Electromechanical Cornea Reshaping for Refractive Vision Therapy.

The corneal stroma consists of orthogonally stacked collagen-fibril lamellae that determine the shape of the cornea and provide most of the refractive power of the eye. We have applied electromechanical reshaping (EMR), an electrochemical platform for remodeling cartilage and other semirigid tissues, to change the curvature of the cornea as a potential procedure for nonsurgical vision correction. EMR relies on short electrochemical pulses to electrolyze water, with subsequent diffusion of protons into the extracellular matrix of collagenous tissues; protonation of immobilized anions within this matrix disrupts the ionic-bonding network, leaving the tissue transiently responsive to mechanical remodeling. Re-equilibration to physiological pH restores the ionic matrix, resulting in persistent shape change of the tissue. Using ex vivo rabbit eyes, we demonstrate here the controlled change of corneal curvature over a wide range of refractive powers with no loss of optical transparency. Optical coherence tomography (OCT), combined with second-harmonic generation (SHG) and confocal microscopy, establish that EMR enables extremely fine control of corneal contouring while maintaining the underlying macromolecular collagen structure and stromal cellular viability, positioning electrochemical vision therapy as a potentially simple and ultralow-cost modality for correcting routine refractive errors.

Simplifying the B Complex: How Vitamins B6 and B9 Modulate One Carbon Metabolism in Cancer and Beyond.

Vitamin B micronutrients are essential regulators of one carbon metabolism that ensures human health. Vitamin B9, or folate, lies at the heart of the folate cycle and converges with the methionine cycle to complete the one carbon pathway. Additionally, vitamin B6 contributes by orchestrating the flux of one carbon cycling. Dysregulation of vitamin B contributes to altered biochemical signaling that manifests in a spectrum of human diseases. This review presents an analysis of the past, present, and future work, highlighting the interplay between folate and vitamin B6 in one carbon metabolism. Emerging insights include advances in metabolomic-based mass spectrometry and the use of live-cell metabolic labeling. Cancer is used as a focal point to dissect vitamin crosstalk and highlight new insights into the roles of folate and vitamin B6 in metabolic control. This collection of vitamin-based research detailing the trends of one carbon metabolism in human disease exemplifies how the future of personalized medicine could unfold using this new base of knowledge and ultimately provide next-generation therapeutics.

Molecular Basis of Nongenomic Actions of Thyroid Hormone.

Nongenomic actions of thyroid hormone are initiated by the hormone at receptors in the plasma membrane, in cytoplasm, or in mitochondria and do not require the interaction of nuclear thyroid hormone receptors (TRs) with their primary ligand, 3,5,3'-triiodo-l-thyronine (T3). Receptors involved in nongenomic actions may or may not have structural homologies with TRs. Certain nongenomic actions that originate at the plasma membrane may modify the state and function of intranuclear TRs. Reviewed here are nongenomic effects of the hormone-T3 or, in some cases, l-thyroxine (T4)-that are initiated at (a) truncated TRα isoforms, e.g., p30 TRα1, (b) cytoplasmic proteins, or (c) plasma membrane integrin αvß3. p30 TRα1 is not transcriptionally competent, binds T3 at the cell surface, and consequently expresses a number of important functions in bone cells. Nongenomic hormonal control of mitochondrial respiration involves a TRα isoform, and another truncated TRα isoform nongenomically regulates the state of cellular actin. Cytoplasmic hormone-binding proteins involved in nongenomic actions of thyroid hormone include ketimine reductase, pyruvate kinase, and TRß that shuttle among intracellular compartments. Functions of the receptor for T4 on integrin αvß3 include stimulation of proliferation of cancer and endothelial cells (angiogenesis) and regulation of transcription of cancer cell survival pathway genes. T4 serves as a prohormone for T3 in genomic actions of thyroid hormone, but T4 is a hormone at αvß3 and more important to cancer cell function than is T3. Thus, characterization of nongenomic actions of the hormone has served to broaden our understanding of the cellular roles of T3 and T4.

