BR3: a biologically inspired fish-like robot actuated by SMA-based artificial muscles

Los peces son animales, donde en la mayoría de los casos, son considerados como nadadores muy eficientes y con una alta capacidad de maniobra. En general los peces se caracterizan por su capacidad de maniobra, locomoción silencioso, giros y partidas rápidas y viajes de larga distancia. Los estudios han identificado varios tipos de locomoción que los peces usan para generar maniobras y natación constante. A bajas velocidades la mayoría de los peces utilizan sus aletas pares y / o impares para su locomoción, que ofrecen una mayor maniobrabilidad y mejor eficiencia de propulsión. A altas velocidades la locomoción implica el cuerpo y / o aleta caudal porque esto puede lograr un mayor empuje y aceleración. Estas características pueden inspirar el diseo y fabricación de una piel muy flexible, una aleta caudal mórfica y una espina dorsal no articulada con una gran capacidad de maniobra. Esta tesis presenta el desarrollo de un novedoso pez robot bio-inspirado y biomimético llamado BR3, inspirado en la capacidad de maniobra y nado constante de los peces vertebrados. Inspirado por la morfología de los peces Micropterus salmoides o también conocido como lubina negra, el robot BR3 utiliza su fundamento biológico para desarrollar modelos y métodos matemáticos precisos que permiten imitar la locomoción de los peces reales. Los peces Largemouth Bass pueden lograr un nivel increíble de maniobrabilidad y eficacia de la propulsión mediante la combinación de los movimientos ondulatorios y aletas morficas. Para imitar la locomoción de los peces reales en una contraparte artificial se necesita del análisis de tecnologías de actuación alternativos, como arreglos de fibras musculares en lugar de servo actuadores o motores DC estándar, así como un material flexible que proporciona una estructura continua sin juntas. Las aleaciones con memoria de forma (SMAs) proveen la posibilidad de construir robots livianos, que no emiten ruido, sin motores, sin juntas y sin engranajes. Asi es como un pez robot submarino se ha desarrollado y cuyos movimientos son generados mediante SMAs. Estos actuadores son los adecuados para doblar la espina dorsal continua del pez robot, que a su vez provoca un cambio en la curvatura del cuerpo. Este tipo de arreglo estructural está inspirado en los músculos rojos del pescado, que son usados principalmente durante la natación constante para la flexión de una estructura flexible pero casi incompresible como lo es la espina dorsal de pescado. Del mismo modo la aleta caudal se basa en SMAs y se modifica para llevar a cabo el trabajo necesario. La estructura flexible proporciona empuje y permite que el BR3 nade. Por otro lado la aleta caudal mórfica proporciona movimientos de balanceo y guiada. Motivado por la versatilidad del BR3 para imitar todos los modos de natación (anguilliforme, carangiforme, subcarangiforme y tunniforme) se propone un controlador de doblado y velocidad. La ley de control de doblado y velocidad incorpora la información del ángulo de curvatura y de la frecuencia para producir el modo de natación deseado y a su vez controlar la velocidad de natación. Así mismo de acuerdo con el hecho biológico de la influencia de la forma de la aleta caudal en la maniobrabilidad durante la natación constante se propone un control de actitud. Esta novedoso robot pescado es el primero de su tipo en incorporar sólo SMAs para doblar una estructura flexible continua y sin juntas y engranajes para producir empuje e imitar todos los modos de natación, así como la aleta caudal que es capaz de cambiar su forma. Este novedoso diseo mecatrónico presenta un futuro muy prometedor para el diseo de vehículos submarinos capaces de modificar su forma y nadar mas eficientemente. La nueva metodología de control propuesto en esta tesis proporcionan una forma totalmente nueva de control de robots basados en SMAs, haciéndolos energéticamente más eficientes y la incorporación de una aleta caudal mórfica permite realizar maniobras más eficientemente. En su conjunto, el proyecto BR3 consta de cinco grandes etapas de desarrollo: • Estudio y análisis biológico del nado de los peces con el propósito de definir criterios de diseño y control. • Formulación de modelos matemáticos que describan la: i) cinemática del cuerpo, ii) dinámica, iii) hidrodinámica iv) análisis de los modos de vibración y v) actuación usando SMA. Estos modelos permiten estimar la influencia de modular la aleta caudal y el doblado del cuerpo en la producción de fuerzas de empuje y fuerzas de rotación necesarias en las maniobras y optimización del consumo de energía. • Diseño y fabricación de BR3: i) estructura esquelética de la columna vertebral y el cuerpo, ii) mecanismo de actuación basado en SMAs para el cuerpo y la aleta caudal, iii) piel artificial, iv) electrónica embebida y v) fusión sensorial. Está dirigido a desarrollar la plataforma de pez robot BR3 que permite probar los métodos propuestos. • Controlador de nado: compuesto por: i) control de las SMA (modulación de la forma de la aleta caudal y regulación de la actitud) y ii) control de nado continuo (modulación de la velocidad y doblado). Está dirigido a la formulación de los métodos de control adecuados que permiten la modulación adecuada de la aleta caudal y el cuerpo del BR3. • Experimentos: está dirigido a la cuantificación de los efectos de: i) la correcta modulación de la aleta caudal en la producción de rotación y su efecto hidrodinámico durante la maniobra, ii) doblado del cuerpo para la producción de empuje y iii) efecto de la flexibilidad de la piel en la habilidad para doblarse del BR3. También tiene como objetivo demostrar y validar la hipótesis de mejora en la eficiencia de la natación y las maniobras gracias a los nuevos métodos de control presentados en esta tesis. A lo largo del desarrollo de cada una de las cinco etapas, se irán presentando los retos, problemáticas y