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E M Levine

Publications and source records attributed to E M Levine.

At least 37 records · Page 2Linked to original sources
PubMed1998

Multipotential stem cells and progenitors in the vertebrate retina.

The vertebrate retina is derived from paired evaginations from the neural tube in embryonic development and is initially produced by progenitor cells similar to those that generate the neurons and glia of other areas of the central nervous system. In some amphibians and fish, the retina continues to grow along with the eye throughout the life of the animal. The new retinal cells are added at the ciliary margin of the eye from the mitotic activity of neural/glial stem cells in a region known as the germinal zone and are seamlessly incorporated into the existing retinal circuitry. Little is known about the cell or molecular biology of these stem cells; however, studies of retinal progenitor cells in chick and mammalian embryos have led to the identification of several factors that control their proliferation. Moreover, studies of retinal regeneration have shown that retinal stem cells can also be derived from two or perhaps three additional sources after retinal damage: (a) the retinal pigmented epithelium (RPE) in amphibians and embryonic chicks and mammals; (b) a specialized rod progenitor in fish; and (c) the Müller glial cells. While there is currently no evidence for a neural/glial stem cell in the adult mammalian retina, and the retina of the mature mammal does not show regenerative capacity after damage, there is a possibility for the reinitiation of stem cell potential at the peripheral retinal margin, from the RPE or from the Müller glial cells. The application of information derived from the studies of retinal progenitor cells in developing organisms should soon provide a test of these possibilities.

Aging↗
PubMed1997

Vsx-1 and Vsx-2: differential expression of two paired-like homeobox genes during zebrafish and goldfish retinogenesis.

Vsx-1 and Vsx-2 are two homeobox genes that were cloned originally from an adult goldfish retinal library. They are members of the paired-like:CVC gene family, which is characterized by the presence of a paired homeodomain and an additional conserved region, termed the CVC domain. To analyze the possible roles for Vsx-1 and Vsx-2 in eye development, we used in situ hybridization to examine their expression patterns in zebrafish and goldfish embryos. Vsx-2 is initially expressed by proliferating neuroepithelial cells of the presumptive neural retina, then it is down-regulated as differentiation begins, and it is finally reexpressed at later stages of differentiation in a subset of cells, presumed to be bipolar cells, in the inner nuclear layer. In contrast, Vsx-1 is expressed only weakly in undifferentiated, presumptive neural retina and is then up-regulated selectively in presumptive bipolar cells at early stages of differentiation (when Vsx-2 is turned off), before decreasing to an intermediate level, which is maintained in the differentiated (adult) retina. The restricted expression patterns of Vsx-2 correspond to the observed phenotypes in mice with the ocular retardation mutation (orJ), further supporting the notion that Vsx-2 and Chx10 are homologues. The sequential complimentary and then corresponding expression patterns of Vsx-1 and Vsx-2 suggest that these similar transcription factors may be recruited for partially overlapping, but distinct, functions during the development of the retina.

Amino Acid Sequence↗
PubMed1997

Vsx-1 and Vsx-2: two Chx10-like homeobox genes expressed in overlapping domains in the adult goldfish retina.

The genetic linkages of the murine ocular retardation mutation with the Chx10 gene and the murine small eye mutation with the Pax-6 gene has demonstrated the importance of Paired class homeobox genes in the development of the mammalian retina. Previously, we identified a Paired-class homeobox gene, Vsx-1, whose expression in the adult goldfish retina is restricted to the inner nuclear layer (INL) and to postmitotic, differentiating progenitor cells in the growth zone at the retinal peripheral margin, where neurogenesis continues throughout life. Here, we report the molecular cloning and expression pattern of a new Paired class homeobox gene, Vsx-2, in the adult goldfish retina. Like Vsx-1, Vsx-2 expression is highly restricted to the retina in the adult goldfish and overlaps with Vsx-1 expression in the mature INL. At the peripheral margin, Vsx-2 is expressed in mitotically active neuronal progenitors and is downregulated as these cells become postmitotic and begin to differentiate. Comparison of the amino acid sequences of Vsx-2, Vsx-1, Chx10, and C. elegans ceh-10 reveal a conserved homeodomain and a unique domain termed the CVC domain. The similarities of the Vsx-2, Vsx-1, and Chx10 expression patterns suggest that genes containing the CVC domain have conserved functions during retinal development in vertebrates.

