Nuclear Ferritin Protects DNA From UV Damage in Corneal Epithelial Cells

Previously, we identified the heavy chain of ferritin as a developmentally regulated nuclear protein of embryonic chicken corneal epithelial cells. The nuclear ferritin is assembled into a supramolecular form indistinguishable from the cytoplasmic form of ferritin found in other cell types and thus most likely has iron-sequestering capabilities. Free iron, via the Fenton reaction, is known to exacerbate UV-induced and other oxidative damage to cellular components, including DNA. Since corneal epithelial cells are constantly exposed to UV light, we hypothesized that the nuclear ferritin might protect the DNA of these cells from free radical damage. To test this possibility, primary cultures of cells from corneal epithelium and stroma, and from skin epithelium and stroma, were UV irradiated, and DNA strand breaks were detected by an in situ 3′-end labeling method. Corneal epithelial cells without nuclear ferritin were also examined. We observed that the corneal epithelial cells with nuclear ferritin had significantly less DNA breakage than other cell types examined. Furthermore, increasing the iron concentration of the culture medium exacerbated the generation of UV-induced DNA strand breaks in corneal and skin fibroblasts, but not in the corneal epithelial cells. Most convincingly, corneal epithelial cells in which the expression of nuclear ferritin was inhibited became much more susceptible to UV-induced DNA damage. Therefore, it seems that corneal epithelial cells have evolved a novel, nuclear ferritin-based mechanism for protecting their DNA against UV damage.

Corneal collagen fibril structure in three dimensions: Structural insights into fibril assembly, mechanical properties, and tissue organization

The ability of the cornea to transmit light while being mechanically resilient is directly attributable to the formation of an extracellular matrix containing orthogonal sheets of collagen fibrils. The detailed structure of the fibrils and how this structure underpins the mechanical properties and organization of the cornea is understood poorly. In this study, we used automated electron tomography to study the three-dimensional organization of molecules in corneal collagen fibrils. The reconstructions show that the collagen molecules in the 36-nm diameter collagen fibrils are organized into microfibrils (≈4-nm diameter) that are tilted by ≈15° to the fibril long axis in a right-handed helix. An unexpected finding was that the microfibrils exhibit a constant-tilt angle independent of radial position within the fibril. This feature suggests that microfibrils in concentric layers are not always parallel to each other and cannot retain the same neighbors between layers. Analysis of the lateral structure shows that the microfibrils exhibit regions of order and disorder within the 67-nm axial repeat of collagen fibrils. Furthermore, the microfibrils are ordered at three specific regions of the axial repeat of collagen fibrils that correspond to the N- and C-telopeptides and the d-band of the gap zone. The reconstructions also show macromolecules binding to the fibril surface at sites that correspond precisely to where the microfibrils are most orderly.

Randomness and degrees of irregularity.

The fundamental question "Are sequential data random?" arises in myriad contexts, often with severe data length constraints. Furthermore, there is frequently a critical need to delineate nonrandom sequences in terms of closeness to randomness--e.g., to evaluate the efficacy of therapy in medicine. We address both these issues from a computable framework via a quantification of regularity. ApEn (approximate entropy), defining maximal randomness for sequences of arbitrary length, indicating the applicability to sequences as short as N = 5 points. An infinite sequence formulation of randomness is introduced that retains the operational (and computable) features of the finite case. In the infinite sequence setting, we indicate how the "foundational" definition of independence in probability theory, and the definition of normality in number theory, reduce to limit theorems without rates of convergence, from which we utilize ApEn to address rates of convergence (of a deficit from maximal randomness), refining the aforementioned concepts in a computationally essential manner. Representative applications among many are indicated to assess (i) random number generation output; (ii) well-shuffled arrangements; and (iii) (the quality of) bootstrap replicates.