Correction: The Dkk3 gene encodes a vital intracellular regulator of cell proliferation.

[This corrects the article DOI: 10.1371/journal.pone.0181724.].

The Dkk3 gene encodes a vital intracellular regulator of cell proliferation.

Members of the Dickkopf (Dkk) family of Wnt antagonists interrupt Wnt-induced receptor assembly and participate in axial patterning and cell fate determination. One family member, DKK3, does not block Wnt receptor activation. Loss of Dkk3 expression in cancer is associated with hyperproliferation and dysregulated ß-catenin signaling, and ectopic expression of Dkk3 halts cancer growth. The molecular events mediating the DKK3-dependent arrest of ß-catenin-driven cell proliferation in cancer cells are unknown. Here we report the identification of a new intracellular gene product originating from the Dkk3 locus. This Dkk3b transcript originates from a second transcriptional start site located in intron 2 of the Dkk3 gene. It is essential for early mouse development and is a newly recognized regulator of ß-catenin signaling and cell proliferation. Dkk3b interrupts nuclear translocation ß-catenin by capturing cytoplasmic, unphosphorylated ß-catenin in an extra-nuclear complex with ß-TrCP. These data reveal a new regulator of one of the most studied signal transduction pathways in metazoans and provides a novel, completely untapped therapeutic target for silencing the aberrant ß-catenin signaling that drives hyperproliferation in many cancers.

A Salmonella nanoparticle mimic overcomes multidrug resistance in tumours.

Salmonella enterica serotype Typhimurium is a food-borne pathogen that also selectively grows in tumours and functionally decreases P-glycoprotein (P-gp), a multidrug resistance transporter. Here we report that the Salmonella type III secretion effector, SipA, is responsible for P-gp modulation through a pathway involving caspase-3. Mimicking the ability of Salmonella to reverse multidrug resistance, we constructed a gold nanoparticle system packaged with a SipA corona, and found this bacterial mimic not only accumulates in tumours but also reduces P-gp at a SipA dose significantly lower than free SipA. Moreover, the Salmonella nanoparticle mimic suppresses tumour growth with a concomitant reduction in P-gp when used with an existing chemotherapeutic drug (that is, doxorubicin). On the basis of our finding that the SipA Salmonella effector is fundamental for functionally decreasing P-gp, we engineered a nanoparticle mimic that both overcomes multidrug resistance in cancer cells and increases tumour sensitivity to conventional chemotherapeutics.

Nongenomic actions of thyroid hormone.

The nongenomic actions of thyroid hormone begin at receptors in the plasma membrane, mitochondria or cytoplasm. These receptors can share structural homologies with nuclear thyroid hormone receptors (TRs) that mediate transcriptional actions of T3, or have no homologies with TR, such as the plasma membrane receptor on integrin αvß3. Nongenomic actions initiated at the plasma membrane by T4 via integrin αvß3 can induce gene expression that affects angiogenesis and cell proliferation, therefore, both nongenomic and genomic effects can overlap in the nucleus. In the cytoplasm, a truncated TRα isoform mediates T4-dependent regulation of intracellular microfilament organization, contributing to cell and tissue structure. p30 TRα1 is another shortened TR isoform found at the plasma membrane that binds T3 and mediates nongenomic hormonal effects in bone cells. T3 and 3,5-diiodo-L-thyronine are important to the complex nongenomic regulation of cellular respiration in mitochondria. Thus, nongenomic actions expand the repertoire of cellular events controlled by thyroid hormone and can modulate TR-dependent nuclear events. Here, we review the experimental approaches required to define nongenomic actions of the hormone, enumerate the known nongenomic effects of the hormone and their molecular basis, and discuss the possible physiological or pathophysiological consequences of these actions.

Data gaps in toxicity testing of chemicals allowed in food in the United States.