soluciones a abordar. Los experimentos en canales de agua estarán orientados a discutir y demostrar cómo la aleta caudal y el cuerpo pueden afectar considerablemente la dinámica / hidrodinámica de natación / maniobras y cómo tomar ventaja de la modulación de curvatura que la aleta caudal mórfica y el cuerpo permiten para cambiar correctamente la geometría de la aleta caudal y del cuerpo durante la natación constante y maniobras. ABSTRACT Fishes are animals where in most cases are considered as highly manoeuvrable and effortless swimmers. In general fishes are characterized for his manoeuvring skills, noiseless locomotion, rapid turning, fast starting and long distance cruising. Studies have identified several types of locomotion that fish use to generate maneuvering and steady swimming. At low speeds most fishes uses median and/or paired fins for its locomotion, offering greater maneuverability and better propulsive efficiency At high speeds the locomotion involves the body and/or caudal fin because this can achieve greater thrust and accelerations. This can inspire the design and fabrication of a highly deformable soft artificial skins, morphing caudal fins and non articulated backbone with a significant maneuverability capacity. This thesis presents the development of a novel bio-inspired and biomimetic fishlike robot (BR3) inspired by the maneuverability and steady swimming ability of ray-finned fishes (Actinopterygii, bony fishes). Inspired by the morphology of the Largemouth Bass fish, the BR3 uses its biological foundation to develop accurate mathematical models and methods allowing to mimic fish locomotion. The Largemouth Bass fishes can achieve an amazing level of maneuverability and propulsive efficiency by combining undulatory movements and morphing fins. To mimic the locomotion of the real fishes on an artificial counterpart needs the analysis of alternative actuation technologies more likely muscle fiber arrays instead of standard servomotor actuators as well as a bendable material that provides a continuous structure without joins. The Shape Memory Alloys (SMAs) provide the possibility of building lightweight, joint-less, noise-less, motor-less and gear-less robots. Thus a swimming underwater fish-like robot has been developed whose movements are generated using SMAs. These actuators are suitable for bending the continuous backbone of the fish, which in turn causes a change in the curvature of the body. This type of structural arrangement is inspired by fish red muscles, which are mainly recruited during steady swimming for the bending of a flexible but nearly incompressible structure such as the fishbone. Likewise the caudal fin is based on SMAs and is customized to provide the necessary work out. The bendable structure provides thrust and allows the BR3 to swim. On the other hand the morphing caudal fin provides roll and yaw movements. Motivated by the versatility of the BR3 to mimic all the swimming modes (anguilliform, caranguiform, subcaranguiform and thunniform) a bending-speed controller is proposed. The bending-speed control law incorporates bend angle and frequency information to produce desired swimming mode and swimming speed. Likewise according to the biological fact about the influence of caudal fin shape in the maneuverability during steady swimming an attitude control is proposed. This novel fish robot is the first of its kind to incorporate only SMAs to bend a flexible continuous structure without joints and gears to produce thrust and mimic all the swimming modes as well as the caudal fin to be morphing. This novel mechatronic design is a promising way to design more efficient swimming/morphing underwater vehicles. The novel control methodology proposed in this thesis provide a totally new way of controlling robots based on SMAs, making them more energy efficient and the incorporation of a morphing caudal fin allows to perform more efficient maneuvers. As a whole, the BR3 project consists of five major stages of development: • Study and analysis of biological fish swimming data reported in specialized literature aimed at defining design and control criteria. • Formulation of mathematical models for: i) body kinematics, ii) dynamics, iii) hydrodynamics, iv) free vibration analysis and v) SMA muscle-like actuation. It is aimed at modelling the e ects of modulating caudal fin and body bend into the production of thrust forces for swimming, rotational forces for maneuvering and energy consumption optimisation. • Bio-inspired design and fabrication of: i) skeletal structure of backbone and body, ii) SMA muscle-like mechanisms for the body and caudal fin, iii) the artificial skin, iv) electronics onboard and v) sensor fusion. It is aimed at developing the fish-like platform (BR3) that allows for testing the methods proposed. • The swimming controller: i) control of SMA-muscles (morphing-caudal fin modulation and attitude regulation) and ii) steady swimming control (bend modulation and speed modulation). It is aimed at formulating the proper control methods that allow for the proper modulation of BR3’s caudal fin and body. • Experiments: it is aimed at quantifying the effects of: i) properly caudal fin modulation into hydrodynamics and rotation production for maneuvering, ii) body bending into thrust generation and iii) skin flexibility into BR3 bending ability. It is also aimed at demonstrating and validating the hypothesis of improving swimming and maneuvering efficiency thanks to the novel control methods presented in this thesis. This thesis introduces the challenges and methods to address these stages. Waterchannel experiments will be oriented to discuss and demonstrate how the caudal fin and body can considerably affect the dynamics/hydrodynamics of swimming/maneuvering and how to take advantage of bend modulation that the morphing-caudal fin and body enable to properly change caudal fin and body’ geometry during steady swimming and maneuvering.

