Tubulin and Glial Fibrillary Acidic Protein Gene Expression in Developing Fetal Human Brain at Midgestation

Developmental alterations in the expression of glial fibrillary acidic protein (GFAP) and -tubulin were examined at the level of mRNA and protein in human fetal brain between weeks 13–23 of gestation. Except for a transient increase at week 15, GFAP expression in the cytoskeletal (CSK) fraction was low until week 17, when it increased steadily to week 23, corresponding to the phase of glial proliferation. The developmental profile of -tubulin in the CSK fraction displayed a biphasic pattern, with an initial rise between weeks 13–16 coinciding with the early phase of neuroblast multiplication, and a second rise between weeks 17–23 corresponding to the phase of glial proliferation. No significant difference in the spatial distribution of -tubulin was found in different region of brain but GFAP expression varied with a higher level in cerebellum than that in cerebrum at late midgestation.     

Thyroid hormone enhances nitric oxide-mediated bacterial clearance and promotes survival after meningococcal infection

Euthyroid sick syndrome characterized by reduced levels of thyroid hormones (THs) is observed in patients with meningococcal shock. It has been found that the level of THs reflects disease severity and is predictive for mortality. The present study was conducted to investigate the impact of THs on host defense during meningococcal infection. We found that supplementation of thyroxine to mice infected with Neisseria meningitidis enhanced bacterial clearance, attenuated the inflammatory responses and promoted survival. In vitro studies with macrophages revealed that THs enhanced bacteria-cell interaction and intracellular killing of meningococci by stimulating inducible nitric oxide synthase (iNos)-mediated NO production. TH treatment did not activate expression of TH receptors in macrophages. Instead, the observed TH-directed actions were mediated through nongenomic pathways involving the protein kinases PI3K and ERK1/2 and initiated at the membrane receptor integrin αvβ3. Inhibition of nongenomic TH signaling prevented iNos induction, NO production and subsequent intracellular bacterial killing by macrophages. These data demonstrate a beneficial role of THs in macrophage-mediated N. meningitidis clearance. TH replacement might be a novel option to control meningococcal septicemia.     

Expression of Organic Anion Transporting Polypeptide 1c1 and Monocarboxylate Transporter 8 in the Rat Placental Barrier and the Compensatory Response to Thyroid Dysfunction

Thyroid hormones (THs) must pass from mother to fetus for normal fetal development and require the expression of placental TH transporters. We investigate the compensatory effect of placental organic anion transporting polypeptide 1c1 (Oatp1c1) and monocarboxylate transporter 8 (Mct8) on maternal thyroid dysfunction. We describe the expressions of these two transporters in placental barriers and trophoblastic cell populations in euthyroidism and thyroid dysfunction resulting from differential iodine nutrition at gestation day (GD) 16 and 20, that is, before and after the onset of fetal thyroid function. Immunohistochemistry revealed that in the blood-placenta barrier, these two TH transporters were strongly expressed in the villous interstitial substance and were weakly expressed in trophoblast cells. Levels of Oatp1c1 protein obviously increased in the placental fetal portion during maternal thyroid deficiency at GD16. Under maternal thyroid deficiency after the production of endogenous fetal TH, quantitative PCR analysis revealed down-regulation of Oatp1c1 occurred along with up-regulation of Mct8 in trophoblast cell populations isolated by laser capture microdissection (LCM); this was consistent with the protein levels in the fetal portion of the placenta. In addition, decreased D3 mRNA at GD16 and increased D2 mRNA on two gestational days were observed in trophoblast cells with thyroid dysfunction. However, levels of Oatp1c1 mRNA at GD16 and D3 mRNA at GD20 were too low to be detectable in trophoblast cells. In conclusion, placental Oatp1c1 plays an essential compensatory role when the transplacental passage of maternal THs is insufficient at the stage before the fetal TH production. In addition, the coordinated effects of Oatp1c1, Mct8, D2 and D3 in the placental barrier may regulate both transplacental TH passage and the development of trophoblast cells during thyroid dysfunction throughout the pregnancy.     

Analysis of the <Emphasis Type="Italic">Rana catesbeiana</Emphasis> tadpole tail fin proteome and phosphoproteome during T<Subscript>3</Subscript>-induced apoptosis: identification of a novel type I keratin

Background  

Thyroid hormones (THs) are vital in the maintenance of homeostasis and in the control of development. One postembryonic developmental process that is principally regulated by THs is amphibian metamorphosis. This process has been intensively studied at the genomic level yet very little information at the proteomic level exists. In addition, there is increasing evidence that changes in the phosphoproteome influence TH action.     