In the United States, chemical additives cannot be used in food without an affirmative determination that their use is safe by FDA or additive manufacturer. Feeding toxicology studies designed to estimate the amount of a chemical additive that can be eaten safely provide the most relevant information. We analyze how many chemical additives allowed in human food have feeding toxicology studies in three toxicological information sources including the U.S. Food and Drug Administration's (FDA) database. Less than 38% of FDA-regulated additives have a published feeding study. For chemicals directly added to food, 21.6% have feeding studies necessary to estimate a safe level of exposure and 6.7% have reproductive or developmental toxicity data in FDA's database. A program is needed to fill these significant knowledge gaps by using in vitro and in silico methods complemented with targeted in vivo studies to ensure public health is protected.

High-fidelity gene synthesis by retrieval of sequence-verified DNA identified using high-throughput pyrosequencing.

The construction of synthetic biological systems involving millions of nucleotides is limited by the lack of high-quality synthetic DNA. Consequently, the field requires advances in the accuracy and scale of chemical DNA synthesis and in the processing of longer DNA assembled from short fragments. Here we describe a highly parallel and miniaturized method, called megacloning, for obtaining high-quality DNA by using next-generation sequencing (NGS) technology as a preparative tool. We demonstrate our method by processing both chemically synthesized and microarray-derived DNA oligonucleotides with a robotic system for imaging and picking beads directly off of a high-throughput pyrosequencing platform. The method can reduce error rates by a factor of 500 compared to the starting oligonucleotide pool generated by microarray. We use DNA obtained by megacloning to assemble synthetic genes. In principle, millions of DNA fragments can be sequenced, characterized and sorted in a single megacloner run, enabling constructive biology up to the megabase scale.

Molecular aspects of thyroid hormone actions.

Cellular actions of thyroid hormone may be initiated within the cell nucleus, at the plasma membrane, in cytoplasm, and at the mitochondrion. Thyroid hormone nuclear receptors (TRs) mediate the biological activities of T(3) via transcriptional regulation. Two TR genes, alpha and beta, encode four T(3)-binding receptor isoforms (alpha1, beta1, beta2, and beta3). The transcriptional activity of TRs is regulated at multiple levels. Besides being regulated by T(3), transcriptional activity is regulated by the type of thyroid hormone response elements located on the promoters of T(3) target genes, by the developmental- and tissue-dependent expression of TR isoforms, and by a host of nuclear coregulatory proteins. These nuclear coregulatory proteins modulate the transcription activity of TRs in a T(3)-dependent manner. In the absence of T(3), corepressors act to repress the basal transcriptional activity, whereas in the presence of T(3), coactivators function to activate transcription. The critical role of TRs is evident in that mutations of the TRbeta gene cause resistance to thyroid hormones to exhibit an array of symptoms due to decreasing the sensitivity of target tissues to T(3). Genetically engineered knockin mouse models also reveal that mutations of the TRs could lead to other abnormalities beyond resistance to thyroid hormones, including thyroid cancer, pituitary tumors, dwarfism, and metabolic abnormalities. Thus, the deleterious effects of mutations of TRs are more severe than previously envisioned. These genetic-engineered mouse models provide valuable tools to ascertain further the molecular actions of unliganded TRs in vivo that could underlie the pathogenesis of hypothyroidism. Actions of thyroid hormone that are not initiated by liganding of the hormone to intranuclear TR are termed nongenomic. They may begin at the plasma membrane or in cytoplasm. Plasma membrane-initiated actions begin at a receptor on integrin alphavbeta3 that activates ERK1/2 and culminate in local membrane actions on ion transport systems, such as the Na(+)/H(+) exchanger, or complex cellular events such as cell proliferation. Concentration of the integrin on cells of the vasculature and on tumor cells explains recently described proangiogenic effects of iodothyronines and proliferative actions of thyroid hormone on certain cancer cells, including gliomas. Thus, hormonal events that begin nongenomically result in effects in DNA-dependent effects. l-T(4) is an agonist at the plasma membrane without conversion to T(3). Tetraiodothyroacetic acid is a T(4) analog that inhibits the actions of T(4) and T(3) at the integrin, including angiogenesis and tumor cell proliferation. T(3) can activate phosphatidylinositol 3-kinase by a mechanism that may be cytoplasmic in origin or may begin at integrin alphavbeta3. Downstream consequences of phosphatidylinositol 3-kinase activation by T(3) include specific gene transcription and insertion of Na, K-ATPase in the plasma membrane and modulation of the activity of the ATPase. Thyroid hormone, chiefly T(3) and diiodothyronine, has important effects on mitochondrial energetics and on the cytoskeleton. Modulation by the hormone of the basal proton leak in mitochondria accounts for heat production caused by iodothyronines and a substantial component of cellular oxygen consumption. Thyroid hormone also acts on the mitochondrial genome via imported isoforms of nuclear TRs to affect several mitochondrial transcription factors. Regulation of actin polymerization by T(4) and rT(3), but not T(3), is critical to cell migration. This effect has been prominently demonstrated in neurons and glial cells and is important to brain development. The actin-related effects in neurons include fostering neurite outgrowth. A truncated TRalpha1 isoform that resides in the extranuclear compartment mediates the action of thyroid hormone on the cytoskeleton.

