Post-translational changes of chromosomal proteins in rat cerebellum during postnatal development

Acetylation, phosphorylation and methylation of nuclear proteins in rat cerebellum at 10 and 30 days of age were investigated in vitro. Isolated nuclei were incubated in the presence of [1-14C]acetyl CoA, S-adenosyl [methyl-3H]methionine and [gamma-32P]ATP and then separated into histones and non histone proteins (NHP), which were further fractionated by polyacrylamide gel electrophoresis. The results obtained indicate that acetylation, phosphorylation and methylation of both basic and acidic proteins decrease from 10 to 30 days of age. Electrophoretic analysis of histones shows that the decrease mainly concerns H1, H3, and H2b fractions. The H3 fraction is always more labeled than the other fractions and shows the major changes during postnatal development. Phosphorylation of H2a and H4 fractions increases from 10 to 30 days of age, whereas acetylation and methylation of these fractions do not show significant changes from 10 to 30 days. The densitometric and radioactive patterns of NHP show considerable changes between 10 and 30 days, especially in the high molecular weight region. The incorporation of 14C-acetyl and 3H-methyl groups and of 32P phosphate appears to be generalized throughout the molecular weight range and decreases from 10 to 30 days of age. The methylation of an as yet unidentified protein with a molecular weight of approximately 110,000 daltons occurred at both ages.     

Expression of mitogen-activated protein kinase pathways during postnatal development of rat heart

The loss of ability to proliferate (terminal differentiation) and reduction in capability to resist ischemia are key phenomena observed during postnatal development of the heart. Mitogen-activated protein kinases (MAPKs) mediate signaling pathways for cell proliferation/differentiation and stress responses such as ischemia. In this study, the expression of these kinases and their associated kinases were investigated in rat heart ventricle. Extracts of 1-, 10-, 20-, 50-, and 365-day-old rat heart ventricles were probed with specific antibodies and their immunoreactivities were quantified by densitometry. Most of the mitogenic protein kinases including Raf1, RafB, Mek1, Erk2, and Rsk1 were significantly down-regulated, whereas the stress signaling kinases, such as Mlk3, Mekk1, Sek1, Mkk3, and Mapkapk2 were up-regulated in expression during postnatal development. Most MAP kinases including Erk1, JNKs, p38 Hog, as well as Rsk2, however, did not exhibit postnatal changes in expression. The proto-oncogene-encoded kinases Mos and Cot/Tpl 2 were up-regulated up to two- and four-fold, respectively, during development. Pak1, which may be involved in the regulation of cytoskeleton as well as in stress signaling, was downregulated with age, but the Pak2 isoform increased only after 50 days. All of these proteins, except RafB, were also detected in the isolated adult ventricular myocytes at comparable levels to those found in adult ventricle. Tissue distribution studies revealed that most of the protein kinases that were up-regulated during heart development tended to be preferentially expressed in heart, whereas the downregulated protein kinases were generally expressed in heart at relatively lesser amounts than in most of other tissues. J. Cell. Biochem. 71:286–301, 1998. © 1998 Wiley-Liss, Inc.     

Polysomes during early postnatal development of brain in the rat

We have attempted to show eventual modifications in the brain protein synthesis apparatus of rat during the first three weeks after birth. Through this time we noted a steady decrease (about 60%) in the free polysomes, when expressed relative to tissue weight. This decrease does not correlate with changes in the polysome profile, indicating that no loss in the efficiency of protein synthesis was involved. Translation in a reticulocyte lysate also failed to reveal differences.     

