Effects of Ultraviolet Light on Melanocyte Differentiation: Studies with Mouse Neural Crest Cells and Neural Crest‐derived Cell Lines

To evaluate the etiologic role of ultraviolet (UV) radiation in acquired dermal melanocytosis (ADM), we investigated the effects of UVA and UVB irradiation on the development and differentiation of melanocytes in primary cultures of mouse neural crest cells (NCC) by counting the numbers of cells positive for KIT (the receptor for stem cell factor) and for the L ‐3,4‐dihydroxyphenylalanine (DOPA) oxidase reaction. No significant differences were found in the number of KIT‐ or DOPA‐positive cells between the UV‐irradiated cultures and the non‐irradiated cultures. We then examined the effects of UV light on KIT‐positive cell lines derived from mouse NCC cultures. Irradiation with UVA but not with UVB inhibited the tyrosinase activity in a tyrosinase‐positive cell line (NCCmelan5). Tyrosinase activity in the cells was markedly enhanced by treatment with α‐melanocyte‐stimulating hormone (α‐MSH), but that stimulation was inhibited by UVA or by UVB irradiation. Irradiation with UVA or UVB did not induce tyrosinase activity in a tyrosinase‐negative cell line (NCCmelb4). Levels of KIT expression in NCCmelan5 cells and in NCCmelb4 cells were significantly decreased after UV irradiation. Phosphorylation levels of extracellular signal‐regulated kinase 1/2 in cells stimulated with stem cell factor were also diminished after UV irradiation. These results suggest that UV irradiation does not stimulate but rather suppresses mouse NCC. Thus if UV irradiation is a causative factor for ADM lesions, it would not act directly on dermal melanocytes but may act in indirect manners, for instance, via the overproduction of melanogenic cytokines such as α‐MSH and/or endothelin‐1.     

A novel antigen is shared by retinal pigment epithelium and pigmented neural crest

 Pigment cells in vertebrate embryos are formed in both the central and peripheral nervous system. The neural crest, a largely pluripotent population of precursor cells derived from the embryonic neural tube, gives rise to pigment cells which migrate widely in head and trunk.The retinal pigment epithelium is derived from the optic cup, which arises from ectoderm of the neural tube. We have generated an antibody, ips6, which stains an antigen common to pigment cells of retinal pigment epithelium and neural crest. Ips6 stains retinal pigment epithelium and choroid as well as a subset of crest cells that migrate in pathways typical of melanoblasts. Immunoreactivity is seen first in the eye and later in a subset of migrating crest cells. Crest cells in the amphibian embryo migrate along specific, stereotyped routes; ips6 immunoreactive cells are found in some but not all of these pathways. In older wild-type embryos, cells expressing ips6 appear coincident with pigment-containing cells in the flank, head, eye and embryonic gut. In older animals, staining in the eye extends to the intraretinal segment of optic nerve and interstices between photoreceptors and cells at the retinal periphery. We suggest that the ips6 antibody defines an antigen common to pigment cells of central and peripheral origin. Received: 22 January 1996/Accepted: 15 July 1996     

一种有效区分移植细胞和宿主细胞脑损伤模型的建立

为了区分移植神经细胞和宿主细胞,便于将来在宿主体内对移植细胞进行在体的电生理记录以及其它方面的研究,通过机械损毁的方法,建立了一种特殊的脑损伤模型。结果发现,通过机械损毁的方法,在大鼠大脑皮层形成形态规则的损伤空洞,其模型稳定,重复性好;在空洞内进行干细胞移植,能够长时间存活,移植神经干细胞绝大部分细胞分化为神经元,只有少量细胞分化为胶质细胞,而且移植细胞与宿主细胞分界明显;对移植细胞进行单细胞电生理记录,记录到神经元放电信号。这些结果表明,通过机械损毁的方法,在大鼠大脑皮层成功建立了一个稳定、精确定位移植细胞与宿主细胞界限的脑损伤模型。     

