Ribonucleic acid transfer from nucleus to cytoplasm during interphase and mitosis in mouse somatic cells cultured in vitro

Summary Kidney cells from primary cultures of 15-day old mouse embryos were incubated for 2, 5 or 10 min with H³-uridine, then either fixed immediately or incubated again for various periods in a chase medium containing an excess of unlabeled uridine and cytidine. The number of grains over the non-nucleolar part of the nucleus (chromatin), the nucleolus and the cytoplasm were counted on the autoradiograms.The grain count showed that both chromatin and nucleolus incorporate very rapidly H³-uridine from the medium, whereas a time lag elapses before any H³-radioactivity above background is detected in the cytoplasm. Incorporation of H³-uridine into the RNA of the nucleus and the nucleolus is not immediately blocked after chase, suggesting that the labeled precursor pool is not completely washed out from the living cell, or diluted by the excess of unlabeled uridine present in the medium. The grain count over the nucleus and the nucleolus rises for a certain time after chase and then gradually declines; H³-radioactivity appears in the cytoplasm 10 min after chase and keeps rising through a 110-min interval. The experiment, then — even though it suggests that the bulk of cellular RNA is synthesized in the chromatin and the nucleolus and then continuously released into the cytoplasm — does not rule out the possibility that some RNA fraction, characterized by a low turnover rate, is synthesized independently in the cytoplasm.Synthesis of RNA is a continuous process throughout the cell cycle, except during metaphase and anaphase. It ceases at prometaphase after the disappearance of the nucleolus and disintegration of the nuclear membrane, and resumes in early telophase. Part of the chromosomal RNA does not remain associated with the chromosomes through division, but is suddenly released into the cytoplasm when the cell enters metaphase.     

A narrow hybrid zone between two crayfish species from a Mexican cave

In Northern Chiapas (Mexico), two newly discovered species of Procambarus crayfish inhabit a subterranean stream. These species can be morphologically distinguished only by comparing extreme phenotypes (dark, thick-eyed, surface dwelling-like individuals vs light, elongated, microphtalmic, cave dwelling-like individuals). Individuals with intermediate phenotypes co-occur with those exhibiting extreme phenotypes. Crayfish were assayed electrophoretically and individual patterns at 23 gene loci were obtained. Unusually high levels of heterozygosity in both species and a clear discrimination between the two gene pools were revealed. The relationships between individuals were investigated by means of multivariate analysis on individual multilocus genotype profiles. Results showed the occurrence of individuals genetically intermediate between the two major clusters, which shared allozymic variants with both species. Due to the occurrence of alternative alleles in the two gene pools, we could quantify patterns of introgression, which revealed asymmetric gene flow between the two species. Moreover, differential levels of introgression in subsamples within the surface-like species were found: most introgressed individuals came from the inner section of the cave, where the two species were greatly mixed. These results are also discussed in reference to the morphometric results from a companion paper. A possible evolutionary pathway, leading to the situation in this cave, and possibly in neighbouring cave systems, is outlined. The hypothesis of a past history of allopatric divergence from a common ancestor and a subsequent secondary contact between these two Procambarus species is supported by geological studies. Crayfish sympatry and competitive exclusion are also discussed.     

24S-hydroxycholesterol in plasma: A marker of cholesterol turnover in neurodegenerative diseases

Brain cholesterol is mainly involved in the cell membrane structure, in signal transduction, neurotransmitter release, synaptogenesis and membrane trafficking. Impairment of brain cholesterol metabolism was described in neurodegenerative diseases, such as Multiple Sclerosis, Alzheimer and Huntington Diseases. Since the blood–brain barrier efficiently prevents cholesterol uptake from the circulation into the brain, de novo synthesis is responsible for almost all cholesterol present there. Cholesterol is converted into 24S-hydroxycholesterol (24OHC) by cholesterol 24-hydroxylase (CYP46A1) expressed in neural cells.     

Metabolic Profiling of Alternative NAD Biosynthetic Routes in Mouse Tissues

NAD plays essential redox and non-redox roles in cell biology. In mammals, its de novo and recycling biosynthetic pathways encompass two independent branches, the “amidated” and “deamidated” routes. Here we focused on the indispensable enzymes gating these two routes, i.e. nicotinamide mononucleotide adenylyltransferase (NMNAT), which in mammals comprises three distinct isozymes, and NAD synthetase (NADS). First, we measured the in vitro activity of the enzymes, and the levels of all their substrates and products in a number of tissues from the C57BL/6 mouse. Second, from these data, we derived in vivo estimates of enzymes''rates and quantitative contributions to NAD homeostasis. The NMNAT activity, mainly represented by nuclear NMNAT1, appears to be high and nonrate-limiting in all examined tissues, except in blood. The NADS activity, however, appears rate-limiting in lung and skeletal muscle, where its undetectable levels parallel a relative accumulation of the enzyme''s substrate NaAD (nicotinic acid adenine dinucleotide). In all tissues, the amidated NAD route was predominant, displaying highest rates in liver and kidney, and lowest in blood. In contrast, the minor deamidated route showed higher relative proportions in blood and small intestine, and higher absolute values in liver and small intestine. Such results provide the first comprehensive picture of the balance of the two alternative NAD biosynthetic routes in different mammalian tissues under physiological conditions. This fills a gap in the current knowledge of NAD biosynthesis, and provides a crucial information for the study of NAD metabolism and its role in disease.     

Macular Microcysts in Mitochondrial Optic Neuropathies: Prevalence and Retinal Layer Thickness Measurements

Purpose

To investigate the thickness of the retinal layers and to assess the prevalence of macular microcysts (MM) in the inner nuclear layer (INL) of patients with mitochondrial optic neuropathies (MON).

Methods

All patients with molecularly confirmed MON, i.e. Leber’s Hereditary Optic Neuropathy (LHON) and Dominant Optic Atrophy (DOA), referred between 2010 and 2012 were enrolled. Eight patients with MM were compared with two control groups: MON patients without MM matched by age, peripapillary retinal nerve fiber layer (RNFL) thickness, and visual acuity, as well as age-matched controls. Retinal segmentation was performed using specific Optical coherence tomography (OCT) software (Carl Zeiss Meditec). Macular segmentation thickness values of the three groups were compared by one-way analysis of variance with Bonferroni post hoc corrections.

Results

MM were identified in 5/90 (5.6%) patients with LHON and 3/58 (5.2%) with DOA. The INL was thicker in patients with MON compared to controls regardless of the presence of MM [133.1±7μm vs 122.3±9μm in MM patients (p<0.01) and 128.5±8μm vs. 122.3±9μm in no-MM patients (p<0.05)], however the outer nuclear layer (ONL) was thicker in patients with MM (101.4±1mμ) compared to patients without MM [77.5±8mμ (p<0.001)] and controls [78.4±7mμ (p<0.001)]. ONL thickness did not significantly differ between patients without MM and controls.

Conclusion

The prevalence of MM in MON is low (5-6%), but associated with ONL thickening. We speculate that in MON patients with MM, vitreo-retinal traction contributes to the thickening of ONL as well as to the production of cystic spaces.