Effect of methyl iodide on deiodinase activity.

Methyl iodide (MeI) has been proposed as an alternative for methyl bromide in pre-plant soil fumigation applications that does not affect stratospheric ozone. Preliminary studies in rabbits noted fetal resorptions if the pregnant does were exposed to MeI during a critical period during gestation. In addition, abnormalities in thyroid hormone parameters were also observed in animals exposed to MeI. Since monodeiodination is the major metabolic pathway of the thyroid hormones, we examined the effect of MeI on deiodinase activity as a possible etiology for the alteration in thyroid hormone parameters and ultimate fetal demise. In vitro studies using tissue microsomes and cell culture showed that MeI has no effect on type I 5'-deiodinase (D1) or type II 5'-deiodinase (D2) at physiologically relevant concentrations. At high concentrations (>10 mM,>10,000 ppm), MeI caused a nonspecific inactivation of D1 and D2. Analysis of D1 and D2 activity in rats exposed by inhalation to increasing concentrations of MeI showed a significant decrease in enzyme activity at 100 ppm, while brain type III 5'-deiodinase (D3) was unaffected by MeI at the exposures studied. While the drop in D1 can be explained by the induction of a hypothyroid state in the exposed rats, there is no clear explanation for the fall in D2 levels. In the rabbit studies, there was a significant decrease in kidney D1 in the adult rabbits exposed to 20 ppm MeI. However, there was no effect on liver D1, brain D2, or placental D3 in the MeI-exposed rabbits. Similarly, there was no effect of MeI on fetal D1 or D2 activity. The lack of a significant direct effect of MeI on deiodinase activity and the absence of a change in placental or fetal deiodinase activity make it unlikely that alterations in deiodinase activity plays a role in the fetal resorptions in the MeI-exposed rabbits.

Medical malpractice reform and employer-sponsored health insurance premiums.

OBJECTIVE: Tort reform may affect health insurance premiums both by reducing medical malpractice premiums and by reducing the extent of defensive medicine. The objective of this study is to estimate the effects of noneconomic damage caps on the premiums for employer-sponsored health insurance. DATA SOURCES/STUDY SETTING: Employer premium data and plan/establishment characteristics were obtained from the 1999 through 2004 Kaiser/HRET Employer Health Insurance Surveys. Damage caps were obtained and dated based on state annotated codes, statutes, and judicial decisions. STUDY DESIGN: Fixed effects regression models were run to estimate the effects of the size of inflation-adjusted damage caps on the weighted average single premiums. DATA COLLECTION/EXTRACTION METHODS: State tort reform laws were identified using Westlaw, LEXIS, and statutory compilations. Legislative repeal and amendment of statutes and court decisions resulting in the overturning or repealing state statutes were also identified using LEXIS. PRINCIPAL FINDINGS: Using a variety of empirical specifications, there was no statistically significant evidence that noneconomic damage caps exerted any meaningful influence on the cost of employer-sponsored health insurance. CONCLUSIONS: The findings suggest that tort reforms have not translated into insurance savings.

