Activity-dependent regulation of N-cadherin in DRG neurons: Differential regulation of N-cadherin,NCAM, and L1 by distinct patterns of action potentials

Cell adhesion molecule (CAM) expression is highly regulated during nervous system development to control cell migration, neurite outgrowth, fasciculation, and synaptogenesis. Using electrical stimulation of mouse dorsal root ganglion (DRG) neurons in cell culture, this work shows that N-cadherin expression is regulated by neuronal firing, and that expression of different CAMs is regulated by distinct patterns of neural impulses. N-cadherin was down-regulated by 0.1 or 1 Hz stimulation, but NCAM mRNA and protein levels were not altered by stimulation. L1 was down-regulated by 0.1 Hz stimulation, but not by 0.3 Hz, 1 Hz, or pulsed stimulation. N-cadherin expression was lowered with faster kinetics than L1 (1 vs. 5 days), and L1 mRNA returned to higher levels after terminating the stimulus. The RSLE splice variant of L1 was not regulated by action potential stimulation, and activity-dependent influences on L1 expression were blocked by target-derived influences. The results are consistent with changes in firing pattern accompanying DRG development and suggest that functional activity can influence distinct developmental processes by regulating the relative abundance of different CAMs. © 1997 John Wiley & Sons, Inc. J Neurobiol 33: 735–748, 1997

1 This is a US Government work and, as such, is in the public domain in the United States of America.

     

Amino acid neurotransmitter transporters: Structure,function, and molecular diversity

Many biologically active compounds including neurotransmitters, metabolic precursors, and certain drugs are accumulated intracellularly by transporters that are coupled to the transmembrane Na⁺ gradient. Amino acid neurotransmitter transporters play a key role in the regulation of extracellular amino acid concentrations and termination of neurotransmission in the CNS

1 Abbreviations: CNS, central nervous system; GABA, γ-aminobutyric acid; cDNA, complementary deoxyribonucleic acid; mRNA, messenger ribonucleic acid; NMDA, N-methyl-D-aspartate; PKC, protein kinase C; PMA, phorbol 12-myristate 13-acetate; DAG, diacyl glycerol; R59022, DAG kinase inhibitor; AA, arachidonic acid; ACHC, cis-3-aminocyclohexanecarboxylic acid; GAT-A, ACHC-sensitive GABA transporter; GAT-B, β-alanine-sensitive GABA transporter; GLY-1 and GLYT-1, glycine transporters; PROT-1, proline transporter; BGT-1, betaine transporter.

. Transporters for the major amino acid neurotransmitters glutamate, GABA, and glycine are found in both neurons and glial cells. Recent work has resulted in the identification of cDNAs encoding several amino acid neurotransmitter transport proteins, all of which belong to the Na⁺-and Cl^?-dependent transporter gene family. The diversity of this family suggests a degree of transporter heterogeneity that is greater than that indicated by biochemical and pharmacological studies.     

Transforming growth factor-β-3 is mitogenic for rat retinal progenitor cells in vitro

Recent data indicate that the process of neurogenesis in the mammalian central nervous system (CNS) may be regulated by peptide growth factors, such as epidermal growth factor, transforming growth factor-alpha, and acidic or basic fibroblast growth factor. We have investigated whether members of the transforming growth factor-beta (TGFβ) family also play a role in this process and have found that TGFβ-3 is mitogenic for embryonic rat retinal cells in vitro. We also show that TGFβ-3 stimulates production of retinal amacrine cells while photoreceptor production remains unchanged. These data demonstrate that TGFβ-3 can regulate cell proliferation in the CNS during development and can also influence commitment or differentiation, or both, of neural progenitor cells to particular retinal fates. © 1995 John Wiley & Sons, Inc.     