The septal organ of the rat during postnatal development

The septal organ of Masera (SO) is a small, isolated patch of olfactory epithelium, located in the ventral part of the nasal septum. We investigated in this systematic study the postnatal development of the SO in histological sections of rats at various ages from the day of birth (P1) to P666. The SO-area increases to a maximum at P66-P105, just as the animals reach sexual maturity, and decreases thereafter, significantly however only in males, indicating a limited neurogenetic capacity for regeneration. In contrast, the main olfactory epithelium area continues to expand beyond P300. The modified respiratory epithelium ('zwischen epithelium') separating the SO and the main olfactory epithelium contains a few olfactory neurons up to age P66. Its length increases postnatally so that the SO becomes more ventral to the OE. Although the position of the SO relative to other anatomical landmarks changes with development it is consistently located just posterior to the opening of the nasopalatine duct (NPAL). Thus, a possible function of the SO is in sensing chemicals in fluids entering the mouth by licking and then delivered to the nasal cavity via the NPAL; therefore the SO may be involved in social/sexual behavior as is the vomeronasal organ (VNO). We suggest that the SO is a separate accessory olfactory organ with properties somewhat different from both OE and VNO and may exist only in species where the NPAL does not open into the VNO.     

Respiratory chain of rat heart mitochondria during heart preservation in formaldehyde

It has been shown by the method of low-temperature ESR-spectroscopy that partial blocking with formaldehyde of electron intake into the respiratory chain promotes the maintenance of a definite level of oxidation in the respiratory chain carriers and preservation of its nativity.     

Differential expression of porcine testis proteins during postnatal development

The development of the testes includes changes in cell morphology and endocrine levels that are essential for the maturation of males. A large number of novel proteins are expressed throughout testis development and play important roles in spermatogenesis. Differences in protein expressions during the development of porcine testes have not been systematically studied. The purpose of this study was to investigate differential protein expression in porcine testes during postnatal development. Testes from four pigs each at 1wk, 3mo, and 1yr of age were used for a proteomic analysis. Expression levels of 264 protein spots were quantified using the Melanie 3 software. In total, 108 protein spots showed more than 2-fold differences (P<0.05) among developmental stages, and 90 of them were successfully identified by mass spectrometry. The proteins were sorted based on whether the expression levels increased with age (36.1%), decreased with age (38.0%), or fluctuated among different developmental stages (25.9%). In total, 69 unique gene products were further classified according to their gene ontology annotations. A majority of the proteins are organelle proteins (41%) with the nucleus and mitochondria being the main organelles. About 45% of the proteins have a protein binding domain and are likely involved in protein-protein interactions. Finally, a large proportion of these differentially expressed proteins are involved in cellular (25%) and metabolic (22%) processes. Identifying these differentially expressed proteins should be valuable for exploring developmental biology and the pathology of male reproduction.     

Effect of hypothyroidism on the biogenesis of free mitochondria in the cerebral hemispheres and in cerebellum of rat during postnatal development

The effect of propylthiouracil-induced neonatal hypothyroidism on some aspects of the biogenesis of free (non-synaptosomal) mitochondria in the cerebral hemispheres and in the cerebellum of developing rat has been studied. The results obtained show that in hypothyroid rats mitochondrial DNA synthesis is delayed, mitochondrial RNA synthesis is not affected and cytochrome aa₃ content of mitochondria is lower than in controls. Furthermore ultrathin sections of 14-and 21-day old hypothyroid rat cerebella show mitochondria with an altered ultrastructural organization and large intracristal spaces.     

The expression pattern of nitric oxide-sensitive guanylyl cyclase in the rat heart changes during postnatal development.

Nitric oxide (NO)-releasing drugs such as glyceryl trinitrate have been used in the treatment of ischemic heart disease for more than a century. Nevertheless, a detailed analysis of the expression of the NO target enzyme soluble guanylyl cyclase (sGC) in the heart is missing. The aim of the current study was to elucidate the expression, cell distribution, and activity of sGC in the rat heart during postnatal development. Using a novel antibody raised against a C-terminal peptide of the rat beta(1)-subunit of sGC, the enzyme was demonstrated in early postnatal and adult hearts by Western blotting analyses, showing maximal expression in 10-day-old animals. Measurements of basal, NO-, and NO/YC-1-stimulated sGC activity revealed an increase of sGC activity in hearts from neonatal to 10-day-old rats, followed by a subsequent decrease in adult animals. As shown by immunohistochemical analysis, sGC expression was present in vascular endothelium and smooth muscle cells in neonatal heart but expression shifted to endothelial cells in adult animals. In isolated cardiomyocytes, sGC activity was not detectable under basal conditions but significant sGC activity could be detected in the presence of NO. An increase in expression during the perinatal period and changes in the cell types expressing sGC at different phases of development suggest dynamic regulation rather than constitutive expression of the NO receptor in the heart.     