Overexpression of thioredoxin in transgenic mice attenuates focal ischemic brain damage

Thioredoxin (TRX) plays important biological roles both in intra- and extracellular compartments, including in regulation of various intracellular molecules via thiol redox control. We produced TRX overexpressing mice and confirmed that there were no anatomical and physiological differences between wild-type (WT) mice and TRX transgenic (Tg) mice. In the present study we subjected mice to focal brain ischemia to shed light on the role of TRX in brain ischemic injury. At 24 hr after middle cerebral artery occlusion, infarct areas and volume were significantly smaller in Tg mice than in WT mice. Moreover neurological deficit was ameliorated in Tg mice compared with WT mice. Protein carbonyl content, a marker of cellular protein oxidation, in Tg mice showed less increase than did that of WT mice after the ischemic insult. Furthermore, c-fos expression in Tg mice was stronger than in WT mice 1 hr after ischemia. Our results suggest that transgene expression of TRX decreased ischemic neuronal injury and that TRX and the redox state modified by TRX play a crucial role in brain damage during stroke.

Regulation of endothelial monocyte-activating polypeptide II release by apoptosis

Endothelial monocyte-activating polypeptide II (EMAP II) is a proinflammatory cytokine and a chemoattractant for monocytes. We show here that, in the mouse embryo, EMAP II mRNA was most abundant at sites of tissue remodeling where many apoptotic cells could be detected by terminal deoxynucleotidyltransferase-mediated dUTP end labeling. Removal of dead cells is known to require macrophages, and these were found to colocalize with areas of EMAP II mRNA expression and programmed cell death. In cultured cells, post-translational processing of pro-EMAP II protein to the mature released EMAP II form (23 kDa) occurred coincidentally with apoptosis. Cleavage of pro-EMAP II could be abrogated in cultured cells by using a peptide-based inhibitor, which competes with the ASTD cleavage site of pro-EMAP II. Our results suggest that the coordinate program of cell death includes activation of a caspase-like activity that initiates the processing of a cytokine responsible for macrophage attraction to the sites of apoptosis.