Laminin SIKVAV peptide-induced angiogenesis in vivo is potentiated by neutrophils

Angiogenesis has been investigated in vivo using subcutaneously injected reconstituted basement membrane (Matrigel) supplemented with angiogenic factors. Previously we found that the laminin-derived synthetic peptide containing SIKVAV (ser-ile-lys-val-ala-val) promoted angiogenesis in vivo. In parallel studies, it was observed that new vessel formation in response to this peptide occurred several days after basic fibroblast growth factor-induced angiogenesis. Since this delay suggested that SIKVAV-induced angiogenesis may be secondary to other events, we investigated here earlier time points to determine if both indirect and direct mechanisms of angiogenesis are involved. We found that neutrophils are continuously recruited to the SIKVAV-containing plugs between 4 hours to 3 days following the initial injection. By day 7, columns of endothelial cells begin to migrate into the plug and form small blood vessels. In contrast, neutropenic mice had a 62% reduction in SIKVAV-induced angiogenesis when compared to control mice. Freshly isolated neutrophils also degraded laminin, the major component of the basement membrane Matrigel. These cells also produced factors in response to SIKVAV peptide which induced proliferation of human umbilical vein endothelial cells relative to a control peptide. In vitro experiments utilizing human neutrophils demonstrated that these cells migrate to the SIKVAV peptide and possess a specific cell surface SIKVAV binding protein of ～56 kD. These data suggest that neutrophils are induced to migrate to the Matrigel plugs, at least in part, by SIKVAV peptide, where they may release their own angiogenic factors and degrade the matrix, thus physically facilitating cell migration and liberating additional angiogenic matrix fragments and/or cytokines. © 1994 Wiley-Liss, Inc.

1 This article is a US Government work and, as such, is in the public domain in the United States of America

     

Increased expression of heme oxygenase-1 in human retinal pigment epithelial cells by transforming growth factor-β

Antibodies specific for heme oxygenase-1 (HO-1) were produced in rabbits, using the multiple antigen peptide (MAP) technique, and were employed to investigate the ability of transforming growth factor-β1 (TGF-β1) to induce the HO-1 protein in cultured human retinal pigment epithelial (RPE) cells. Western blot analyses showed that the cytokine induced HO-1 in these cells in a time- and dose-dependent manner. TGF-β1 also increased the mRNA for HO-1 in treated cells prior to the increase in HO-1 protein. The induction was effectively blocked by a neutralizing antibody preparation against TGF-β1. When tested under similar conditions, other growth factors such as basic fibroblast growth factor-I, plateletderived growth factor, insulin-like growth factor, transforming growth factor-α, and epidermal growth factor did not show appreciable induction of HO-1. Lipopolysaccharide, tumor necrosis factor-α, and interferon-γ were also not inducers, although TGF-β2 effectively induced HO-1. Heavy metal ions and thiol reagents were also highly potent inducers of HO-1 in human RPE cells. The induction of HO-1 by TGF-β1 was also observed in bovine choroid fibroblasts, but not in HELA, HEL or bovine corneal fibroblasts. Our results demonstrate for the first time that HO-1 can be induced by an important cytokine, TGF-β1, causing an increase in the expression of both HO-1 message and protein in specific neuroepithelial and fibroblast cells. © 1994 wiley-Liss, Inc.

1 This article is a US Government work and, as such, is in the public domain in the United States of America.

     

c-Myc inactivation by mutant max alters growth and morphology of NCI-H-630 colon cancer cells

The myc gene family has been implicated in multiple cell processes including proliferation, differentiation, tumorigenesis, and apoptosis. For its cellular growth promoting function, Myc must heterodimerize with Max. To study the effect of Myc inactivation on the growth and differentiation properties of epithelial tumor cells, we transfected the H-630 human colon cancer cell line with bm-max, a mutant Max protein in which DNA-binding activity has been abolished. Cells expressing high levels of bm-Max grow poorly, and the morphology of both colonies and single cells is altered. Moreover, increased bm-Max expression results in a prolonged G₀/G₁ phase accompanied by induced expression of p21 (WAF1/CIP1), elevated levels of alkaline phosphatase (ALP) activity, and accumulation of large fat granuli within the cells. These distinctive cell characteristics are associated with differentiation processes in numerous malignant cell lines. The results of this study support a model in which sequestering of endogenous Myc and Max proteins into “basic mutant” dimers lacking DNA-binding activity is sufficient both to inhibit proliferation and to induce changes in cell behavior consistent with differentiation. © 1996 Wiley-Liss, Inc.