Ontogenesis of alpha-amylase in rat parotid gland during postnatal development

Changes in alpha-amylase (alpha-1,4-glucan-4-glucanohydrolase, EC 3.2.1.1) of parotid gland were investigated during postnatal development of the rat. Modifications in amylase activity after birth allow the distinction of three stages which can be correlated with the morphologic development of the parotid gland. Significant sexual differences in the evolution of alpha-amylase activity were found. During the first stage (from birth to the 20th day) there is a higher increase in females, while males have a more pronounced increment in the second stage (from the 20th to the 30th day). By means of gel electrophoresis of parotid extracts, four molecular forms of amylase can be separated. The slowest migrating band (Form 1) is not detected at the initial stage.     

Expression of protein phosphatases during postnatal development of rabbit heart

Protein phosphatases play a major role in the regulation of L-type calcium current (I_Ca) in heart cells. We previously showed developmental differences in the effects of inhibitors of protein phosphatases (PP's) on the modulation of I_Ca, with greater stimulatory effects on I_Ca observed in newborn than in adult ventricular cells. We hypothesized that this developmental difference might be due to greater expression and levels of PP 1 and PP 2A in newborn than in adult ventricular cells. We thus determined the mRNA expression of and subunits of PP 1 and the subunit of PP 2A in adult and newborn rabbit ventricles and levels of PP 1 and PP 2A in total homogenates, particulate membranes, and in soluble fraction prepared from isolated ventricular myocytes from adult and newborn rabbits. RT-PCR analysis demonstrated the presence of mRNA of these subunits of PP's in both newborn and adult ventricles. Northern blot analysis using ³²P labeled cDNA probes specific for PP 1, PP 1 and PP 2A showed that the expression of steady state mRNA levels for PP 1, PP 1 and PP 2A were much higher in newborn compared to adult rabbit ventricles. mRNA for glyceraldehyde-3-phosphate dehydrogenase (GAPDH) and for sarcoplasmic reticulum Ca²⁺-ATPase (SERCA) in rabbit ventricles were measured as controls. GAPDH did not show significant developmental changes while mRNA for SERCA was higher in adult compared to newborns. Western blot analysis showed that PP 1 and PP 2A protein levels were also much higher in newborn compared to adult rabbit ventricular cells. Immunoblot analysis in particulate membranes and soluble fraction showed that PP1 was mainly membrane bound while PP 2 was present only in soluble fraction. These findings suggest that the two major protein phosphatases (PP 1 and PP 2A) in heart are expressed at much higher levels in newborn and decline to lower levels in adult ventricular myocytes. The presence of high levels of PP's and particularly PP 1 in newborn cells may be responsible for the greater dependence of newborn cells on the inhibition of PP as a mechanism of action of -agonist isoproterenol on I_Ca.     

Expression and localization of caveolins during postnatal development in rat heart: implication of thyroid hormone.