A versatile synthetic dimerizer for the regulation of protein–protein interactions

The use of low molecular weight organic compounds to induce dimerization or oligomerization of engineered proteins has wide-ranging utility in biological research as well as in gene and cell therapies. Chemically induced dimerization can be used to activate intracellular signal transduction pathways or to control the activity of a bipartite transcription factor. Dimerizer systems based on the natural products cyclosporin, FK506, rapamycin, and coumermycin have been described. However, owing to the complexity of these compounds, adjusting their binding or pharmacological properties by chemical modification is difficult. We have investigated several families of readily prepared, totally synthetic, cell-permeable dimerizers composed of ligands for human FKBP12. These molecules have significantly reduced complexity and greater adaptability than natural product dimers. We report here the efficacies of several of these new synthetic compounds in regulating two types of protein dimerization events inside engineered cells—–induction of apoptosis through dimerization of engineered Fas proteins and regulation of transcription through dimerization of transcription factor fusion proteins. One dimerizer in particular, AP1510, proved to be exceptionally potent and versatile in all experimental contexts tested.

Up-regulation of specific tyrosinase mRNAs in mouse melanomas with the c2j gene substituted for the wild-type tyrosinase allele: Utilization in design of syngeneic immunotherapy models

The expression of cell-specialization genes is likely to be changing in tumor cells as their differentiation declines. Functional changes in these genes might yield unusual peptide epitopes with anti-tumor potential and could occur without modification in the DNA sequence of the gene. Melanomas undergo a characteristic decline in melanization that may reflect altered contributions of key melanocytic genes such as tyrosinase. Quantitative reverse transcriptase–PCR of the wild-type (C) tyrosinase gene in transgenic (C57BL/6 strain) mouse melanomas has revealed a shift toward alternative splicing of the pre-mRNA that generated increased levels of the Δ1b and Δ1d mRNA splice variants. The spontaneous c2j albino mutation of tyrosinase (in the C57BL/6 strain) changes the pre-mRNA splicing pattern. In c2j/c2j melanomas, alternative splicing was again increased. However, while some mRNAs (notably Δ1b) present in C/C were obligatorily absent, others (Δ3 and Δ1d) were elevated. In c2j/c2j melanomas, the percentage of total tyrosinase transcripts attributable to Δ3 reached approximately 2-fold the incidence in c2j/c2j or C/C skin melanocytes. The percentage attributable to Δ1d rose to approximately 2-fold the incidence in c2j/c2j skin, and to 10-fold that in C/C skin. These differences provide a basis for unique mouse models in which the melanoma arises in skin grafted from a C/C or c2j/c2j transgenic donor to a transgenic host of the same or opposite tyrosinase genotype. Immunotherapy designs then could be based on augmenting those antigenic peptides that are novel or overrepresented in a tumor relative to the syngeneic host.

Population regulation in snowshoe hare and Canadian lynx: Asymmetric food web configurations between hare and lynx

The snowshoe hare and the Canadian lynx in the boreal forests of North America show 9- to 11-year density cycles. These are generally assumed to be linked to each other because lynx are specialist predators on hares. Based on time series data for hare and lynx, we show that the dominant dimensional structure of the hare series appears to be three whereas that of the lynx is two. The three-dimensional structure of the hare time series is hypothesized to be due to a three-trophic level model in which the hare may be seen as simultaneously regulated from below and above. The plant species in the hare diet appear compensatory to one another, and the predator species may, likewise, be seen as an internally compensatory guild. The lynx time series are, in contrast, consistent with a model of donor control in which their populations are regulated from below by prey availability. Thus our analysis suggests that the classic view of a symmetric hare–lynx interaction is too simplistic. Specifically, we argue that the classic food chain structure is inappropriate: the hare is influenced by many predators other than the lynx, and the lynx is primarily influenced by the snowshoe hare.