Convergence of normal stem cell and cancer stem cell developmental stage: Implication for differential therapies

Increased evidence shows that normal stem cells may contribute to cancer development and progression by acting as cancer-initiating cells through their interactions with abnormal environmental elements.We postulate that normal stem cells and cancer stem cells (CSC) possess similar mechanisms of self-renewal and differentiation.CSC can be the key to the elaboration of anti-cancer-based therapy.In this article,we focus on a controversial new theme relating to CSC.Tumorigenesis may have a critical stage characterized as a "therapeutic window",which can be identified by asso-ciation of molecular,biochemical and biological events.Identifying such a stage can allow the production of more effective therapies (e.g.manipulated stem cells) to treat several cancers.More importantly,confirming the existence of a similar therapeutic window during the conversion of normal stem cells to malignant CSC may lead to targeted therapy specifically against CSC.This conversion information may be derived from investigating the biological behaviour of both normal stem cells and cancerous stem cells.Currently,there is little knowledge about the cellular and molecular mechanisms that govern the initiation and maintenance of CSC.Studies on co-evolution and interdependence of cancer with normal tissues may lead to a useful treatment paradigm of cancer.The crosstalk between normal stem cells and cancer formation may converge developmental stages of different types of stem cells (e.g.normal stem cells,CSC and embryonic stem cells).The differential studies of the convergence may result in novel therapies for treating cancers.     

A two-state cell damage model under hyperthermic conditions: theory and in vitro experiments

The ultimate goal of cancer treatment utilizing thermotherapy is to eradicate tumors and minimize damage to surrounding host tissues. To achieve this goal, it is important to develop an accurate cell damage model to characterize the population of cell death under various thermal conditions. The traditional Arrhenius model is often used to characterize the damaged cell population under the assumption that the rate of cell damage is proportional to exp(-EaRT), where Ea is the activation energy, R is the universal gas constant, and T is the absolute temperature. However, this model is unable to capture transition phenomena over the entire hyperthermia and ablation temperature range, particularly during the initial stage of heating. Inspired by classical statistical thermodynamic principles, we propose a general two-state model to characterize the entire cell population with two distinct and measurable subpopulations of cells, in which each cell is in one of the two microstates, viable (live) and damaged (dead), respectively. The resulting cell viability can be expressed as C(tau,T)=exp(-Phi(tau,T)kT)(1+exp(-Phi(tau,T)kT)), where k is a constant. The in vitro cell viability experiments revealed that the function Phi(tau,T) can be defined as a function that is linear in exposure time tau when the temperature T is fixed, and linear as well in terms of the reciprocal of temperature T when the variable tau is held as constant. To determine parameters in the function Phi(tau,T), we use in vitro cell viability data from the experiments conducted with human prostate cancerous (PC3) and normal (RWPE-1) cells exposed to thermotherapeutic protocols to correlate with the proposed cell damage model. Very good agreement between experimental data and the derived damage model is obtained. In addition, the new two-state model has the advantage that is less sensitive and more robust due to its well behaved model parameters.     

A microscale model for prediction of breast cancer cell damage during cryosurgery

The morphology of cancerous breast tissue is characterized by tightly packed groups of small malignant cells, as found in most duct cell carcinoma. This special structure affects the osmotic responses of the cells to freezing and hence their probability of damage from cellular dehydration or intracellular ice formation. A mathematical model has been developed to study the microscale damage to these breast cancer cells during cryosurgery by accounting for their special structure. The model is based on a spherical unit comprised of an extracellular region that surrounds several layers of cancer cells, as experimentally observed of breast duct cell carcinoma by other researchers. Temperature transients in the breast cancer undergoing cryosurgery are calculated numerically using the Pennes equation. When subjected to various thermal histories, both cellular dehydration and intracellular ice formation in the unit structure are examined by considering the cell-to-cell contact and water transport at the microscale level. It is found that the cells in the inner layers hardly dehydrated while those in the outermost layer do greatly. The results help interpret the previously observed experimental phenomena that breast cancer tissues exhibit intracellular ice formation even at a slow cooling rate of -3 degrees C/min. In the attempt to better define an optimal procedure for breast cancer cryosurgery, various freezing protocols are simulated. The constant heat flux protocol induces greater cellular dehydration and higher intracellular ice formation probability simultaneously compared to the other protocols studied.     