The effects of human cytomegalovirus infection on cytoskeleton-associated polysomes

Human cytomegalovirus (HCMV) infection induces disruption of the host cell's cytoskeleton (CSK). This disruption is accompanied by three transient phases of actin depolymerization that occur at 20 min, 5 to 10 h and 48 to 72 h post infection (pi). During the 20 min peak of actin depolymerization, the level of cellular polysomes associated with the CSK was reduced, due to release of ribosomes from CSK-associated polysomes. Cellular mRNAs previously existing in these polysomes, however, remained associated with the CSK. Also during this period, nuclear to cytoplasmic transport of host cellular mRNA as well as the association of newly synthesized mRNA with the CSK was temporarily delayed. By 60 min pi, ribosomes, preexisting host cellular mRNA, and newly synthesized mRNAs (host and viral) had reestablished a distribution in the infected cell comparable to that of uninfected cells. Sedimentation profiles of soluble and CSK fractions at various times throughout the viral infection indicated that, although the amount of polysomes associated with the CSK at 20 min pi was reduced, essentially all HCMV and all host cell polysomes present were associated with the CSK. The majority of HCMV DNA hybridizable poly(A)+ RNAs were associated with the CSK throughout the viral infection. These early events appear to correlate with a transient interruption of host cellular mRNA translation early in infection and may represent a process whereby HCMV gene expression becomes competitive with that of the host cell.     

Born to be young? Prenatal thyroid hormones increase early-life telomere length in wild collared flycatchers

The underlying mechanisms of the lifelong consequences of prenatal environmental condition on health and ageing remain little understood. Thyroid hormones (THs) are important regulators of embryogenesis, transferred from the mother to the embryo. Since prenatal THs can accelerate early-life development, we hypothesized that this might occur at the expense of resource allocation in somatic maintenance processes, leading to premature ageing. Therefore, we investigated the consequences of prenatal TH supplementation on potential hallmarks of ageing in a free-living avian model in which we previously demonstrated that experimentally elevated prenatal TH exposure accelerates early-life growth. Using cross-sectional sampling, we first report that mitochondrial DNA (mtDNA) copy number and telomere length significantly decrease from early-life to late adulthood, thus suggesting that these two molecular markers could be hallmarks of ageing in our wild bird model. Elevated prenatal THs had no effect on mtDNA copy number but counterintuitively increased telomere length both soon after birth and at the end of the growth period (equivalent to offsetting ca 4 years of post-growth telomere shortening). These findings suggest that prenatal THs might have a role in setting the ‘biological'' age at birth, but raise questions about the nature of the evolutionary costs of prenatal exposure to high TH levels.     

Thyroid hormone-induced morphological differentiation and maturation of astrocytes are mediated through the beta-adrenergic receptor

The molecular mechanisms associated with thyroid hormone (TH)-induced maturation of astrocytes have been studied using primary cultures. We have previously demonstrated that unlike normal astrocyte cultures, hypothyroid cultures fail to differentiate from flat polygonal cells with epithelioid morphology into mature process-bearing cells with stellate morphology. Addition of TH to the hypothyroid cells reverses the effect, and astrocytes transform into stellate cells. The beta-adrenergic receptor (beta-AR) agonist isoproterenol (ISP) has a similar effect, whereas simultaneous addition of the beta-adrenergic antagonist propranolol blocks the differentiation induced by TH or ISP. Addition of TH or ISP to hypothyroid cultures is also associated with a decrease in the level of filamentous cytoskeletal (F(i)) actin and an increase in the level of actin mRNA. Although addition of propranolol inhibited the decline in the level of F(i) actin in the TH- or ISP-supplemented cells as well as the induction of actin mRNA by TH, it partially inhibited the ISP-induced actin mRNA in these cultures. The hormone-induced maturation appears to be selectively regulated through the beta(2)-AR. The overall results indicate that the beta-adrenergic system plays an obligatory role in promoting TH-induced differentiation and maturation of astrocytes and in regulating the hormone-induced expression of actin and its intracellular organization in a way conducive to the morphological differentiation of the cells.     

