Neuroglobin in Neurons of the Frontal and Parietal Cortex of White Rats in Cholestasis

The aim of the study was to investigate the distribution and content of neuroglobin in the pyramidal neurons of the frontal and parietal cortex of white rats during simulated cholestasis.Material and methods. The study included 60 outbred white male rats weighed 200–250 g. Cholestasis was simulated by ligation of the common bile duct in the porta hepatis area (main group, n=30). Animals of the control group (n=30) were performed a false operation preserving physiological bile outflow. Sections of the frontal and parietal cortex of white rats were selected for investigation. The content and distribution of neuroglobin was detected immunohistochemically on paraffin sections using mouse monoclonal primary antibodies Anti- Neuroglobin antibody (Abcam). Cytophotometric analysis was used to perform quantitative assessment of the content of the studied molecular marker; after that statistical analysis was performed.Results. It was found that the content of neuroglobin in the pericarions of neurons of the frontal and parietal cortex modified in a wave-like mode. After ligation of the common bile duct, the content of neuroglobin significantly decreased in 2, 10 and 45 days (minimum on the 10th day) in the frontal cortex and in 90 days in the parietal cortex, and increased in 5 and 20 days of the experiment (maximum on the 20th day).Conclusion. In cholestasis, the content of neuroglobin in the pericarions of neurons of the frontal and parietal cortex modifies in waves: a decrease is observed on the 2nd, 10th, 45th day; an increase is observed on the 5th and 20th day; a recovery is observed on the 90th day.

Heterochromatin organization in metaphase chromosomes and interphase nuclei of Dasypyrum breviaristatum (Lindb) Frederiksen

Metaphase chromosomes of <em>Dasypyrum breviaristatum</em> (Lindb f) Frederiksen, a tetraploid wild species, were differentially stained with C-banding and fluorochromes in order to aquire information on heterochromatin chromosomal distribution and composition. DNA content and relative amount of nuclear heterochromatin were determined by cytophotometric analysis after Feulgen reaction. The results were compared to those of <em>Dasypyrum villosum</em> (L.) P. Candargy, a diploid species of the same genus. The achieved information indicate that <em>D. breviaristatum</em> and <em>D. villosum</em> differ in the composition, organization and distribution of heterochromatin, and may suggest that the telomeric regions of the chromosomes of the two species are more differentiated than the centromeric ones, as a result of a long lasting divergence between the two species.

Genome size variation in Pisum sativum

Pisum sativum L. is one of the plant species where infraspecific genome size variation, up to 1.29-fold between cultivars, has been reported. The present investigation deals with a Feulgen cytophotometric analysis of this phenomenon in 25 wild accessions, landraces, and cultivars of widely different geographic origin. Differences between accessions were maximally 1.054-fold in single experiments but proved to be nonreproducible upon repeated measurements. Seedlings of the same accession often differed significantly, up to 1.056-fold, but values from root and shoot tips in one individual were not significantly correlated, indicating the absence of true genome size variation between plants. Upon calibration against Allium cepa a 1C value of 4.42 pg is estimated for Pisum sativum. Altogether the data suggest that, contrary to the divergence in the literature data and recent reports on DNA content variation, the pea has a stable genome size.Key words: Pisum sativum L., genome size variation, Feulgen cytophotometry.

Characterization of factors in routine laboratory protocols that significantly influence the Feulgen reaction.

We investigated the parameters that could affect the cytophotometric analysis of cell nuclei stained by the Feulgen reaction. These parameters included: the hydrolysis temperature (in the normal "room temperature" range); the composition of the Schiff's reagent; the speed of centrifugation of the cell suspensions; the mode of preservation [air-drying or ethanol-formalin-acetic acid (EFA) fixation]; the fixation time; the pronase digestion time; and the concentration of pronase used to obtain cell suspensions from archival (formalin-fixed, paraffin-embedded) materials. Relatively homogeneous material was studied: the MXT mouse mammary adenocarcinoma growing in vivo as tumors with both small and hyperchromatic cell nuclei and in vitro as monolayers with larger and less hyperchromatic cell nuclei. The results of these investigations demonstrate the necessity for the precise definition of a protocol for such procedures as sampling, fixation, and staining of cell nuclei if computerized cell image analyses are to be objective and reproducible. For present purposes this protocol differs depending on whether fresh or archival material is studied. For fresh tissue the protocol is immersion of the sample in EFA within 10 sec, fixation for 30 min, and staining by the Feulgen reaction in which hydrolysis is performed with 6 N HCl at 24 degrees C for 60 min. For archival tissue, the protocol becomes fixation in formol (or EFA), embedding, sectioning at 80 microns, digestion with 0.05% pronase for 2 hr, centrifugation at 1200 x g on glass slides, and staining by the Feulgen reaction in which hydrolysis is performed with 6 N HCl for 60 min at 24 degrees C.