Melanocyte stem cells: a melanocyte reservoir in hair follicles for hair and skin pigmentation

Most mammals are coated with pigmented hair. Melanocytes in each hair follicle produce melanin pigments for the hair during each hair cycle. The key to understanding the mechanism of cyclic melanin production is the melanocyte stem cell (MelSC) population, previously known as 'amelanotic melanocytes'. The MelSCs directly adhere to hair follicle stem cells, the niche cells for MelSCs and reside in the hair follicle bulge-subbulge area, the lower permanent portion of the hair follicle, to serve as a melanocyte reservoir for skin and hair pigmentation. MelSCs form a stem cell system within individual hair follicles and provide a 'hair pigmentary unit' for each cycle of hair pigmentation. This review focuses on the identification of MelSCs and their characteristics and explains the importance of the MelSC population in the mechanisms of hair pigmentation, hair greying, and skin repigmentation.     

Freeze-thaw damage in isolated lobster sarcoplasmic reticulum membranes: A model system for membrane damage

Sarcoplasmic reticulum membrane vesicles (SRV), isolated from the abdominal muscle of Maine lobsters, were put through a freeze-thaw cycle in order to study membrane freezing damage on a molecular basis, The major membrane protein in SRV is a (Ca²⁺ − Mg²⁺) ATPase capable of accumulating Ca²⁺ with the concomitant hydrolysis of ATP, After being frozen and thawed in the presence of NaCl, the SRV showed an increased ATPase activity and a decreased ability to accumulate Ca²⁺. The degree of increased ATPase activity and decreased Ca²⁺ accumulation was dependent upon the NaCl concentration (damage increased with increased NaCl concentration) and cooling rate (damage was only observed at slow cooling rates, i.e., less than 10 °C/min). Slow thawing rates also increased the amount of damage.The freeze-thaw damage of the SRV membranes is probably not due to osmotic shock, since the vesicles are quite resistant to osmotic stress and are highly permeable to small molecules and monovalent ions. Incubation of the SRV in 2 NaCl at 22 °C has no effect on Ca²⁺ accumulation whereas freezing in 0.25 NaCl totally abolishes their ability to take up Ca²⁺. Thus, a combination of salt and low temperature is necessary for damage. The freeze-thaw damage can be largely prevented by the addition of DMSO, glycerol, or PVP. The factors above have implications for the storage of tissue or membranes for subsequent analysis of membrane-bound enzymes. The SRV mimic the behavior of cells in their response to cooling and thawing rates, salts and cryoprotectants.     

A Sizer model for cell differentiation in Arabidopsis thaliana root growth

Plant roots grow due to cell division in the meristem and subsequent cell elongation and differentiation, a tightly coordinated process that ensures growth and adaptation to the changing environment. How the newly formed cells decide to stop elongating becoming fully differentiated is not yet understood. To address this question, we established a novel approach that combines the quantitative phenotypic variability of wild‐type Arabidopsis roots with computational data from mathematical models. Our analyses reveal that primary root growth is consistent with a Sizer mechanism, in which cells sense their length and stop elongating when reaching a threshold value. The local expression of brassinosteroid receptors only in the meristem is sufficient to set this value. Analysis of roots insensitive to BR signaling and of roots with gibberellin biosynthesis inhibited suggests distinct roles of these hormones on cell expansion termination. Overall, our study underscores the value of using computational modeling together with quantitative data to understand root growth.     