Non-genomic actions of thyroid hormone in brain development.

Thyroid hormone (TH) is essential for neuronal migration and synaptogenesis in the developing brain. Assembly of neuronal circuits depends on guidance cues provided by the extracellular matrix. These cues are interpreted by the migrating neuron and its growing neurites through transmembrane signaling proteins anchored in place by the actin cytoskeleton. One of the best examples of a non-genomic action of thyroid hormone is its dynamic regulation of the number and quantity of actin fibers in astrocytes. Thyroxine (T4) and its transcriptionally inactive metabolite, 3',5',3-triiodothyronine (reverse T3) are responsible for modulating microfilament organization, while the transcriptional activator, 3',3,5-triiodothyronine (T3) is inert. The biological consequence of the loss of the actin filaments in astrocytes is the inability of the cell to anchor laminin, to its cell surface, and the loss of this key guidance molecule interrupts neurite pathfinding and neuronal migration. These data provide the essentials to construct a physiological pathway where TH-dependent regulation of the polymerization state of actin in the astrocyte and the developing neuron modulates the production and recognition of guidance cues--cues that if disrupted lead to abnormal neuronal migration and neuronal process formation--and lead to the morphological deficits observed in the cretinous brain.

Mechanisms of nongenomic actions of thyroid hormone.

The nongenomic actions of thyroid hormone require a plasma membrane receptor or nuclear receptors located in cytoplasm. The plasma membrane receptor is located on integrin alphaVbeta3 at the Arg-Gly-Asp recognition site important to the binding by the integrin of extracellular matrix proteins. l-Thyroxine (T(4)) is bound with greater affinity at this site than 3,5,3'-triiodo-l-thyronine (T(3)). Mitogen-activated protein kinase (MAPK; ERK1/2) transduces the hormone signal into complex cellular/nuclear events including angiogenesis and tumor cell proliferation. Acting at the integrin receptor and without cell entry, thyroid hormone can foster ERK1/2-dependent serine phosphorylation of nuclear thyroid hormone receptor-beta1 (TRbeta1) and de-repress the latter. The integrin receptor also mediates actions of the hormone on intracellular protein trafficking and on plasma membrane ion pumps, including the sodium/protein antiporter. Tetraiodothyroacetic (tetrac) is a T(4) analog that inhibits binding of iodothyronines to the integrin receptor and is a probe for the participation of this receptor in cellular actions of the hormone. Tetrac blocks thyroid hormone effects on angiogenesis and cancer cell proliferation. Acting on a truncated form of nuclear TRalpha1 (TRDeltaalpha1) located in cytoplasm, T(4) and 3,3',5'-triiodothyronine (reverse T(3)), but not T(3), cause conversion of soluble actin to fibrous (F) actin that is important to cell motility, e.g., in cells such as glia and neurons. Normal development of the central nervous system requires such motility. TRbeta1 in cytoplasm mediates action of T(3) on expression of certain genes via phosphatidylinositol 3-kinase (PI 3-K) and the protein kinase B/Akt pathway. PI 3-K and, possibly, cytoplasmic TRbeta1 are involved in stimulation by T(3) of insertion of Na,K-ATPase in the plasma membrane and of increase in activity of this pump. Because ambient thyroid hormone levels are constant in the euthyroid intact organism, these nongenomic hormone actions are likely to be contributors to basal rate-setting of transcription of certain genes and of complex cellular events such as angiogenesis and cancer cell proliferation.

Identification of the key residues responsible for the assembly of selenodeiodinases.