1 This article is a US Government work and, as such, is in the public domain in the United States of America.

     

Environmental magnetic fields inhibit the antiproliferative action of tamoxifen and melatonin in a human breast cancer cell line

We have previously reported that environmental-level magnetic fields (1.2 μT [12 milligauss], 60 Hz) block the growth inhibition of the hormone melatonin (10⁻⁹ M) on MCF-7 human breast cancer cells in vitro. We now report that the same 1.2 μT, 60 Hz magnetic fields significantly block the growth inhibitory action of pharmacological levels of tamoxifen (10⁻⁷ M). In biophysical studies we have taken advantage of Faraday's Law of Current Induction and tested whether the 1.2 μT magnetic field or the associated induced electric field is responsible for this field effect on melatonin and tamoxifen. We observe that the magnetic field component is associated with the field blocking effect on melatonin and tamoxifen function. To our knowledge the tamoxifen studies represent the first experimental evidence for an environmental-level magnetic field modification of drug interaction with human breast cancer cells. Together, these findings provide support to the theory that environmental-level magnetic fields can act to modify the action of a drug or hormone on regulation of cell proliferation. Melatonin and tamoxifen may act through different biological pathways to down-regulate cell growth, and further studies are required to identify a specific biological site of interaction for the 1.2 μT magnetic field. Bioelectromagnetics 18:555–562, 1997. Published 1997 Wiley-Liss, Inc.

1 This article is a US Government work and, as such, is in the public domain in the United States of America.

     

Identification and characterization of an ecdysiotropic peptide from brain extracts of the gypsy moth,Lymantria dispar

A peptide (Lymantria TE) was isolated from brains of the gypsy moth, Lymantria dispar, which stimulates synthesis of ecdysteroid in the testes of larval and pupal insects. This ecdysiotropic peptide was purified and its structure determined to be NH₂-IIe-Ser-Asp-Phe-Asp-Glu-Tyr-Glu-Pro-Leu-Asn-Asp-Ala-Asp-Asn-Asn-Glu-Val-Leu-Asp-Phe-OH using protein sequence analysis and electrospray mass spectrometry. The peptide was biphasic in activity, with maximal activity in the pupal testes at 10⁻¹³ M and 10⁻⁹ M, with a minimum at 10⁻¹⁰ M, and with maxima at 10⁻¹⁵ M and 10⁻¹⁰ M and minimum at 10⁻¹³ M for larval testes. Arch. Insect Biochem. Physiol. 34:175–189, 1997. © 1997 Wiley-Liss, Inc.

1 This article is a US Government work and, as such, is in the public domain in the United States of America.

     

Phenotypic consequences of transforming growth factor β1 gene ablation in murine embryonic fibroblasts: Autocrine control of cell proliferation and extracellular matrix biosynthesis