A computational model to link psychophysics and cortical cell activation patterns in human texture processing

The human visual system uses texture information to automatically, or pre-attentively, segregate parts of the visual scene. We investigate the neural substrate underlying human texture processing using a computational model that consists of a hierarchy of bi-directionally linked model areas. The model builds upon two key hypotheses, namely that (i) texture segregation is based on boundary detection--rather than clustering of homogeneous items--and (ii) texture boundaries are detected mainly on the basis of a large scenic context that is analyzed by higher cortical areas within the ventral visual pathway, such as area V4. Here, we focus on the interpretation of key results from psychophysical studies on human texture segmentation. In psychophysical studies, texture patterns were varied along several feature dimensions to systematically characterize human performance. We use simulations to demonstrate that the activation patterns of our model directly correlate with the psychophysical results. This allows us to identify the putative neural mechanisms and cortical key areas which underlie human behavior. In particular, we investigate (i) the effects of varying texture density on target saliency, and the impact of (ii) element alignment and (iii) orientation noise on the detectability of a pop-out bar. As a result, we demonstrate that the dependency of target saliency on texture density is linked to a putative receptive field organization of orientation-selective neurons in V4. The effect of texture element alignment is related to grouping mechanisms in early visual areas. Finally, the modulation of cell activity by feedback activation from higher model areas, interacting with mechanisms of intra-areal center-surround competition, is shown to result in the specific suppression of noise-related cell activities and to improve the overall model capabilities in texture segmentation. In particular, feedback interaction is crucial to raise the model performance to the level of human observers.     

Relationship of the cell cycle to xylem cell differentiation: A new model

Abstract. Conflicting data have appeared in the literature concerning the necessity for DNA synthesis prior to xylem cell differentiation. In some systems DNA synthesis is not required before differentiation, while in other systems DNA synthesis appears to be an absolute necessity. The construction of a model for the cell cycle in which the G1 phase is subdivided into a separate 'early' and 'late' phase can resolve this apparent conflict.     

The 180-kD component of the neural cell adhesion molecule N-CAM is involved in cell-cell contacts and cytoskeleton-membrane interactions

Summary N-CAM₁₈₀, the molecular form of the three neural cell adhesion molecules (N-CAM) with the largest cytoplasmic domain, is accumulated at sites of cell-cell contact (cell bodies, neurites, growth cones) in cultures of neuroblastoma and cerebellum. At these sites the cytoskeletonmembrane linker protein brain spectrin and actin are also accumulated. Brain spectrin copurifies with N-CAM₁₈₀ by immunoaffinity chromatography and binds specifically to N-CAM₁₈₀ but not to N-CAM₁₄₀ or N-CAM₁₂₀ in a solid-phase binding test. These observations indicate an association of N-CAM₁₈₀ with the cytoskeleton in vivo. This association may underlie the reduced lateral mobility of N-CAM₁₈₀ in the surface membrane compared to N-CAM₁₄₀ (Pollerberg et al. 1986). Together with the fact that N-CAM₁₈₀ is only expressed after termination of neuron migration in vivo (Persohn and Schachner, unpublished) these results suggest a role for N-CAM₁₈₀ in stabilization of cell contacts.     

An appetite for destruction: From self-eating to cell cannibalism as a neuronal survival strategy

Autophagy plays an important role in cellular survival by resupplying cells with nutrients during starvation or by clearing misfolded proteins and damaged organelles and thereby preventing degenerative diseases. Conversely, the autophagic process is also recognized as a cellular death mechanism. The circumstances that determine whether autophagy has a beneficial or a detrimental role in cellular survival are currently unclear. We recently showed that autophagy induction is detrimental in neurons that lack a functional AMPK enzyme (AMP-activated protein kinase) and that suffer from severe metabolic stress. We further demonstrated that autophagy and AMPK are interconnected in a negative feedback loop that prevents excessive and destructive stimulation of the autophagic process. Finally, we uncovered a new survival mechanism in AMPK-deficient neurons-cell cannibalism.     