Evolutionary roots of iodine and thyroid hormones in cell-cell signaling

In vertebrates, thyroid hormones (THs, thyroxine, and triiodothyronine) are critical cell signaling molecules. THs regulate and coordinate physiology within and between cells, tissues, and whole organisms, in addition to controlling embryonic growth and development, via dose-dependent regulatory effects on essential genes. While invertebrates and plants do not have thyroid glands, many utilize THs for development, while others store iodine as TH derivatives or TH precursor molecules (iodotyrosines)-or produce similar hormones that act in analogous ways. Such common developmental roles for iodotyrosines across kingdoms suggest that a common endocrine signaling mechanism may account for coordinated evolutionary change in all multi-cellular organisms. Here, I expand my earlier hypothesis for the role of THs in vertebrate evolution by proposing a critical evolutionary role for iodine, the essential ingredient in all iodotyrosines and THs. Iodine is known to be crucial for life in many unicellular organisms (including evolutionarily ancient cyanobacteria), in part, because it acts as a powerful antioxidant. I propose that during the last 3-4 billion years, the ease with which various iodine species become volatile, react with simple organic compounds, and catalyze biochemical reactions explains why iodine became an essential constituent of life and the Earth's atmosphere-and a potential marker for the origins of life. From an initial role as membrane antioxidant and biochemical catalyst, spontaneous coupling of iodine with tyrosine appears to have created a versatile, highly reactive and mobile molecule, which over time became integrated into the machinery of energy production, gene function, and DNA replication in mitochondria. Iodotyrosines later coupled together to form THs, the ubiquitous cell-signaling molecules used by all vertebrates. Thus, due to their evolutionary history, THs, and their derivative and precursors molecules not only became essential for communicating within and between cells, tissues and organs, and for coordinating development and whole-body physiology in vertebrates, but they can also be shared between organisms from different kingdoms.     

Thyroid hormones increase inducible nitric oxide synthase gene expression downstream from PKC-zeta in murine tumor T lymphocytes

Regulation of cell proliferation by thyroid hormone (TH) has been demonstrated, but the effect of THs and the mechanisms involved in lymphocyte activity have not been elucidated. Differential expression of PKC isoenzymes and high nitric oxide synthase (NOS) activity have been described in tumor T lymphocytes. We have analyzed the direct actions of TH on normal T lymphocytes and BW5147 T lymphoma cells in relation to PKC and NOS activities. THs increased tumor and mitogen-induced normal T lymphocyte proliferation. PKC isoenzyme-selective blockers impaired these effects in both cell types, indicating the participation of Ca2+-dependent and -independent isoenzymes in normal and tumor cells, respectively. TH actions were blunted by extra- and intracellular Ca2+ blockers only in normal T lymphocytes, whereas NOS blockers impaired TH-induced proliferation in T lymphoma cells. Incubation for 24 h with TH induced a rise in total and membrane-associated PKC activities in both cell types and led to a rapid and transient effect only in tumor cells. THs increased atypical PKC-zeta expression in BW5147 cells and classical PKC isoenzymes in mitogen-stimulated normal T cells. TH augmented NOS activity and inducible NOS protein and gene expression only in tumor cells. Blockade of PKC and the atypical PKC-zeta isoform inhibited TH-mediated stimulation of inducible NOS and cell proliferation. These results show, for the first time, that differential intracellular signals are involved in TH modulation of lymphocyte physiology and pathophysiology.     

Cooperative nongenomic and genomic actions on thyroid hormone mediated-modulation of T cell proliferation involve up-regulation of thyroid hormone receptor and inducible nitric oxide synthase expression

Thyroid hormones (THs) exert a broad range of actions on development, growth, and cell differentiation by both genomic and nongenomic mechanisms. THs regulate lymphocyte function, but the participation of nongenomic actions is still unknown. Here the contribution of both genomic and nongenomic effects on TH-induced division of T cells was studied by using free and noncell permeable THs coupled to agarose (TH-ag). THs-ag led to cell division, but to a lesser extent than free hormones. THs induced nongenomically the rapid translocation of protein kinase C (PKC) ζ isoform to cell membranes, extracellular-signal-regulated kinases (ERK1/2) phosphorylation and nuclear factor-κB (NF-κB) activation. The signaling cascade include sphingomyelinases acting up-stream the activation of PKCζ isoform, while ERK and NF-κB are activated downstream this PKC isoenzyme. Both free and THs-ag increased the protein and mRNA levels of TH nuclear receptor TRα1, while only free hormones incremented the inducible NOS gene and protein levels as well as a calcium independent NOS activity. Both effects were blunted by PKCζ inhibition. These results indicate that THs, by triggering a nongenomic signaling cascade that involves Smases-mediated activation of PKCζ, lead to ERK 1/2 and NF-κB activation and to the genomic increase of TRs and the inducible nitric oxide synthase protein and mRNA levels, improving T lymphocyte proliferation. These finding not only contribute to the understanding of the mechanisms involved in TH modulation of lymphocyte physiology, but would also point out for the first time the interplay between genomic and nongenomic TH actions in T cells.     