Rapid down-regulation of mammalian Period genes during behavioral resetting of the circadian clock

The pervasive role of circadian clocks in regulating physiology and behavior is widely recognized. Their adaptive value is their ability to be entrained by environmental cues such that the internal circadian phase is a reliable predictor of solar time. In mammals, both light and nonphotic behavioral cues can entrain the principal oscillator of the hypothalamic suprachiasmatic nuclei (SCN). However, although light can advance or delay the clock during circadian night, behavioral events trigger phase advances during the subjective day, when the clock is insensitive to light. The recent identification of Period (Per) genes in mammals, homologues of dperiod, which encodes a core element of the circadian clockwork in Drosophila, now provides the opportunity to explain circadian timing and entrainment at a molecular level. In mice, expression of mPer1 and mPer2 in the SCN is rhythmic and acutely up-regulated by light. Moreover, the temporal relations between mRNA and protein cycles are consistent with a clock based on a transcriptional/translational feedback loop. Here we describe circadian oscillations of Per1 and Per2 in the SCN of the Syrian hamster, showing that PER1 protein and mRNA cycles again behave in a manner consistent with a negative-feedback oscillator. Furthermore, we demonstrate that nonphotic resetting has the opposite effect to light: acutely down-regulating these genes. Their sensitivity to nonphotic resetting cues supports their proposed role as core elements of the circadian oscillator. Moreover, this study provides an explanation at the molecular level for the contrasting but convergent effects of photic and nonphotic cues on the clock.

Distinct, Constitutively Active MAPK Phosphatases Function in Xenopus Oocytes: Implications for p42 MAPK Regulation In Vivo

Xenopus oocyte maturation requires the phosphorylation and activation of p42 mitogen-activated protein kinase (MAPK). Likewise, the dephosphorylation and inactivation of p42 MAPK are critical for the progression of fertilized eggs out of meiosis and through the first mitotic cell cycle. Whereas the kinase responsible for p42 MAPK activation is well characterized, little is known concerning the phosphatases that inactivate p42 MAPK. We designed a microinjection-based assay to examine the mechanism of p42 MAPK dephosphorylation in intact oocytes. We found that p42 MAPK inactivation is mediated by at least two distinct phosphatases, an unidentified tyrosine phosphatase and a protein phosphatase 2A–like threonine phosphatase. The rates of tyrosine and threonine dephosphorylation were high and remained constant throughout meiosis, indicating that the dramatic changes in p42 MAPK activity seen during meiosis are primarily attributable to changes in MAPK kinase activity. The overall control of p42 MAPK dephosphorylation was shared among four partially rate-determining dephosphorylation reactions, with the initial tyrosine dephosphorylation of p42 MAPK being the most critical of the four. Our findings provide biochemical and kinetic insight into the physiological mechanism of p42 MAPK inactivation.

Selenium redox biochemistry of zinc–sulfur coordination sites in proteins and enzymes

Selenium has been increasingly recognized as an essential element in biology and medicine. Its biochemistry resembles that of sulfur, yet differs from it by virtue of both redox potentials and stabilities of its oxidation states. Selenium can substitute for the more ubiquitous sulfur of cysteine and as such plays an important role in more than a dozen selenoproteins. We have chosen to examine zinc–sulfur centers as possible targets of selenium redox biochemistry. Selenium compounds release zinc from zinc/thiolate-coordination environments, thereby affecting the cellular thiol redox state and the distribution of zinc and likely of other metal ions. Aromatic selenium compounds are excellent spectroscopic probes of the otherwise relatively unstable functional selenium groups. Zinc-coordinated thiolates, e.g., metallothionein (MT), and uncoordinated thiolates, e.g., glutathione, react with benzeneseleninic acid (oxidation state +2), benzeneselenenyl chloride (oxidation state 0) and selenocystamine (oxidation state −1). Benzeneseleninic acid and benzeneselenenyl chloride react very rapidly with MT and titrate substoichiometrically and with a 1:1 stoichiometry, respectively. Selenium compounds also catalyze the release of zinc from MT in peroxidation and thiol/disulfide-interchange reactions. The selenoenzyme glutathione peroxidase catalytically oxidizes MT and releases zinc in the presence of t-butyl hydroperoxide, suggesting that this type of redox chemistry may be employed in biology for the control of metal metabolism. Moreover, selenium compounds are likely targets for zinc/thiolate coordination centers in vivo, because the reactions are only partially suppressed by excess glutathione. This specificity and the potential to undergo catalytic reactions at low concentrations suggests that zinc release is a significant aspect of the therapeutic antioxidant actions of selenium compounds in antiinflammatory and anticarcinogenic agents.