Caveolins modulate signaling pathways involved in cardiac development. Caveolin-1 exists in two isoforms: the beta-isoform derivates from an alternative translational start site that creates a protein truncated by 31 amino acids, mainly expressed in endothelial cells, whereas caveolin-3 is present in muscle cells. Our aim was to define caveolin distribution and expression during cardiac postnatal development using immunofluorescence and Western blotting. Caveolin-3 sarcolemmal labeling appeared as dotted lines from days 1 to 5 and as continuous lines after 14 days of age. Caveolin-3 expression, low at birth, increased (4-fold) to reach a maximum (P < 0.05) by day 5 and then decreased to stabilize in adults. Total caveolin-1 and its alpha-isoform were codistributed at birth in endothelial and smooth muscle cells; afterward, only the caveolin-1alpha labeling became limited to endothelium. Quantitative analysis indicated a similar temporal pattern of both total caveolin-1 and caveolin-1alpha expression, suggesting that caveolin-1alpha and -1beta are coregulated; the caveolin-1alpha levels increased fourfold by day 5 to reach a maximum by day 14 (P < 0.05). Tyrosine-14-caveolin-1 phosphorylation, low at birth, increased suddenly around day 14 (8-fold vs. day 1) and returning afterward to basal level. Because the T3/T4 level is maximal by day 14, caveolin-1 expression/phosphorylation profiles were analyzed in hypothyroid heart. The levels of caveolin-1alpha and consequently tyrosine-14-caveolin-1 phosphorylation, but not that of caveolin-3, decreased (50%) in hypothyroid 14-day-old rats. Our data demonstrate that, during postnatal cardiac growth, 1) caveolins are distinctly regulated, and 2) thyroid hormones are involved in caveolin-1alpha expression.     

Growth patterns of rat hepatocytes during postnatal development.

During postnatal growth in the liver of the rat, a characteristic shift towards binuclear cells and cells of higher ploidy class occurs. When the protein content of individual isolated hepatocytes of different ploidy classes is analysed cytophotometrically using the specific protein stain Naphthol Yellow S, it appears that the growth in mass in the period 30-99 days is due mainly to increase of protein content of binuclear diploid (BD) and mononuclear tetraploid (MT) cells. The mononuclear diploid (MD) cells play a quickly diminishing role in the parenchymal population after the initial growth phase and cells of highest ploidy degree remain unimportant quantitatively. The quickly growing BD and MT cells only reach a Naphthol Yellow S protein value twice that of MD cells after a certain period of growth, whereas changes in protein content are slight or absent from 99 days onwards in all cell types investigated.     

Changes of hippocampal protein levels during postnatal brain development in the rat

Information on postnatal brain protein expression is very limited, and we therefore compared hippocampal protein levels in rat hippocampus at different developmental time points using two-dimensional gel electrophoresis followed by mass spectrometrical protein identification and specific software for quantification. Proteins from several cascades as e.g., antioxidant, metabolic, cytoskeleton, proteasomal, and chaperone pathways were developmentally regulated, which is relevant for design and interpretation of protein chemical studies in the mammalian brain.     

Diversification of the myofibrillar M-band in rat skeletal muscle during postnatal development

Summary The fine structure of the M-band in soleus (SOL) and extensor digitorum longus (EDL) muscles in newborn and four-week-old rats was studied using electron-microscopic techniques. In newborn rats, all myotubes and fibres in both muscles had an identical myofibrillar appearance. A five-line M-band pattern was seen in longitudinal sections and distinct M-bridges in cross-sections. The Z-discs were of medium width. On the other hand, in four-week-old rats, different muscle fibre types were observed on the basis of their myofibrillar pattern. In SOL two fibre types were distinguished in longitudinal sections. One had a four-line M-band pattern and very broad Z-discs, whereas the other type had five lines in the M-band and broad Z-discs. In EDL, three different myofibrillar patterns were observed. The M-bands were composed of three, four or five lines. Fibres had either thin, broad or medium Z-disc widths, respectively. In cross-sections of the SOL muscle one group of fibres showed indistinct M-bridges, whereas distinct M-bridges were seen in the other fibres and in all observed EDL muscle fibres. We conclude that initially there seems to be a single intrinsic program for M-band genesis; this program becomes modified upon the induction of functionally differentiated fibres.