Antigen receptor regulation of phosphoinositide-dependent kinase 1 pathways during thymocyte development

Phosphoinositide-dependent kinase 1 (PDK1) is essential for T cell development but little is know about the stimuli that regulate PDK1 signaling in vivo. The thymus contains a heterogeneous mixture of cells at different stages of development making it difficult to use biochemical techniques to examine the activity of PDK1 pathways as thymocytes develop in situ. Herein, we use a single cell assay to quantify activation of the PDK1 target kinase ribosomal S6 kinase 1 (S6K1) in different murine thymocyte subsets immediately ex vivo. This technique allows an assessment of S6K1 activation as thymocytes respond to developmental stimuli in vivo. These studies reveal that only a small percentage of thymocytes show evidence for activation of PDK1 mediated signaling in situ. The thymic subpopulations that contain active PDK1/S6K1 are those known to be responding to signaling by the pre T cell receptor and the mature alpha/beta T cell antigen receptor (TCR). Moreover, loss of antigen receptor signaling in T cell progenitors that cannot rearrange their TCR beta locus prevents in vivo activation of S6K1. The present data identifying antigen receptor signaling as a key activator of PDK1 mediated signaling afford a molecular explanation for the important role of this molecule in T cells.     

Iron at the cell surface controls both DNA synthesis and plasma membrane redox system

Summary Addition of the impermeable iron II chelator bathophenanthroline disulfonate (BPS) to cultured Chinese hamster lung fibroblast (CCL 39 cells) inhibits DNA synthesis but not protein synthesis or cytoplasmic alkalinization, when cell growth is initiated with growth factors such as EGF plus insulin, thrombin, or ceruloplasmin. The BPS inhibition is reversed by addition of stoichiometric ferrous iron at stoichiometric concentration. BPS does not inhibit cell growth stimulated by fetal calf serum. The effect of the BPS differs from the inhibition of growth by hydroxyurea which acts on the ribonucleotide reductase. The BPS treatment leads to release of iron from the cells as determined by BPS iron II complex formation over 90 min. Cells treated with BPS just during starvation period cannot re-initiate DNA synthesis after mitogen stimulation even if BPS is removed from the medium and cells are previously washed. BPS treatment also inhibits transplasma membrane electron which is restored by incubation of cells with 10 M ferric ammonium citrate. Growth factor stimulation of DNA synthesis is restored by addition of 1 M ferrous ammonium sulfate or ferric ammonium citrate, or 0.1 M diferric transferrin. Copper, cobalt, nickel, zinc, gallium, aluminum, or apotransferrin cannot restore the activity. The BPS effect is consistent with removal of iron from a site on the cell surface which controls electron transport and DNA synthesis.Abbreviations BCS bathocuproine disulfonate - BPS bathophenan-throline disulfonate - CUP ceruloplasmin - FCS fetal calf serum - Fe₂Tf diferric transferrin - EGF epidermal growth factor - HU hydroxyurea - THR -thrombin     

The regulation of DNA repair during development

DNA repair is important in such phenomena as carcinogenesis and aging. While much is known about DNA repair in single-cell systems such as bacteria, yeast, and cultured mammalian cells, it is necessary to examine DNA repair in a developmental context in order to completely understand its processes in complex metazoa such as man. We present data to support the notion that proliferating cells from organ systems, tumors, and embryos have a greater DNA repair capacity than terminally differentiated, nonproliferating cells. Differential expression of repair genes and accessibility of chromatin to repair enzymes are considered as determinants in the developmental regulation of DNA repair.     

Changes in heat shock protein synthesis and heat sensitivity during mouse thymocyte development

Heat shock protein synthesis was examined in mouse thymocytes at three stages of development: early embryonic thymocytes, which are CD4^?CD8^?, adult thymocytes, which are primarily CD4⁺CD8⁺, and mature spleen T cells, which are CD4⁺CD8^? or CD4^?CD8⁺. After either a 41°C or 42°C heat shock, the synthesis of the maior heat-inducible protein (hsp68) was elevated during the first hour of recovery but then decreased abruptly in thymocytes from adult mice. In contrast, the synthesis of hsp68 continued for up to 4 h after heating embryonic mouse thymocytes or mature spleen T cells. The more rapid termination ofthe heat shock response in the adult thymocytes was not the result of eitherless heat damage or more rapid repair since the recovery of general protein synthesis was more severely delayed in these cells. As well, the double positive CD4⁺CD8⁺ cells were more sensitive to hyperthermia than either the double negative CD4^?CD8^? or single positive CD4⁺CD8^? or CD4^?CD8⁺ cells. Exposure of fetal thymus organ cultures to elevated temperature revealed that the double negative thymocytes were able to survive and differentiate normally following a heat shock treatment that was lethal for the double positive thymocytes. Exposure of thymocytes from adult mice to elevated temperatures induced apoptotic cell death. This was evident by the cleavage of DNA into oligonucleosome-sized fragments. Quantitation of the extent of DNA fragmentation and the number of apoptotic cells by flow cytometry demonstrated that the extent of apoptotic cell death was related to the severity of the heat stress. Double positive (CD4+CD8+) thymocytes are selected on the basis of their T-cell antigen receptor (TCR). Most of these cells are negatively selected and die within the thymus by an active process of cell deletion known as apoptosis. Restricting hsp synthesis in response to stress might be essential during developmental processes in which cell maturation is likely to result in death rather than functional differentiation. © 1993Wiley-Liss, Inc.     