Regulation of the OxyR transcription factor by hydrogen peroxide and the cellular thiol—disulfide status

The Escherichia coli transcription factor OxyR is activated by the formation of an intramolecular disulfide bond and subsequently is deactivated by enzymatic reduction of the disulfide bond. Here we show that OxyR can be activated by two possible pathways. In mutants defective in the cellular disulfide-reducing systems, OxyR is constitutively activated by a change in the thiol—disulfide redox status in the absence of added oxidants. In wild-type cells, OxyR is activated by hydrogen peroxide. By monitoring the presence of the OxyR disulfide bond after exposure to hydrogen peroxide in vivo and in vitro, we also show that the kinetics of OxyR oxidation by low concentrations of hydrogen peroxide is significantly faster than the kinetics of OxyR reduction, allowing for transient activation in an overall reducing environment. We propose that the activity of OxyR in vivo is determined by the balance between hydrogen peroxide levels and the cellular redox environment.

Multistep regulation of DNA replication by Cdk phosphorylation of HsCdc6

We have characterized HsCdc6, a human protein homologous to the budding yeast Cdc6p that is essential for DNA replication. We show that, unlike Cdc6p, the levels of HsCdc6 protein remain constant throughout the cell cycle in human cells. However, phosphorylation of HsCdc6 is regulated during the cell cycle. HsCdc6 is an excellent substrate for Cdk2 in vitro and is phosphorylated in vivo at three sites (Ser-54, Ser-74, and Ser-106) that are phosphorylated by Cdk2 in vitro, strongly suggesting that HsCdc6 is an in vivo Cdk substrate. HsCdc6 is nuclear in G1, but translocates to the cytoplasm at the start of S phase via Crm1-dependent export. An HsCdc6A1A2A3 mutant, which mimics unphosphorylated HsCdc6, is exclusively nuclear, and its expression inhibits initiation of DNA replication. An HsCdc6E1E2E3 mutant, which mimics phosphorylated HsCdc6, is exclusively cytoplasmic and is not associated with the chromatin/nuclear matrix fraction. Based on these results, we propose that phosphorylation of HsCdc6 by Cdks regulates DNA replication of at least two steps: first, by promoting initiation of DNA replication and, second, through nuclear exclusion preventing DNA rereplication.

Crystal structures of bovine milk xanthine dehydrogenase and xanthine oxidase: Structure-based mechanism of conversion

Mammalian xanthine oxidoreductases, which catalyze the last two steps in the formation of urate, are synthesized as the dehydrogenase form xanthine dehydrogenase (XDH) but can be readily converted to the oxidase form xanthine oxidase (XO) by oxidation of sulfhydryl residues or by proteolysis. Here, we present the crystal structure of the dimeric (Mr, 290,000) bovine milk XDH at 2.1-Å resolution and XO at 2.5-Å resolution and describe the major changes that occur on the proteolytic transformation of XDH to the XO form. Each molecule is composed of an N-terminal 20-kDa domain containing two iron sulfur centers, a central 40-kDa flavin adenine dinucleotide domain, and a C-terminal 85-kDa molybdopterin-binding domain with the four redox centers aligned in an almost linear fashion. Cleavage of surface-exposed loops of XDH causes major structural rearrangement of another loop close to the flavin ring (Gln 423—Lys 433). This movement partially blocks access of the NAD substrate to the flavin adenine dinucleotide cofactor and changes the electrostatic environment of the active site, reflecting the switch of substrate specificity observed for the two forms of this enzyme.

