Association of poly(adenylate)-deficient messenger ribonucleic acid with membranes in mouse kidney

To describe further the metabolism of messenger ribonucleic acid (mRNA) in mouse kidney, we examined newly synthesized mRNA deficient in poly(adenylate) [poly(A)]. Approximately 50% of renal polysomal mRNA that labeled selectively in the presence of the pyrimidine analogue 5-fluoroorotic acid lacks or is deficient in poly(A) as defined by its ability to bind to poly(A) affinity columns. Nearly one-half of this poly(A)-deficient mRNA is associated uniquely with a cellular membrane fraction detected by sedimentation of renal cytoplasm in sucrose density gradients containing EDTA and nonionic detergents. Poly(A+) mRNA and poly(A)-deficient mRNA [poly(A-) mRNA] have similar modal sedimentation coefficients (20-22 S) and similar cytoplasmic distribution. Although 95% of newly synthesized poly(A+) mRNA is released in 10 mM EDTA as 20-90 S ribonucleoproteins from polysomes greater than 80 S, only 55% of poly(A)-deficient mRNA is released under the same conditions. Poly(A)-deficient mRNA recovered from greater than 80 S ribonucleoproteins resistant to EDTA treatment lacks ribosomal RNA, is similar in size to poly(A+) mRNA, and is associated with membranous structures, since 70% of poly(A)-deficient mRNA in EDTA-resistant ribonucleoproteins is released into the 20-80 S region by solubilizing membranes with 1% Triton X-100. These membrane-associated renal poly(A-) mRNAs could have unique coding or regulatory functions.     

Physical aspects and cytoplasmic distribution of messenger RNA in mouse kidney.

As a prerequisite to examining mRNA metabolism in compensatory renal hypertrophy, polyadenylated RNA has been purified from normal mouse kidney polysomal RNA by selection on oligo(dT)-cellulose. Poly(A)-containing RNA dissociated from polysomes by treatment with 10 mM EDTA and sedimented heterogeneously in dodecyl sulfate-containing sucrose density gradients with a mean sedimentation coefficient of 20 S. Poly(A) derived from this RNA migrated at the rate of 6-7 S RNA in dodecyl sulfate-containing 10% polyacrylamide gels. Coelectrophoresis of poly(A) labeled for 90 min with poly(A) labeled for 24 h indicated the long-term labeled poly(A) migrated faster than pulse-labeled material. Twenty percent of the cytoplasmic poly(A)-containing mRNA was not associated with the polysomes, but sedimented in the 40-80 S region (post-polysomal). Messenger RNA from the post-polysomal region had sedimentation properties similar to those of mRNA prepared from polysomes indicating post-polysomal mRNA was not degraded polysomal mRNA. Preliminary labeling experiments indicated a rapid equilibration of radioactivity between the polysomal and post-polysomal mRNA populations, suggesting the post-polysomal mRNA may consist of mRNA in transit to the polysomes.     

Messenger ribonucleoprotein complexes isolated with oligo(dT)-cellulose chromatography from kidney polysomes

As an initial step towards understanding the role of mRNP complexes in translational regulation during compensatory renal hypertrophy, characteristics of polysome-associated mRNP isolated by affinity chromatography were studied. Renal mRNP contained 15–30% of the counts after a 1 hr pulse with ³H-orotic acid; it sedimented mainly between 10S and 100S and had a buoyant density of 1.42–1.44 g/cm³. RNA derived from the mRNP sedimented between 5S and 40S on sucrose density gradients, with the greatest radioactivity in the region of 15S. After labeling with ³H-adenine for 1 hr, up to 17% of the radioactivity present in the mRNP-associated RNA was resistant to digestion by pancreatic and T1 ribonucleases. The mRNP protein moiety contained six polypeptides with molecular weights 69,000, 75,000, 80,000, 100,000, 109,000, and 118,000 daltons, which were undetected in the material not binding to oligo(dT)-cellulose.     

Widespread Changes in White Matter Microstructure after a Day of Waking and Sleep Deprivation

BackgroundElucidating the neurobiological effects of sleep and waking remains an important goal of the neurosciences. Recently, animal studies indicated that sleep is important for cell membrane and myelin maintenance in the brain and that these structures are particularly susceptible to insufficient sleep. Here, we tested the hypothesis that a day of waking and sleep deprivation would be associated with changes in diffusion tensor imaging (DTI) indices of white matter microstructure sensitive to axonal membrane and myelin alterations.MethodsTwenty-one healthy adult males underwent DTI in the morning [7:30AM; time point (TP)1], after 14 hours of waking (TP2), and then after another 9 hours of waking (TP3). Whole brain voxel-wise analysis was performed with tract based spatial statistics.ResultsA day of waking was associated with widespread increases in white matter fractional anisotropy, which were mainly driven by radial diffusivity reductions, and sleep deprivation was associated with widespread fractional anisotropy decreases, which were mainly explained by reductions in axial diffusivity. In addition, larger decreases in axial diffusivity after sleep deprivation were associated with greater sleepiness. All DTI changes remained significant after adjusting for hydration measures.ConclusionsThis is the first DTI study of sleep deprivation in humans. Although previous studies have observed localized changes in DTI indices of cerebral microstructure over the course of a few hours, further studies are needed to confirm widespread DTI changes within hours of waking and to clarify whether such changes in white matter microstructure serve as neurobiological substrates of sleepiness.     

Biochemical and Molecular Responses to Loss of Renal Mass: Membranes and Protein Synthesis: Macromolecular Metabolism in Compensatory Renal Hypertrophy

1. The initial biochemical changes of compensatory hypertrophy occur well within 1 hour of unilateral nephrectomy and perhaps within the first few minutes.2. The initial increment in rRNA is from decreased metabolism rather than from increased synthesis.3. Changes in the processing of mRNA precursors are probably also important.4. Compensatory hypertrophy is regulated by a humoral stimulus or stimuli.5. The stimulus needs to be present virtually all of the time during the early phases of compensatory hypertrophy.6. The stimulus is related to loss of renal mass, not to loss of renal function.     

Development of the metanephric kidney. Protein and nucleic acid synthesis

The metanephric kidney was studied in fetal and older mice beginning at 16 days after mating of the parents. Polyribosomes from fetal kidneys labeled in vitro with 14C-labeled amino acids had 10-20 times more acid-precipitable radioactivity associated with them than polysomes from adult kidneys similarly labeled. Between 3 and 6 days after birth the rate incorporation of labeled amino acids by polyribosomes from neonatal kidneys declined sharply to only twice the value found for adult kidneys. There was no change in the shape of the polyribosome profile with increasing age, but before birth few, if any, ribosomes were bound to membranes compared with 20% 2 days after birth and between 20 and 30% in the adult. Total protein represented less than 10% of the wet weight in the fetal kidney but increased to 17% of the wet weight in the adult kidney. There was a steady decline in the concentration of RNA and DNA with respect to dry weight throughout kidney development. DNA concentration declined more rapidly than RNA concentration, so that the milligram to milligram ratio of RNA to DNA increased. In males the RNA/DNA ratio was stable at 1.3 at 40 days after birth; but in females the decline in DNA concentration was more protracted, and at 200 days after birth the RNA/DNA ratio was only 0.99. Thus, total nucleic acids show only gradual changes in concentration throughout development of the kidney, but a sharp change in the synthetic activity of the ribosomes and in their binding to membranes occurs in kidneys soon after birth.