Type I deiodinase is the best characterized member of a small family of selenoenzymes catalyzing the bioactivation and disposal of thyroid hormone. This enzyme is an integral membrane protein composed of two 27-kDa subunits that assemble into a functional enzyme after translation using a highly conserved sequence of 16 amino acids in the C-terminal half of the polypeptide, (148)DFLXXYIXEAHXXDGW(163). In this study, we used alanine scanning mutagenesis to identify the key residues in this domain required for holoenzyme assembly. Overexpression of sequential alanine-substituted mutants of a dimerization domain-green fluorescent protein fusion showed that sequence (152)IYI(154) was required for type I enzyme assembly and that a catalytically active monomer was generated by a single I152A substitution. Overexpression of the sequential alanine-substituted dimerization domain mutants in type II selenodeiodinase-expressing cells showed that five residues ((153)FLIVY(157)) at the beginning and three residues ((164)SDG(166)) at the end of this region were required for the assembly of the type II enzyme. In vitro binding analysis revealed a free energy of association of -60 +/- 5 kJ/mol for the noncovalent interaction between dimerization domain monomers. These data identify and characterize the essential residues in the dimerization domain that are responsible for the post-translational assembly of selenodeiodinases.

Dynamic nongenomic actions of thyroid hormone in the developing rat brain.

Two well-characterized nongenomic actions of thyroid hormone in cultured brain tissues are: 1) regulation of type 2 iodothyronine 5'deiodinase (D2) activity and 2) regulation of actin polymerization. In particular, the latter is likely to have profound effects on neuronal migration in the developing brain. In this study, we determined whether these nongenomic actions also occurred in vivo during brain development. Neonatal hypothyroidism was induced by propylthiouracil given to pregnant dams beginning on d17 of gestation and continued throughout the neonatal period. On postnatal d 14, rats were injected with either cold or [(125)I]-labeled iodothyronines and killed sequentially after injection. In contrast to reports in the adult rat, all three iodothyronines readily and equally entered developing brain tissues. As expected, cerebrocortical D2 activity was markedly elevated in the hypothyroid brain and both reverse T(3) (rT(3)) and T(4) rapidly decreased D2 to euthyroid levels within 3 h. Furthermore, cerebellar G-actin content in the hypothyroid rat was approximately 5-fold higher than in the euthyroid rat. Again, both rT(3) and T(4) rapidly decreased the G-actin content by approximately 50%, with a reciprocal increase in F-actin content to euthyroid levels without altering total actin. Neither T(3) nor vehicle had any effect on D2 activity in the cortex or G- or F-actin content in the cerebellum. The thyroid hormone-dependent regulation of actin polymerization in the rat brain provides a mechanism by which this morphogenic hormone can influence neuronal migration independent of the need for altered gene transcription. Furthermore, these data suggest a prominent role for rT(3) during brain development.

Neuroanatomical pathways for thyroid hormone feedback in the human hypothalamus.

CONTEXT: Recent findings point to an increasing number of hypothalamic proteins involved in the central regulation of thyroid hormone feedback. The functional neuroanatomy of these proteins in the human hypothalamus is largely unknown at present. OBJECTIVE: The aim of this study was to report the distribution of type II and type III deiodinase (D2 and D3) as well as the recently identified T(3) transporter, monocarboxylate transporter 8 (MCT8), in the human hypothalamus. DESIGN: The study included enzyme activity assays, immunocytochemical studies, and mRNA in situ hybridizations in postmortem human hypothalamus (n = 9). RESULTS: D2 immunoreactivity is prominent in glial cells of the infundibular nucleus/median eminence, blood vessels, and cells lining the third ventricle. By contrast, both D3 and MCT8 are expressed by neurons of the paraventricular (PVN), supraoptic, and infundibular nucleus (IFN). In support of these immunocytochemical data, D2 and D3 enzyme activities are detectable in the mediobasal human hypothalamus. Combined D2, D3, MCT8, and thyroid hormone receptor immunohistochemistry and TRH mRNA in situ hybridization clearly showed that D3, MCT8, and thyroid hormone receptor isoforms are all expressed in TRH neurons of the PVN, whereas D2 is not. CONCLUSIONS AND IMPLICATIONS: Based on these findings, we propose three possible routes for thyroid hormone feedback on TRH neurons in the human PVN: 1) local thyroid hormone uptake from the vascular compartment within the PVN, 2) thyroid hormone uptake from the cerebrospinal fluid in the third ventricle followed by transport to TRH neurons in the PVN or IFN neurons projecting to TRH neurons in the PVN, and 3) thyroid hormone sensing in the IFN of the mediobasal hypothalamus by neurons projecting to TRH neurons in the PVN.