Specific atomic groups and RNA helix geometry in acceptor stem recognition by a tRNA synthetase

Oligonucleotides that recapitulate the acceptor stems of tRNAs are substrates for aminoacylation by many tRNA synthetases in vitro, even though these substrates are missing the anticodon trinucleotides of the genetic code. In the case of tRNAAla a single acceptor stem G⋅U base pair at position 3·70 is essential, based on experiments where the wobble pair has been replaced by alternatives such as I⋅U, G⋅C, and A⋅U, among others. These experiments led to the conclusion that the minor-groove free 2-amino group (of guanosine) of the G⋅U wobble pair is essential for charging. Moreover, alanine-inserting tRNAs (amber suppressors) that replace G⋅U with mismatches such as G⋅A and C⋅A are partially active in vivo and can support growth of an Escherichia coli tRNAAla knockout strain, leading to the hypothesis that a helix irregularity and nucleotide functionalities are important for recognition. Herein we investigate the charging in vitro of oligonucleotide and full-length tRNA substrates that contain mismatches at the position of the G⋅U pair. Although most of these substrates have undetectable activity, G⋅A and C⋅A variants retain some activity, which is, nevertheless, reduced by at least 100-fold. Thus, the in vivo assays are much less sensitive to large changes in aminoacylation kinetic efficiency of 3·70 variants than is the in vitro assay system. Although these functional data do not clarify all of the details, it is now clear that specific atomic groups are substantially more important in determining kinetic efficiency than is a helical distortion. By implication, the activity of mutant tRNAs measured in the in vivo assays appears to be more dependent on factors other than aminoacylation kinetic efficiency.

Electric Field–directed Cell Motility Involves Up-regulated Expression and Asymmetric Redistribution of the Epidermal Growth Factor Receptors and Is Enhanced by Fibronectin and Laminin

Wounding corneal epithelium establishes a laterally oriented, DC electric field (EF). Corneal epithelial cells (CECs) cultured in similar physiological EFs migrate cathodally, but this requires serum growth factors. Migration depends also on the substrate. On fibronectin (FN) or laminin (LAM) substrates in EF, cells migrated faster and more directly cathodally. This also was serum dependent. Epidermal growth factor (EGF) restored cathodal-directed migration in serum-free medium. Therefore, the hypothesis that EGF is a serum constituent underlying both field-directed migration and enhanced migration on ECM molecules was tested. We used immunofluorescence, flow cytometry, and confocal microscopy and report that 1) EF exposure up-regulated the EGF receptor (EGFR); so also did growing cells on substrates of FN or LAM; and 2) EGFRs and actin accumulated in the cathodal-directed half of CECs, within 10 min in EF. The cathodal asymmetry of EGFR and actin staining was correlated, being most marked at the cell–substrate interface and showing similar patterns of asymmetry at various levels through a cell. At the cell–substrate interface, EGFRs and actin frequently colocalized as interdigitated, punctate spots resembling tank tracks. Cathodal accumulation of EGFR and actin did not occur in the absence of serum but were restored by adding ligand to serum-free medium. Inhibition of MAPK, one second messenger engaged by EGF, significantly reduced EF-directed cell migration. Transforming growth factor β and fibroblast growth factor also restored cathodal-directed cell migration in serum-free medium. However, longer EF exposure was needed to show clear asymmetric distribution of the receptors for transforming growth factor β and fibroblast growth factor. We propose that up-regulated expression and redistribution of EGFRs underlie cathodal-directed migration of CECs and directed migration induced by EF on FN and LAM.

A recipe for randomness

Despite many diverse theories that address closely related themes—e.g., probability theory, algorithmic complexity, cryptoanalysis, and pseudorandom number generation—a near-void remains in constructive methods certified to yield the desired “random” output. Herein, we provide explicit techniques to produce broad sets of both highly irregular finite and normal infinite sequences, based on constructions and properties derived from approximate entropy (ApEn), a computable formulation of sequential irregularity. Furthermore, for infinite sequences, we considerably refine normality, by providing methods for constructing diverse classes of normal numbers, classified by the extent to which initial segments deviate from maximal irregularity.

Aldehyde dehydrogenase class 3 expression: Identification of a cornea-preferred gene promoter in transgenic mice