A model for control of breathing in mammals: coupling neural dynamics to peripheral gas exchange and transport

A new model for aspects of the control of respiration in mammals has been developed. The model integrates a reduced representation of the brainstem respiratory neural controller together with peripheral gas exchange and transport mechanisms. The neural controller consists of two components. One component represents the inspiratory oscillator in the pre-Bötzinger complex (pre-BötC) incorporating biophysical mechanisms for rhythm generation. The other component represents the ventral respiratory group (VRG), which is driven by the pre-BötC for generation of inspiratory (pre)motor output. The neural model was coupled to simplified models of the lungs incorporating oxygen and carbon dioxide transport. The simplified representation of the brainstem neural circuitry has regulation of both frequency and amplitude of respiration and is done in response to partial pressures of oxygen and carbon dioxide in the blood using proportional (P) and proportional plus integral (PI) controllers. We have studied the coupled system under open and closed loop control. We show that two breathing regimes can exist in the model. In one regime an increase in the inspiratory frequency is accompanied by an increase in amplitude. In the second regime an increase in frequency is accompanied by a decrease in amplitude. The dynamic response of the model to changes in the concentration of inspired O₂ or inspired CO₂ was compared qualitatively with experimental data reported in the physiological literature. We show that the dynamic response with a PI-controller fits the experimental data better but suggests that when high levels of CO₂ are inspired the respiratory system cannot reach steady state. Our model also predicts that there could be two possible mechanisms for apnea appearance when 100% O₂ is inspired following a period of 5% inspired O₂. This paper represents a novel attempt to link neural control and gas transport mechanisms, highlights important issues in amplitude and frequency control and sets the stage for more complete neurophysiological control models.     

Butyrylcholinesterase is present in the human epidermis and is regulated by H2O2: more evidence for oxidative stress in vitiligo

The human epidermis holds the capacity for autocrine cholinergic signal transduction, but the presence of butyrylcholinesterase (BchE) has not been shown so far. Our results demonstrate that this compartment transcribes a functional BchE. Its activity is even higher compared to acetylcholinesterase (AchE). Moreover, we show that BchE is subject to regulation by H(2)O(2) in a concentration-dependent manner as it was recently described for AchE. Epidermal BchE protein expression and enzyme activities are severely affected by H(2)O(2) in vitiligo as previously demonstrated for AchE. Removal/reduction of H(2)O(2) by a pseudocatalase PC-KUS yields normal/increased protein expression and activities. H(2)O(2)-mediated oxidation of methionine residues in BchE was confirmed by FT-Raman spectroscopy. Computer simulation supported major alteration of the enzyme active site and its tetramerisation domain suggesting deactivation of the enzyme due to H(2)O(2)-mediated oxidation. Based on our results we conclude that H(2)O(2) is a major player in the regulation of the cholinergic signal via both AchE and BchE and this signal is severely affected in the epidermis of patients with active vitiligo.     

A quantitative model for cyclin-dependent kinase control of the cell cycle: revisited

The eukaryotic cell division cycle encompasses an ordered series of events. Chromosomal DNA is replicated during S phase of the cell cycle before being distributed to daughter cells in mitosis. Both S phase and mitosis in turn consist of an intricately ordered sequence of molecular events. How cell cycle ordering is achieved, to promote healthy cell proliferation and avert insults on genomic integrity, has been a theme of Paul Nurse's research. To explain a key aspect of cell cycle ordering, sequential S phase and mitosis, Stern & Nurse proposed 'A quantitative model for cdc2 control of S phase and mitosis in fission yeast'. In this model, S phase and mitosis are ordered by their dependence on increasing levels of cyclin-dependent kinase (Cdk) activity. Alternative mechanisms for ordering have been proposed that rely on checkpoint controls or on sequential waves of cyclins with distinct substrate specificities. Here, we review these ideas in the light of experimental evidence that has meanwhile accumulated. Quantitative Cdk control emerges as the basis for cell cycle ordering, fine-tuned by cyclin specificity and checkpoints. We propose a molecular explanation for quantitative Cdk control, based on thresholds imposed by Cdk-counteracting phosphatases, and discuss its implications.     