Amino Acid Sequence↗
PubMed1997

Sonic hedgehog promotes rod photoreceptor differentiation in mammalian retinal cells in vitro.

The hedgehog gene family encodes secreted proteins important in many developmental patterning events in both vertebrates and invertebrates. In the Drosophila eye disk, hedgehog controls the progression of photoreceptor differentiation in the morphogenetic furrow. To investigate whether hedgehog proteins are also involved in the development of the vertebrate retina at stages of photoreceptor differentiation, we analyzed expression of the three known vertebrate hedgehog genes. We found that Sonic hedgehog and Desert hedgehog are expressed in the developing retina, albeit at very low levels, whereas Indian hedgehog (Ihh) is expressed in the developing and mature retinal pigmented epithelium, beginning at embryonic day 13. To determine whether hedgehog proteins have activities on developing retinal cells, we used an in vitro system in which much of retinal histogenesis is recapitulated. N-terminal recombinant Sonic Hedgehog protein (SHH-N) was added to rat retinal cultures for 3-12 d, and the numbers of retinal cells of various phenotypes were analyzed by immunohistochemistry. We found that SHH-N caused a transient increase in the number of retinal progenitor cells, and a 2- to 10-fold increase in the number of photoreceptors differentiating in the cultures when analyzed with three different photoreceptor-specific antigens. In contrast, the numbers of retinal ganglion cells and amacrine cells were similar to those in control cultures. These results show that Hedgehog proteins can regulate mitogenesis and photoreceptor differentiation in the vertebrate retina, and Ihh is a candidate factor from the pigmented epithelium to promote retinal progenitor proliferation and photoreceptor differentiation.

Animals↗
PubMed1995

Heparin and the phenotype of adult human vascular smooth muscle cells.

To study mechanisms controlling growth and phenotype in human vascular smooth muscle cells, we established culture conditions under which these cells proliferate rapidly and achieve life-spans of 50-60 population doublings. In medium containing heparin and heparin-binding growth factors, growth rate and life-span of human vascular smooth muscle cells increased more than 50% relative to cultures with neither supplement, and more than 20% compared to cultures supplemented only with heparin-binding growth factors. In contrast to observations made in rat vascular smooth muscle cells, smooth muscle-specific alpha-actin in the human cells was expressed only in the presence of heparin and colocalized with beta/gamma nonmuscle actins in stress fibers, not in adhesion plaques. Heparin, in the presence of heparin-binding growth factors, also caused more than 170% stimulation of tracer glucosamine incorporation into hyaluronic acid and a 7.5-fold increase in hyaluronic acid accumulation. In comparison, total sulfate incorporation into sulfated glycosaminoglycans increased by less than 40%. In light of our previous findings that heparin suppresses collagen gene expression, we conclude that heparin induces human vascular smooth muscle cells exposed to heparin-binding growth factors to remodel their extracellular matrix by altering the relative rates of hyaluronic acid (HA) and collagen synthesis. The resulting hyaluronic-acid-rich, collagen-poor matrix may enhance infiltration of CD44/hyaluronate-receptor-bearing T-lymphocytes and monocytes into the vascular wall, an early event in atherogenesis.

Actin Cytoskeleton↗
PubMed1994

Restricted expression of a new paired-class homeobox gene in normal and regenerating adult goldfish retina.

We describe the cloning and expression pattern of a new paired-class homeobox gene, Vsx-1, in the continuously growing retina of the goldfish. Vsx-1 belongs to a subset of paired-class homeobox genes that lack a second DNA binding domain, the paired-domain, and is closely related to the C. elegans ceh-10 gene. In the adult goldfish, Vsx-1 expression is restricted to the neural retina. In the central, mature retina, Vsx-1 mRNA is synthesized in a subset of differentiated cells in the inner nuclear layer in a pattern suggestive of bipolar cells. In immature retina, adjacent to the retinal margin, Vsx-1 is expressed in a relatively broader subset of newly postmitotic cells but is downregulated in some of these cells to form the mature expression pattern. Following retinal injury, during the early phase of regeneration, Vsx-1 mRNA synthesis appears to be upregulated in cells in the inner nuclear layer and is expressed de novo in cells outside this layer. By virtue of its identity as a transcriptional regulatory gene and its patterns of expression, we speculate that Vsx-1 may stabilize the differentiated state of a subset of cells in the inner nuclear layer and may be involved in cellular differentiation during retinal development and regeneration.