Aldehyde dehydrogenase class 3 (ALDH3) constitutes 20–40% of the total water-soluble proteins in the mammalian cornea. Here, we show by Northern blot analysis that ALDH3 expression in the mouse is at least 500-fold higher in the cornea than in any other tissue examined, with very low levels of expression detected in the stomach, urinary bladder, ocular lens, and lung. Histochemical localization reveals that this exceptional level of expression in the mouse cornea occurs in the anterior epithelial cells and that little ALDH3 is present in the keratocytes or corneal endothelial cells. A 13-kbp mouse ALDH3 promoter fragment containing >12 kbp of the 5′ flanking sequence, the 40-bp untranslated first exon, and 29 bp of intron 1 directed cat reporter gene expression to tissues that express the endogenous ALDH3 gene, except that transgene promoter activity was higher in the stomach and bladder than in the cornea. By contrast, when driven by a 4.4-kbp mouse ALDH3 promoter fragment [1,050-bp 5′ flanking region, exon 1, intron 1 (3.4 kbp), and 7 bp of exon 2] expression of the cat reporter gene was confined to the corneal epithelial cells, except for very low levels in the liver, effectively reproducing the corneal expression pattern of the endogenous ALDH3 gene. These results indicate that tissue-specific expression of ALDH3 is determined by positive and negative elements in the 5′ flanking region of the gene and suggests putative silencers located in intron 1. We demonstrate regulatory sequences capable of directing cornea-specific gene expression, affording the opportunity for genetic engineering in this transparent tissue.

p63 identifies keratinocyte stem cells

The proliferative compartment of stratified squamous epithelia consists of stem and transient amplifying (TA) keratinocytes. Some polypeptides are more abundant in putative epidermal stem cells than in TA cells, but no polypeptide confined to the stem cells has yet been identified. Here we show that the p63 transcription factor, a p53 homologue essential for regenerative proliferation in epithelial development, distinguishes human keratinocyte stem cells from their TA progeny. Within the cornea, nuclear p63 is expressed by the basal cells of the limbal epithelium, but not by TA cells covering the corneal surface. Human keratinocyte stem and TA cells when isolated in culture give rise to holoclones and paraclones, respectively. We show by clonal analysis that p63 is abundantly expressed by epidermal and limbal holoclones, but is undetectable in paraclones. TA keratinocytes, immediately after their withdrawal from the stem cell compartment (meroclones), have greatly reduced p63, even though they possess very appreciable proliferative capacity. Clonal evolution (i.e., generation of TA cells from precursor stem cells) is promoted by the sigma isoform of the 14-3-3 family of proteins. Keratinocytes whose 14-3-3σ has been down-regulated remain in the stem cell compartment and maintain p63 during serial cultivation. The identification of p63 as a keratinocyte stem cell marker will be of practical importance for the clinical application of epithelial cultures in cell therapy as well as for studies on epithelial tumorigenesis.

Comparative evaluation of the antitumor activity of antiangiogenic proteins delivered by gene transfer

Although the systemic administration of a number of different gene products has been shown to result in the inhibition of angiogenesis and tumor growth in different animal tumor models, the relative potency of those gene products has not been studied rigorously. To address this issue, recombinant adenoviruses encoding angiostatin, endostatin, and the ligand-binding ectodomains of the vascular endothelial growth factor receptors Flk1, Flt1, and neuropilin were generated and used to systemically deliver the different gene products in several different preexisting murine tumor models. Single i.v. injections of viruses encoding soluble forms of Flk1 or Flt1 resulted in ≈80% inhibition of preexisting tumor growth in murine models involving both murine (Lewis lung carcinoma, T241 fibrosarcoma) and human (BxPC3 pancreatic carcinoma) tumors. In contrast, adenoviruses encoding angiostatin, endostatin, or neuropilin were significantly less effective. A strong correlation was observed between the effects of the different viruses on tumor growth and the activity of the viruses in the inhibition of corneal micropocket angiogenesis. These data underscore the need for comparative analyses of different therapeutic approaches that target tumor angiogenesis and provide a rationale for the selection of specific antiangiogenic gene products as lead candidates for use in gene therapy approaches aimed at the treatment of malignant and ocular disorders.

Functional and structural mapping of human cerebral cortex: Solutions are in the surfaces

The human cerebral cortex is notorious for the depth and irregularity of its convolutions and for its variability from one individual to the next. These complexities of cortical geography have been a chronic impediment to studies of functional specialization in the cortex. In this report, we discuss ways to compensate for the convolutions by using a combination of strategies whose common denominator involves explicit reconstructions of the cortical surface. Surface-based visualization involves reconstructing cortical surfaces and displaying them, along with associated experimental data, in various complementary formats (including three-dimensional native configurations, two-dimensional slices, extensively smoothed surfaces, ellipsoidal representations, and cortical flat maps). Generating these representations for the cortex of the Visible Man leads to a surface-based atlas that has important advantages over conventional stereotaxic atlases as a substrate for displaying and analyzing large amounts of experimental data. We illustrate this by showing the relationship between functionally specialized regions and topographically organized areas in human visual cortex. Surface-based warping allows data to be mapped from individual hemispheres to a surface-based atlas while respecting surface topology, improving registration of identifiable landmarks, and minimizing unwanted distortions. Surface-based warping also can aid in comparisons between species, which we illustrate by warping a macaque flat map to match the shape of a human flat map. Collectively, these approaches will allow more refined analyses of commonalities as well as individual differences in the functional organization of primate cerebral cortex.