Enrichment for murine keratinocyte stem cells based on cell surface phenotype

The identification and physical isolation of epithelial stem cells is critical to our understanding of their growth regulation during homeostasis, wound healing, and carcinogenesis. These stem cells remain poorly characterized because of the absence of specific molecular markers that permit us to distinguish them from their progeny, the transit amplifying (TA) cells, which have a more restricted proliferative potential. Cell kinetic analyses have permitted the identification of murine keratinocyte stem cells (KSCs) as slowly cycling cells that retain [3H]thymidine ([3H]Tdr) label, termed label-retaining cells (LRCs), whereas TA cells are visualized as rapidly cycling cells after a single pulse of [3H]Tdr, termed pulse-labeled cells (PLCs). Here, we report on the successful separation of KSCs from TA cells through the combined use of in vivo cell kinetic analysis and fluorescence-activated cell sorting. Specifically, we demonstrate that murine dorsal keratinocytes characterized by their high levels of α6 integrin and low to undetectable expression of the transferrin receptor (CD71) termed α6briCD71dim cells, are enriched for epithelial stem cells because they represent a minor (≈8%) and quiescent subpopulation of small blast-like cells, with a high nuclear:cytoplasmic ratio, containing ≈70% of label-retaining cells, the latter being a well documented characteristic of stem cells. Conversely, TA cells could be enriched in a phenotypically distinct subpopulation termed α6briCD71bri, representing the majority (≈60%) of basal keratinocytes that are actively cycling, and importantly contain ≈70% of [3H]Tdr pulse-labeled cells. Importantly, immunostaining of dorsal skin revealed the presence of CD71dim cells in the hair follicle bulge region, a well documented location for KSCs.

Vitamin B2-based blue-light photoreceptors in the retinohypothalamic tract as the photoactive pigments for setting the circadian clock in mammals

In mammals the retina contains photoactive molecules responsible for both vision and circadian photoresponse systems. Opsins, which are located in rods and cones, are the pigments for vision but it is not known whether they play a role in circadian regulation. A subset of retinal ganglion cells with direct projections to the suprachiasmatic nucleus (SCN) are at the origin of the retinohypothalamic tract that transmits the light signal to the master circadian clock in the SCN. However, the ganglion cells are not known to contain rhodopsin or other opsins that may function as photoreceptors. We have found that the two blue-light photoreceptors, cryptochromes 1 and 2 (CRY1 and CRY2), recently discovered in mammals are specifically expressed in the ganglion cell and inner nuclear layers of the mouse retina. In addition, CRY1 is expressed at high level in the SCN and oscillates in this tissue in a circadian manner. These data, in conjunction with the established role of CRY2 in photoperiodism in plants, lead us to propose that mammals have a vitamin A-based photopigment (opsin) for vision and a vitamin B2-based pigment (cryptochrome) for entrainment of the circadian clock.

Sulfur K-edge x-ray absorption spectroscopy: A spectroscopic tool to examine the redox state of S-containing metabolites in vivo

The sulfur K-edge x-ray absorption spectra for the amino acids cysteine and methionine and their corresponding oxidized forms cystine and methionine sulfoxide are presented. Distinct differences in the shape of the edge and the inflection point energy for cysteine and cystine are observed. For methionine sulfoxide the inflection point energy is 2.8 eV higher compared with methionine. Glutathione, the most abundant thiol in animal cells, also has been investigated. The x-ray absorption near-edge structure spectrum of reduced glutathione resembles that of cysteine, whereas the spectrum of oxidized glutathione resembles that of cystine. The characteristic differences between the thiol and disulfide spectra enable one to determine the redox status (thiol to disulfide ratio) in intact biological systems, such as unbroken cells, where glutathione and cyst(e)ine are the two major sulfur-containing components. The sulfur K-edge spectra for whole human blood, plasma, and erythrocytes are shown. The erythrocyte sulfur K-edge spectrum is similar to that of fully reduced glutathione. Simulation of the plasma spectrum indicated 32% thiol and 68% disulfide sulfur. The whole blood spectrum can be simulated by a combination of 46% disulfide and 54% thiol sulfur.