Amino Acid Sequence↗
PubMed1994

Complex expression of keratins in goldfish optic nerve.

Keratins are the predominant intermediate filament proteins in the nonneuronal cells of the goldfish optic nerve. At least three different keratin pairs are expressed in this tissue, indicating an unexpected complexity. Expression of the type II keratin ON3 in goldfish optic nerve astrocytes predicts the expression of a type I keratin partner. Here we report the cDNA sequence and predicted amino acid sequence of two type I keratins from the goldfish optic nerve, designated GK48 and GK49. The GK48 protein is the goldfish equivalent of mammalian keratin 18 (K18) and is the most likely type I keratin partner to the ON3 protein. The GK49 protein is similar to the GK50 protein, a type I keratin characterized previously from the goldfish optic nerve. The GK48 and ON3 mRNAs are expressed in a variety of goldfish tissues, whereas the expression of GK49 mRNA has a more limited expression. In addition, in situ hybridization experiments show that the expression of the GK48 and ON3 mRNAs are evenly distributed throughout the optic nerve, while the GK49 mRNA is expressed along longitudinal lines. These results show that there is a diversity of keratin expression within different cell types in the goldfish optic nerve.

Amino Acid Sequence↗
PubMed1994

Cloning of multiple forms of goldfish vimentin: differential expression in CNS.

In efforts to determine the primary structure of intermediate filament proteins in the goldfish visual pathway, we isolated clones from a retinal lambda gt11 cDNA expression library that represent goldfish vimentin. We show that there are at least two forms of goldfish vimentin, designated as vimentin alpha and vimentin beta. RNase protection assays indicate that vimentin alpha mRNA is expressed in low amounts in retina, optic nerve, and brain and in higher amounts in spinal cord. In contrast, vimentin beta mRNA is expressed in low amounts in retina, optic nerve, brain, and spinal cord and in very high amounts in eye lens. Immunohistochemical studies show that in the optic nerve, vimentin alpha is mainly restricted to blood vessels, meninges, and septa. Light staining is observed with this antibody in an astrocytic glial pattern throughout the optic nerve. Two-dimensional gel analysis shows that all of these goldfish vimentins are low abundant components of optic nerve cytoskeletal preparations.

Amino Acid Sequence↗
PubMed1993

Homeobox genes are expressed in the retina and brain of adult goldfish.

The goldfish (Carassius auratus) visual pathway displays continuous growth and plasticity throughout life. Since homeobox genes are important transcriptional regulators in development, we searched for homeobox genes in the adult goldfish retina and brain. Using the PCR, we discovered a repertoire of homeobox sequences expressed in these tissues. In addition to isolating homeodomain sequences found in the vertebrate Hox gene clusters, a sequence identical to the chicken CHox7 homeodomain was characterized. Furthermore, a sequence with significant homologies to the Xenopus XIHbox8 and leech Htr-A2 homeodomains was identified, and these sequences may define an additional class of homeodomain. Finally, a sequence belonging to the paired class (prd) of homeodomains is reported. Homeobox gene expression in the adult goldfish retina and brain may be associated with the persistent developmental features of these tissues.

Amino Acid Sequence↗
PubMed1992

Karyotypic and phenotypic changes during in vitro aging of human endothelial cells.

Karyotypic and phenotypic changes were found in human adult endothelial cells (EC) during aging in vitro. A trisomy of chromosome 11 was found in 11 out of 12 EC cultures examined, derived from 9 cell lines from 8 donors. The incidence of this trisomy in some cell lines increased over time from 0% to as much as 100% near the end of their in vitro life span. A number of oncogenes and other important genes are on chromosome 11. These genes might play a role in the changes observed. An increase in the percentage of polyploid cells was also found near the end of the in vitro life span in 6 lines. The cellular levels of two gene products characteristic of the EC, von Willebrand factor (vWF) or Factor VIII, and angiotensin converting enzyme (ACE) were also monitored. vWf was studied in 2 lines and was decreased in both with serial passage. ACE decreased in three out of the four lines examined. These chromosomal and phenotypic changes which occur with increasing age in vitro make the endothelial cell a suitable model to study in vitro culture-related changes, senescence, cardiovascular disease, and tumorigenesis.

