortion of specimens during tomogram acquisition and from the presence of a region of missing data in the Fourier space, the "missing wedge". The ray artefacts caused by the presence of the missing wedge can be attenuated by the angular image filter, which attenuates the transition between the data and the missing wedge regions. In this work, we present an analysis of the influence of angular filtering on the resolution of averaged repetitive structural motives extracted from three-dimensional reconstructions of tomograms acquired in the single-axis tilting geometry.

The synthesis and characterization of biotin-silver-dendrimer nanocomposites as novel bioselective labels.

This paper presents a synthesis of a novel nanoparticle label with selective biorecognition properties based on a biotinylated silver-dendrimer nanocomposite (AgDNC). Two types of labels, a biotin-AgDNC (bio-AgDNC) and a biotinylated AgDNC with a poly(ethylene)glycol spacer (bio-PEG-AgDNC), were synthesized from a generation 7 (G7) hydroxyl-terminated ethylenediamine-core-type (2-carbon core) PAMAM dendrimer (DDM) by an N,N'-dicyclohexylcarbodiimide (DDC) biotin coupling and a NaBH(4) silver reduction method. Synthesized conjugates were characterized by several analytical methods, such as UV-vis, FTIR, AFM, TEM, ELISA, HABA assay and SPR. The results show that stable biotinylated nanocomposites can be formed either with internalized silver nanoparticles (AgNPs) in a DMM polymer backbone ('type I') or as externally protected ('type E'), depending on the molar ratio of the silver/DMM conjugate and type of conjugate. Furthermore, the selective biorecognition function of the biotin is not affected by the AgNPs' synthesis step, which allows a potential application of silver nanocomposite conjugates as biospecific labels in various bioanalytical assays, or potentially as fluorescence cell biomarkers. An exploitation of the presented label in the development of electrochemical immunosensors is anticipated.

Positioning of the NOR-bearing chromosomes in relation to nucleoli in daughter cells after mitosis.

It is known that chromosomes occupy non-random positions in the cell nucleus. However, it is not clear to what extent their nuclear positions, together with their neighborhood, are conserved in daughter cells. To address specific aspects of this problem, we used the model of the chromosomes carrying ribosomal genes that are organized in clusters termed Nucleolus Organizer Regions (NORs). We compared the association of chosen NOR-bearing chromosomes (NOR-chromosomes) with nucleoli, as well as the numbers of nucleoli, in the pairs of daughter cells, and established how frequently the daughter cells had equal numbers of the homologs of certain NOR-chromosomes associated with individual nucleoli. The daughter cells typically had different numbers of nucleoli. At the same time, using immuno-FISH with probes for chromosomes 14 and 15 in HeLa cells, we found that the cell pairs with identical combinations appeared significantly more frequently than predicted by the random model. Thus, although the total number of chromosomes associated with nucleoli is variable, our data indicate that the position of the NOR-bearing chromosomes in relation to nucleoli is partly conserved through mitosis.

[Spectrum and dynamics of free amino acids in bovine ocular tissue and their interaction with pilocarpine. IV. The sclera]. / Spektrum a dynamika vol'ných amínokyselín tkanív hovädzieho oka a ich interakcia s pilokarpínom. IV. Sklera.

Two-dimensional ascending chromatographic analysis revealed that a 3% aqueous pilocarpine solution dropped into the conjunctival sac of cattle 2.5 hours after the last instillation shifts in a specific qualitative and quantitative way the levels of different free amino acids in the anterior, equatorial and posterior portion of the sclera. This alkaloid in the anterior and posterior part reduces and in the equatorial part of the sclera increases the total levels of free amino acids. The greatest drop in the anterior portion occurred in leucine + isoleucine methionine + valine and glutamine, in the posterior part threonine and leucine + isoleucine. The greatest rise in the equatorial part was observed in glutamine, leucine + isoleucine and taurine.

[The spectrum and dynamics of free amino acids in the tissues of the bovine eye and their interaction with pilocarpine. III. The lens]. / Spektrum a dynamika vol'ných amínokyselín tkanív hovädzieho oka a ich interakcia s pilokarpínom. III. Sosovka.

The authors revealed by chromatographic analysis that in the investigated tissues of the bovine eye a 3% aqueous pilocarpine solution 2.5 hours after the last instillations alters specifically the levels of different free amino acids. In the capsule and anterior coat an increase was observed and in the nucleus and posterior coat a decline of the total amount of these components. In the control and pilocarpine-treated capsule and in the lenticular nucleus glutamic acid, is present in the highest concentration and in the anterior and posterior coat of the lens aspartic acid and lysine.

[The spectrum and dynamics of free amino acids in bovine ocular tissue and their interactions with pilocarpine. II. The aqueous humor, choroid epithelium and the choroid]. / Spektrum a dynamika vol'ných amínokyselín tkanív hovädzieho oka a ich interakcia s pilokarpínom. II. Komorová voda, epitel vráskovca a vráskovec.

The authors revealed by two-dimensional ascendent chromatography on paper and thin-layer chromatography that a 3% aqueous solution of pilocarpine acts specifically on the free amino acids in the choroid, in the choroid epithelium and the aqueous humour of the bovine eye and that it reduces the total free amino acid level more in the choroid epithelium than in the aqueous humour. Specific changes of free amino acid levels caused by pilocarpine indicate the routes of transport, distribution and metabolic changes of pilocarpine and free amino acids. Based on these changes it is possible to evaluate also the physiological and morphological condition of ocular tissues and the organ of vision resp.

[The action of instilled physostigmine on the acetylcholine content of the bovine ciliary body (author's transl)]. / Action de la physostigmine instillée sur le taux d'acétylcholine du corps ciliaire des bovins.

The authors have compared the content of acetylcholine (ACh) of the two ciliary bodies (150 microgram of tissue) after the instillation of a stimulating dose of a 1% aqueous solution of physostigmine (10 applications of three drops at intervals of three minutes) into the conjunctival fornix of one eye. Kymographic measurements on guinea-pig ileum demonstrated an average elevation of the ACh content in the instilled ciliary bodies (0.48 microgram) against the controls (0.33 micron). The eyes were enucleated one hour after the last application.

[Action of physostigmine on the acetylcholine content of the bovine choroid (author's transl)]. / Action de la physostigmine sur le taux d'acétylcholine dans la choroïde des bovins.

The measurement of the acetylcholine (ACh) content of 12 choroids influenced by a 1% solution of physostigmine salicylate (10 applications of three drops at three minute intervals) into the conjunctival fornix of one eye demonstrated elevated average values for ACh content (0.87 microgram) compared with those for the control eye (0.75 microgram). The eyes were enucleated one hour after the last application.

[The action of instilled physostigmine on the acetylcholine content of the bovine retina]. / Action de la physostigmine instillée sur le taux d'acétylcholine dans la rétine des bovins.

Kymographic measurements of acetylcholine (ACh) in bovine retinal tissue carried out on guinea-pig ileum do not indicate a significant elevated concentration of ACh in the eye instilled with physostingmine salicylate (1% aqueous solution, 10 applications of three drops at intervals of 3 minutes). The eyes were enucleated one hour after the last application.