Characterization of the protein dimerization domain responsible for assembly of functional selenodeiodinases.

Thyroid hormone metabolism is catalyzed by a small family of selenoenzymes. Type I deiodinase (D1) is the best characterized family member and is an integral membrane protein composed of two 27-kDa subunits that assemble to a functional holoenzyme after translation. To characterize the protein domain(s) responsible for this post-translational assembly event, we used deletion/truncation analysis coupled with immune depletion assays to map the dimerization domain of D1. The results of our studies show that a highly conserved sequence of 16 amino acids in the C-terminal half of the D1 subunit, -D148FL-YI-EAH-DGW163-, serves as the dimerization domain. Based on the high conservation of this domain, we synthesized a novel bait peptide-green fluorescent protein fusion probe (DDD(GFP)) to examine holoenzyme assembly of other family members. Overexpression of either the DDD(GFP) or an inert D1 subunit (M4) into SeD2 (accession number U53505)-expressing C6 cells specifically led to the loss of >90% of the catalytic activity. Catalytically inactive D2 heterodimers composed of SeD2: DDD(GFP) subunits were rescued by specific immune precipitation with anti-SeD2 IgG, suggesting that SeD2 requires two functional subunits to assemble a catalytically active holoenzyme. These findings identify and characterize the essential dimerization domain responsible for post-translational assembly of selenodeiodinases and show that family members can intermingle through this highly conserved protein domain.

Regulation of cerebellar neuronal migration and neurite outgrowth by thyroxine and 3,3',5'-triiodothyronine.

The timing of granule cell migration in the developing cerebellum is regulated by thyroid hormone. Granule cell migration depends on the recognition of extracellular neuronal guidance molecule(s), such as laminin, and this, in turn, requires cell surface adhesion molecules (integrins) that are anchored on the cell membrane by the actin cytoskeleton. While many of the actions of thyroid hormone, specifically 3,5,3'-triiodothyronine (T3), are mediated by regulated gene expression, both thyroxine (T4) and 3,3',5'-triiodothyronine (rT3) also exert direct, positive control of the quantity of polymerized actin in cultured astrocytes without affecting gene expression. T4-dependent actin polymerization has been shown to (i) participate in the immobilization of laminin to the cell surface, (ii) help deposit laminin in the molecular layer of the developing cerebellum, and (iii) anchor integrin(s) that recognize laminin present in the extracellular matrix. In this study, we show that both T4 and rT3, but not T3, directly regulate the F-actin content of elongating neurites of cerebellar neurons. T4 and rT3 also promoted extensive granule cell migration from cerebellar explants, as well as, dense cell clustering and extensive neuronal process formation when granule cells were grown on a laminin-coated surface. Both granule cell migration and neuronal process outgrowth were markedly attenuated by the addition of integrin-blocking antibodies or binding peptides, by the absence of thyroid hormone or the presence of T3. These data suggest that the T4-dependent actin polymerization in developing neurons is necessary for these migrating cells to recognize the laminin guidance molecule, thereby providing a novel molecular mechanism for the profound influence of thyroid hormone on brain development that is independent of regulated gene expression.

Ultrafiltration for proteomic sample preparation.

Proteome analysis represents significant challenges to the existing sample preparation techniques. Traditional methods, such as two-dimensional electrophoresis, typically separate high-molecular-weight proteins while discarding low-molecular-weight species. This approach is well justified considering the complexity of any proteome. However, it is desirable to extract the maximum amount of information from each sample to investigate the entire range of biomolecules. We have demonstrated that ultrafiltration not only improves two-dimensional electrophoresis (2-DE) resolution of the protein fraction but also yields the low-molecular-weight fraction amenable for further analysis by high-resolution mass spectrometry. This approach was successfully adapted to the variety of biological samples including cell and tissue lysates and serum. Therefore, ultrafiltration offers an alternative sample preparation technique that enables more thorough analysis of a proteome.