The redox state of transglutaminase 2 controls arterial remodeling

While inward remodeling of small arteries in response to low blood flow, hypertension, and chronic vasoconstriction depends on type 2 transglutaminase (TG2), the mechanisms of action have remained unresolved. We studied the regulation of TG2 activity, its (sub) cellular localization, substrates, and its specific mode of action during small artery inward remodeling. We found that inward remodeling of isolated mouse mesenteric arteries by exogenous TG2 required the presence of a reducing agent. The effect of TG2 depended on its cross-linking activity, as indicated by the lack of effect of mutant TG2. The cell-permeable reducing agent DTT, but not the cell-impermeable reducing agent TCEP, induced translocation of endogenous TG2 and high membrane-bound transglutaminase activity. This coincided with inward remodeling, characterized by a stiffening of the artery. The remodeling could be inhibited by a TG2 inhibitor and by the nitric oxide donor, SNAP. Using a pull-down assay and mass spectrometry, 21 proteins were identified as TG2 cross-linking substrates, including fibronectin, collagen and nidogen. Inward remodeling induced by low blood flow was associated with the upregulation of several anti-oxidant proteins, notably glutathione-S-transferase, and selenoprotein P. In conclusion, these results show that a reduced state induces smooth muscle membrane-bound TG2 activity. Inward remodeling results from the cross-linking of vicinal matrix proteins, causing a stiffening of the arterial wall.     

DNA methylation controls the timing of astrogliogenesis through regulation of JAK-STAT signaling

DNA methylation is a major epigenetic factor that has been postulated to regulate cell lineage differentiation. We report here that conditional gene deletion of the maintenance DNA methyltransferase I (Dnmt1) in neural progenitor cells (NPCs) results in DNA hypomethylation and precocious astroglial differentiation. The developmentally regulated demethylation of astrocyte marker genes as well as genes encoding the crucial components of the gliogenic JAK-STAT pathway is accelerated in Dnmt1-/- NPCs. Through a chromatin remodeling process, demethylation of genes in the JAK-STAT pathway leads to an enhanced activation of STATs, which in turn triggers astrocyte differentiation. Our study suggests that during the neurogenic period, DNA methylation inhibits not only astroglial marker genes but also genes that are essential for JAK-STAT signaling. Thus, demethylation of these two groups of genes and subsequent elevation of STAT activity are key mechanisms that control the timing and magnitude of astroglial differentiation.     

Telomere length regulation during postnatal development and ageing in Mus spretus.

Telomere shortening has been causally implicated in replicative senescence in humans. To examine the relationship between telomere length and ageing in mice, we have utilized Mus spretus as a model species because it has telomere lengths of approximately the same length as humans. Telomere length and telomerase were analyzed from liver, kidney, spleen, brain and testis from >180 M.spretus male and female mice of different ages. Although telomere lengths for each tissue were heterogeneous, significant changes in telomere lengths were found in spleen and brain, but not in liver, testis or kidney. Telomerase activity was abundant in liver and testis, but weak to non-detectable in spleen, kidney and brain. Gender differences in mean terminal restriction fragment length were discovered in tissues from M.spretus and from M.spretus xC57BL/6 F1 mice, in which a M. spretus -sized telomeric smear could be measured. The comparison of the rank order of tissue telomere lengths within individual M. spretus showed that certain tissues tended to be longer than the others, and this ranking also extended to tissues of the M.spretus xC57BL/6 F1 mice. These data suggest that telomere lengths within individual tissues are regulated independently and are genetically controlled.     

Osteopontin expression and regulation in the testis, efferent ducts, and epididymis of rats during postnatal development through to adulthood

Osteopontin (OPN), a multifunctional phosphoprotein found in both hard and soft tissues, was examined in the male reproductive tract. The expression and regulation of OPN in the rat testis, efferent ducts, and epididymis was examined during postnatal development through to adulthood using immunocytochemistry at the light- and electron-microscopic level. Immunoblot analysis revealed a major 30-kDa band for epididymal tissue and a major 60-kDa band for the testis. In the testis, immunostaining of OPN was noted in early germ cells from spermatogonia to early pachytene spermatocytes, suggesting a role for OPN as an adhesive protein binding these cells to the basement membrane and adjacent Sertoli cells. Nonciliated cells of the efferent ducts expressed OPN, whereas a cell- and region-specific distribution of OPN was observed in the epididymis. Reactivity of OPN in the apical region of the cell corresponded to labeling of microvilli, small endocytic vesicles, and endosomes, where OPN may serve to remove calcium from the epididymal lumen and, thus, prevent mineral accumulation and subsequent decrease in sperm fertility. Regulation and postnatal studies revealed that circulating androgens regulate OPN expression in principal cells of the epididymis only. Taken together, the data reveal cell- and region-specific expression and regulation of OPN in the epididymis.     