El Niño and the related phenomenon Southern Oscillation (ENSO): The largest signal in interannual climate variation

El Niño and the related phenomenon Southern Oscillation (ENSO) is the strongest signal in the interannual variation of ocean-atmosphere system. It is mainly a tropical event but its impact is global. ENSO has been drawing great scientific attention in many international research programs. There has been an observational system for the tropical ocean, and scientists have known the climatologies of the upper ocean, developed some theories about the ENSO cycle, and established coupled ocean-atmosphere models to give encouraging predictions of ENSO for a 1-year lead. However, questions remain about the physical mechanisms for the ENSO cycle and its irregularity, ENSO-monsoon interactions, long-term variation of ENSO, and increasing the predictive skill of ENSO and its related climate variations.

Myofibroblasts differentiate from fibroblasts when plated at low density.

Myofibroblasts, defined by their expression of smooth muscle alpha-actin, appear at corneal and dermal incisions and promote wound contraction. We report here that cultured fibroblasts differentiate into myofibroblasts by a cell density-dependent mechanism. Fibroblasts seeded at low density (5 cells per mm2) produced a cell culture population consisting of 70-80% myofibroblasts, 5-7 days after seeding. In contrast, fibroblasts seeded at high density (500 cells per mm2) produced cultures with only 5-10% myofibroblasts. When the myofibroblast-enriched cultures were subsequently passaged at high density, the smooth muscle alpha-actin phenotype was lost within 3 days. Furthermore, initially 60% of the low density-cultured cells incorporated BrdUrd compared to 30% of cells passaged at high density. Media from myofibroblast-enriched cultures had more latent and active transforming growth factor beta (TGF-beta) than did media from fibroblast-enriched cultures. Although there was a trend towards increased numbers of myofibroblasts after addition of exogenous TGF-beta, the results did not reach statistical significance. We conclude that myofibroblast differentiation can be induced in fibroblasts by plating at low density. We propose a cell density-dependent model of myofibroblast differentiation during wounding and healing in which at least two factors interact: loss of cell contact and the presence of TGF-beta.

Functional anatomy of human eyeblink conditioning determined with regional cerebral glucose metabolism and positron-emission tomography.

Relative cerebral glucose metabolism was examined with positron-emission tomography (PET) as a measure of neuronal activation during performance of the classically conditioned eyeblink response in 12 young adult subjects. Each subject received three sessions: (i) a control session with PET scan in which unpaired presentations of the tone conditioned stimulus and corneal airpuff unconditioned stimulus were administered, (ii) a paired training session to allow associative learning to occur, and (iii) a paired test session with PET scan. Brain regions exhibiting learning-related activation were identified as those areas that showed significant differences in glucose metabolism between the unpaired control condition and well-trained state in the 9 subjects who met the learning criterion. Areas showing significant activation included bilateral sites in the inferior cerebellar cortex/deep nuclei, anterior cerebellar vermis, contralateral cerebellar cortex and pontine tegmentum, ipsilateral inferior thalamus/red nucleus, ipsilateral hippocampal formation, ipsilateral lateral temporal cortex, and bilateral ventral striatum. Among all subjects, including those who did not meet the learning criterion, metabolic changes in ipsilateral cerebellar nuclei, bilateral cerebellar cortex, anterior vermis, contralateral pontine tegmentum, ipsilateral hippocampal formation, and bilateral striatum correlated with degree of learning. The localization to cerebellum and its associated brainstem circuitry is consistent with neurobiological studies in the rabbit model of eyeblink classical conditioning and neuropsychological studies in brain-damaged humans. In addition, these data support a role for the hippocampus in conditioning and suggest that the ventral striatum may also be involved.