Adult↗
PubMed1992

Plasticin, a novel type III neurofilament protein from goldfish retina: increased expression during optic nerve regeneration.

The goldfish visual pathway displays a remarkable capacity for continued development and plasticity. The intermediate filament proteins in this pathway are unexpected and atypical, suggesting these proteins provide a structure that supports growth and plasticity. Using a goldfish retina lambda gt10 library, we have isolated a full-length cDNA clone that encodes a novel type III intermediate filament protein. The mRNA for this protein is located in retinal ganglion cells, and its level dramatically increases during optic nerve regeneration. The protein is transported into the optic nerve within the slow phase of axonal transport. We have named this protein plasticin because it was isolated from a neuronal pathway well known for its plasticity.

Amino Acid Sequence↗
PubMed1992

Cloning of a type I keratin from goldfish optic nerve: differential expression of keratins during regeneration.

We report the cDNA sequence and predicted amino acid sequence of a novel type I keratin, designated as GK50, and show that keratin expression in the goldfish optic nerve is highly complex. The GK50 protein is one of at least three type I keratins expressed in goldfish optic nerve based on both antibody reactivity and blot-binding to the type II keratin ON3. After optic nerve crush in situ hybridization shows a localized increase in GK50 mRNA expression in the crush zone. This is in contrast to ON3 mRNA which shows a localized increase that is limited to the proximal and distal margins of the crush zone, suggesting a diversity of keratin expression in different cell types of the goldfish optic nerve.

Amino Acid Sequence↗
PubMed1991

Culture of human adult endothelial cells on liquid-liquid interfaces: a new approach to the study of cell-matrix interactions.

Human adult endothelial cells (ECs) were cultured on liquid-liquid interface formed when aqueous culture medium is overlaid onto a fluorocarbon solvent. When ECs were seeded on untreated interfaces, some cells seemed to attach but they did not spread or grow. In contrast, when ECs were seeded on interfaces pretreated with such proteins as collagen type IV (COL), laminin (LN), fibronectin (FN), and fibrinogen (FG) the cells spread and proliferated until they formed confluent monolayers. Proteins such as bovine serum albumin (BSA) or gelatin (GN) were not as effective in providing surfaces for vigorous growth. Cells grown on fluorocarbon interfaces expressed specialized characteristics exhibited by endothelial cells grown under the usual culture conditions; they grew in a cobblestone monolayer, stained positively for Factor VIII-related antigen, and produced angiotensin-converting enzyme. The growth rate of ECs was the same whether they were cultured on treated fluorocarbon interfaces or on the usual tissue culture plastic surfaces. Using this culture system, the interactions of ECs with various adhesive proteins used as substrata was examined. ECs were observed to attach readily to the interfaces coated with GN, COL, LN, FN, and FG, but poorly to those coated with BSA. All the substrates tested, with the exception of BSA, promoted EC growth on fluorocarbon interfaces; ECs tended to grow more rapidly on COL- or FG-coated interfaces than on LN-, FN-, or GN-coated interfaces.

Animals↗
PubMed1991

Zidovudine-associated embryonic toxicity in mice.

A novel toxicity associated with zidovudine (AZT), the standard antiviral therapy for infection with human immunodeficiency virus, is described. When AZT was administered to mice to evaluate its safety during gestation, the animals failed to complete pregnancy successfully. Mice receiving AZT during gestation yielded fewer fetuses (P = .003) and greater numbers of resorptions (P = .003) per pregnant mouse compared with untreated animals. Drug effects on adult mice were assessed to determine if toxicity could account for the pregnancy failures. However, adult animals receiving AZT demonstrated no adverse effects with regard to growth, food consumption, activity, or ovarian histology. A direct toxic effect of AZT on the mouse embryo was tested by cultivating single-cell fertilized oocytes in vitro in the presence of increasing concentrations of drug. Exposure to AZT was highly correlated with failure to develop to the blastocyst stage (P less than .001). These data indicate that AZT has a direct toxic effect on the developing mouse embryo. Further analysis of the nature of this toxicity may be important in designing less toxic antiretroviral agents and in planning future uses of AZT.

Animals↗