Thyroidal regulation of different isoforms of NaKATPase in glial cells of developing rat brain.

The developmental profile of the different isoforms of NaKATPase have been investigated during the first three weeks of postnatal development using primary cultures of isolated glial cells derived from neonatal rat cerebra. Northern and Western blot analysis show that the expression of four isoforms (alpha1, alpha2, beta1 and beta2) in these cells increases progressively between 5 to 20 days of culture. Comparison of the mRNA levels of these isoforms in thyroid hormone deficient (TH def) and thyroid hormone supplemented (TH sup) cells cultured for 5-10 days, revealed for the first time that all four isoforms are sensitive to T3 in the glial cells. Furthermore immunocytochemical staining of these cells with isoform specific NaKATPase antibodies also showed that the localization of the different isoforms in the TH def cells were altered in comparison to that in the TH sup cells. These results establish glial cells as the target cells for the regulation of NaKATPase by TH in the developing brain.     

Transient growth‐enhancing effects of elevated maternal thyroid hormones at no apparent oxidative cost during early postnatal period

Maternal thyroid hormones (THs) have been proven crucial for embryonic development in humans, but their influence within the natural variation on wild animals remains unknown. So far the only two studies that experimentally investigated the potential fitness consequences of maternal THs in birds found inconsistent results. More studies are thus required to assess the general effects of maternal THs and their influences on more behavioral and physiological parameters. In this study, we experimentally elevated yolk TH content in a wild migratory passerine species, the collared flycatcher Ficedula albicollis, to investigate the effects on hatching success, nestling growth and oxidative stress. We found that TH‐injected eggs had a higher hatching success, and the nestlings hatched from TH‐injected eggs were heavier and larger than control nestlings, but only during the early postnatal period. These differences vanished by fledging. Nestlings from TH‐injected eggs exhibited lower activity of the glutathione‐s‐transferase, a major antioxidant enzyme, than control nestlings at day 12, a few days before fledging, but they did not differ in oxidative damage and overall intracellular oxidative state. These results suggest that the early growth‐enhancing effects incurred no observable oxidative stress. We hypothesize that such a transient growth‐enhancing effect might be adaptive in advancing the development and maturation of the offspring so they are well‐prepared in time for the upcoming migration. Further studies investigating whether such advancing effects can influence long‐term fitness, will be more than valuable.     

Translational control and the cytoskeleton in Physarum polycephalum

Translationally active plasmodia of the syncytial slime mold Physarum polycephalum develop into translationally dormant sclerotia during starvation. Although functional mRNA and ribosomes exist in sclerotia, protein synthesis is suppressed at the level of initiation. To test the possibility that alterations in the cytoskeleton may limit protein synthesis, we have examined the distribution of polysomes and actin mRNA in the cytoskeletal (CSK) and soluble (SOL) fractions of Triton X-100-extracted plasmodia and sclerotia. Most of the polysomes and actin mRNA were located in the CSK of plasmodia, while most of the ribosomes and actin mRNA were located in the SOL of sclerotia. The results suggest that ribosomes and mRNA shift from the CSK to the SOL as protein synthesis is suppressed during starvation. Plasmodia and sclerotia can be induced to accumulate excess polysomes by treatment with low levels of the elongation inhibitor cycloheximide. Treatment of plasmodia with cycloheximide caused excess polysomes to accumulate in the SOL, suggesting that the CSK contains a limited capacity for binding translational components and that the association of polysomes with the cytoskeleton is not required for protein synthesis. Treatment of sclerotia with cycloheximide, however, caused polysomes and actin mRNA to accumulate in the CSK, suggesting that the sclerotial cytoskeleton, although depleted in ribosomes and mRNA, is capable of binding translational components. It is concluded that alterations in the sclerotial cytoskeleton are not involved in translational control.     