Hydrocortisone and triiodothyronine regulation of malate-aspartate shuttle enzymes during postnatal development of chicken.

The normal endogenous level of malate-aspartate shuttle enzymes and its regulation by hydrocortisone and triiodothyronine were studied in the liver and kidney of 0-, 30- and 60-day old male Rhode Island Red (RIR) chicken. The endogenous activity of cytosolic malate dehydrogenase (c-MDH) was significantly higher in the liver of day 30 as compared to day 0 and 60. In contrast, mitochondrial malate dehydrogenase (m-MDH) activity decreased at day 60 in the liver. However, both c- and m-MDH had significantly lower activities at day 0, which increased sharply at day 30 and 60 in the kidney. On the other hand, activity of both cytosolic and mitochondrial aspartate aminotransferase (c- and m-AsAT) showed peak value at day 30 in both liver and kidney. Hydrocortisone administration induced c-MDH in the liver at all the ages studied, but did not influence the activity of the isoenzymes in the kidney whereas, it induced m-MDH in the liver at day 0 and in kidney at day 30. Administration of hydrocortisone, however, did not influence AsAT isoenzymes (c- and m-AsAT) in either of the tissues at any of the postnatal ages. Triiodothyronine induced c-MDH in the liver at all the ages whereas kidney isoenzyme was induced only at day 60. In contrast, m-MDH was induced by triiodothyronine in both liver and kidney at day 30 and 60. Administration of triiodothyronine did not influence c-AsAT of liver and kidney at either of the ages, whereas it induced m-AsAT of only liver at day 0 and 60. These findings indicated a tissue- and age-specific expression of the malate-aspartate shuttle enzymes in chicken and difference in the regulation exerted by hydrocortisone and triiodothyronine during postnatal development of chicken.     

Sterol synthesis by the ocular lens of the rat during postnatal development

Great amounts of plasma membranes are formed during early postnatal development of the ocular lens as lens epithelial cells differentiate into fiber cells. Little information is available on the source of the lipids, and particularly cholesterol, required for formation of these plasma membranes. The present study measured the capacity of the lens of the rat to synthesize cholesterol during this dynamic period of growth. Incorporation by lens of (3)H(2)O into total fatty acids was also examined. Absolute rates of cholesterol synthesis per whole lens were estimated in vitro from incorporation of (3)H from (3)H(2)O into digitonide precipitable sterols (DPS) by intact lenses of 6- to 30-day old rats. Rates of cholesterol synthesis were calculated which were adequate to furnish from either 50-100% or 20-40% of the cholesterol required by the lens for growth, depending upon the animal's age and upon whether one considered NADPH to be generated by the pentose phosphate pathway or by oxidative enzymatic processes (NADPH from the pentose pathway is not labeled from (3)H(2)O). Generation of the NADPH necessary for cholesterol synthesis principally by the pentose pathway would support the higher percent contribution of synthesis to the total growth requirement. The pentose pathway was clearly active in the young rat lens, since between 7.5 to 9.0 times more [1-(14)C]glucose than [6-(14)C]glucose was oxidized in vitro to (14)CO(2) by 6- and 22-day old lenses. Incorporation of (3)H(2)O into DPS decreases sharply after 2 weeks of age in spite of a constant rate of cholesterol accumulation by the lens. These results indicate that the ocular lens of the rat can furnish most if not all of its cholesterol requirements by synthesis de novo during the first 2 weeks of life, and imply a contribution from another source at older ages. Whether lipoproteins can supply cholesterol to the lens is still unclear, although neither HDL nor LDL altered the incorporation in vitro of [U-(14)C]glucose into DPS by lens.-Cenedella, R. J. Sterol synthesis by the ocular lens of the rat during postnatal development.     

Conduction of excitation through the superior cervical ganglion during early postnatal development

The action of hexamethonium, D-tubocurarine, phentolamine, and atropine on synaptic transmission in the superior cervical ganglion was studied in the early stage of postnatal development (1–8 days after birth) and in the adult period in cats, rabbits, and rats. Hexamethonium and D-tubocurarine, if injected intravenously or added to the Krebs' solution surrounding the ganglion, were shown to inhibit the conduction of excitation through the ganglion effectively in both newborn and adult animals. No significant difference in the action of phentolamine and atropine on synaptic transmission in the ganglia could be found in these groups of animals. It is concluded that synaptic transmission in sympathetic ganglia is cholinergic in the early stage of postnatal development of animals blind at birth.