Differentiation and mineralization in chick chondrocytes maintained in a high cell density culture: A model for endochondral ossification

Summary Chondrocytes isolated from the proliferative and differentiating zones of 3-wk-old chick growth plates were cultured in the presence of 10% fetal bovine serum (FBS) and ascorbic acid for up to 21 d in a high cell density culture within Eppendorf tubes. The proliferative, differentiating, and calcification properties of the chondrocytes were examined by immunolocalization and by enzyme histochemical and biochemical methods. The cells maintained a chondrocyte phenotype throughout culture: they were round in shape and synthesized both collagen type II and proteoglycans. The expression of a hypertrophic phenotype was evident by Day 3 of culture and from this time onwards characteristics of terminal differentiation were observed. The cells were positive for both alkaline phosphatase (ALP) activity and c-myc protein and the surrounding matrix stained strongly for collagen type X. Small foci of mineralization associated with individual chondrocytes were first evident by Day 6 and more widespread areas of mineralization occupying large areas of matrix were present by Day 15. Mineralization occurred without the addition of exogenous phosphate to the medium. This culture system displays characteristics that are similar in both morphological and developmental terms to that of chick chondrocyte differentiation and calcification in vivo and therefore offers an excellent in vitro model for endochondral ossification.     

Interplay between autophagy and programmed cell death in mammalian neural stem cells

Mammalian neural stem cells (NSCs) are of particular interest because of their role in brain development and function. Recent findings suggest the intimate involvement of programmed cell death (PCD) in the turnover of NSCs. However, the underlying mechanisms of PCD are largely unknown. Although apoptosis is the best-defined form of PCD, accumulating evidence has revealed a wide spectrum of PCD encompassing apoptosis, autophagic cell death (ACD) and necrosis. This mini-review aims to illustrate a unique regulation of PCD in NSCs. The results of our recent studies on autophagic death of adult hippocampal neural stem (HCN) cells are also discussed. HCN cell death following insulin withdrawal clearly provides a reliable model that can be used to analyze the molecular mechanisms of ACD in the larger context of PCD. More research efforts are needed to increase our understanding of the molecular basis of NSC turnover under degenerating conditions, such as aging, stress and neurological diseases. Efforts aimed at protecting and harnessing endogenous NSCs will offer novel opportunities for the development of new therapeutic strategies for neuropathologies. [BMB Reports 2013; 46(8): 383-390]     

A three-dimensional vertex dynamics cell model of space-filling polyhedra simulating cell behavior in a cell aggregate

We developed a three-dimensional (3D) cell model of a multicellular aggregate consisting of several polyhedral cells to investigate the deformation and rearrangement of cells under the influence of external forces. The polyhedral cells fill the space in the aggregate without gaps or overlaps, consist of contracting interfaces and maintain their volumes. The interfaces and volumes were expressed by 3D vertex coordinates. Vertex movements obey equations of motion that rearrange the cells to minimize total free energy, and undergo an elementary process that exchanges vertex pair connections when vertices approach each other. The total free energy includes the interface energy of cells and the compression or expansion energy of cells. Computer simulations provided the following results: An aggregate of cells becomes spherical to minimize individual cell surface areas; Polygonal interfaces of cells remain flat; Cells within the 3D cell aggregate can move and rearrange despite the absence of free space. We examined cell rearrangement to elucidate the viscoelastic properties of the aggregate, e.g. when an external force flattens a cell aggregate (e.g. under centrifugation) its component cells quickly flatten. Under a continuous external force, the cells slowly rearrange to recover their original shape although the cell aggregate remains flat. The deformation and rearrangement of individual cells is a two-step process with a time lag. Our results showed that morphological and viscoelastic properties of the cell aggregate with long relaxation time are based on component cells where minimization of interfacial energy of cells provides a motive force for cell movement.