Lymphocyte cytotoxicity in x-irradiation-induced rat small bowel adenocarcinoma. III. Blocking by 3 M KCL extract

Hypertonic salt extracts (3 M KCl) of x-irradiation-induced Holtzman rat small bowel adenocarcinomas blocked the in vitro destruction of allogeneic cultured cells of this malignancy by sensitized lymphoid cells obtained from tumor-bearing animals. The protective effect were mediated by a blocking action at both the effector and the target cell level. The extracts were separated into 50% ammonium sulfate soluble and insoluble fractions with the soluble fraction being more effective in blocking the cytotoxic responses through interaction with the lymphoid cells whereas the insoluble one had a greater effect upon tumor target cells. Associated with both fractions was the oncofetal glycoprotein previously identified with the cellular membrane of this x-ray-induced malignancy. Immunoglobulins were identified with insoluble fraction; some were able to bind the oncofetal protein, thus clasifying it as a fetal antigen. The protective effects of the soluble fraction and this neoantigen were found to be citric acid labile, whereas the effects due to the insoluble fraction were unchanged.     

The role of thyroid hormones during the development of eye pigmentation in the Pacific bluefin tuna (Thunnus orientalis)

Thyroid hormones (THs) are essential for the embryonic and post-embryonic development of fish. We studied the role of THs during the early, post-embryonic, development of Pacific bluefin tuna. Embryos were treated with L-thyroxine (T(4)) or the anti-thyroid drug methimazole (MMI), and reared in microtitre plates for 3 days. Immersion in MMI, but not T(4), led to retardation of retinal pigment epithelium (RPE) pigmentation 3 days post-hatching (dph). Concurrent immersion in T(4) and MMI had no effect of RPE pigmentation. We also measured the expression of TRalphaA, TRalphaB, and TRbeta mRNA using real-time RT-PCR. Treatment with MMI significantly reduced TRbeta mRNA expression. Taken together these results suggest that the development of RPE pigmentation is mediated by TH, most likely via TRbeta.     

Effects of experimentally manipulated yolk thyroid hormone levels on offspring development in a wild bird species

Maternal effects are a crucial mechanism in a wide array of taxa to generate phenotypic variation, thereby affecting offspring development and fitness. Maternally derived thyroid hormones (THs) are known to be essential for offspring development in mammalian and fish models, but have been largely neglected in avian studies, especially in respect to natural variation and an ecological context. We studied, for the first time in a wild species and population, the effects of maternally derived THs on offspring development, behavior, physiology and fitness-related traits by experimental elevation of thyroxine and triiodothyronine in ovo within the physiological range in great tits (Parus major). We found that elevated yolk TH levels had a sex-specific effect on growth, increasing male and decreasing female growth, relative to controls, and this effect was similar throughout the nestling period. Hatching or fledging success, motor coordination behavior, stress reactivity and resting metabolic rate were not affected by the TH treatment. We conclude that natural variation in maternally derived THs may affect some offspring traits in a wild species. As this is the first study on yolk thyroid hormones in a wild species and population, more such studies are needed to investigate its effects on pre-hatching development, and juvenile and adult fitness before generalizations on the importance of maternally derived yolk thyroid hormones can be made. However, this opens a new, interesting avenue for further research in the field of hormone mediated maternal effects.     

Synthesis and Turnover of Cytoskeletal Proteins in Cultured Astrocytes

Abstract: We previously reported that the cytoskeleton of rat astrocytes in primary culture contains vimentin, glial fibrillary acidic protein (GFAP), and actin. These proteins were found in a fraction insoluble in Triton X-100 and thought to be assembled in filamentous structures. We now used primary astrocyte cultures to study the kinetics of synthesis and turnover of these cytoskeletal proteins. The intermediate filament proteins were among the most actively synthesized by astrocytes. High levels of synthesis were detectable by the third day of culture in the early log phase of growth, and the pattern of labeling at day 3 was similar to that at 14 days when the cultures had reached confluency. In short-term incorporation experiments vimentin, GFAP, and actin in the Triton-insoluble fraction were labeled within 5 min after exposure of the cultures to radioactive leucine. We did not detect any saturation of labeling for up to 6 h of incubation. The turnover of filament proteins studied by following the decay of radioactivity from prelabeled vimentin, GFAP, and cytoskeletal actin displayed biphasic decay kinetics for all three proteins. In the initial phase a fast-decaying pool with a half-life of 12–18 h contributed about 40% of the total activity in each protein. A major portion, about 60%, of each protein, however, decayed much more slowly, exhibiting a half-life of about 8 days.