The profound effects of transforming growth factor β1 (TGF-β1) on the immune system, cardiogenesis, in yolk sac hematopoeisis and in differentiation of endothelium have been demonstrated by detailed analyses of TGF-β1 knockout mice during embryogenesis. We have systematically examined the autocrine and paracrine roles of TGF-β1 in cell proliferation and in its ability to modulate the gene expression of selected components of extracellular matrix (ECM) using embryonic fibroblasts from TGF-β1 null mice (TGF-β1^−/−). The rates of cell proliferation of embryonic fibroblasts from normal mice (TGF-β1^+/+) and TGF-β1 null mice were compared by cell counting, by ³H thymidine incorporation, and by measuring the fraction of cells in the G1, S, and G2/M phases of the cell cycle by fluorescent activated cell sorting (FACS). Concurrently, the expression of pro-α1(I) collagen, fibronectin, and plasminogen activator inhibitor-1 (PAI-1) was also quantified by hybridization of total mRNA from TGF-β1^+/+ and TGF-β1^−/− embryonic fibroblasts. We report that TGF-β1^−/− cells proliferated at about twice the rate of TGF-β1^+/+ cells. Further, TGF-β1 null fibroblasts accumulated and synthesized lower constitutive levels of pro-α1(I) collagen, fibronectin, and PAI-1 mRNA. The quantitative differences in the rates of cell proliferation and ECM gene expression between TGF-β1^+/+ and TGF-β1^−/− cells could be eliminated by treatment of TGF-β1^+/+ cells with a neutralizing antibody of TGF-β1. Thus, our results are consistent with the hypothesis that TGF-β1 acts as a negative autocrine regulator of growth and a positive autocrine regulator of ECM biosynthesis in embryonic fibroblasts. 176:67–75, 1998. Published 1998 Wiley-Liss, Inc.

1 This article was prepared by a group of United States government employees and non-United States government employees, and as such is subject to 17 U.S.C. Sec. 105.

     

The viscostat: Productstat method of feed-rate control in continuous fermentations

Xanthan biopolymer has been produced in a single-stage continuous fermentation with Xanthomonas campestirs NRRL B-1459, using a viscostal control method instead of the conventional chemostat method. A Bendix Ultraviscoson

1 The mention of firm names or trade products does not imply that they are endorsed or recommended by the U.S. Department of Agriculture over other firms or similar products not mentioned.

sensed the fermentor viscosity, and the recorder–controller actuated the feed medium pump in an on–off control mode. Since all continuous fermentations eventually become contaminated or suffer culture variation, this work served also to demonstrate the effectiveness of the viscostat control. Neither the presence of a mold contaminant with specific growth rates lower than that of X. campestris, nor the presence of a bacterial contaminant of specific growth rate greater than X. campestris, affected the maintenance of constant viscosity in this control system.     

Crystal structure of TGF-β2 refined at 1.8 Å resolution

The crystal structure of TGF-β2 has been refined using data collected with synchrotron radiation (CHESS) to 1.8 Å resolution with a residual R (= ∑ | |F_o| ? |F_c| | /∑ |F_o|) factor of 17.3%. The model consists of 890 protein atoms from all 112 residues and 59 water molecules. The monomer of TGF-β2 assumes a rather extended conformation and lacks a well-defined hydrophobic core. Surface accessibility calculations show only 44% of the nonpolar surface is buried in the monomer. In contrast, 55.8% of the nonpolar surface area is buried when the two monomers from a dimer, a typical value for globular proteins. This includes a 1300 Ų buried interface area that is largely hydrophobic. Sequence comparisons using a profile derived from the refined TGF-β2 structure suggest that the cluster of four disulfides (three intramonomeric disulfide bonds 15–78, 44–109, 48–111 forming a disulfide knot, and one intermonomeric disulfide 77–77) together with the extended β strand region constitutes the conserved structural motif for the TGF-β superfamily. This structural motif, without the 77–77 disulfide bond, defines also the common fold for a general family of growth factors, including the nerve growth factor and platelet-derived growth factor families. The fold is conserved only at the monomer level, while the active forms are dimers, suggesting that dimerization plays an important role in regulating the binding of these cytokines to their receptors and in modulating the biological responses. © 1993 Wiley-Liss, Inc.

1 This article is a US Government work and, as such, is in the public domain in the United States of America.

     

Multicellular-vesicle-promoting polypeptide from Trichoplusia ni: Tissue distribution and N-terminal sequence

An N-terminal amino acid sequence of a 16.9 kDa hemolymph polypeptide, “Vesicle Promoting Factor” (VPF) from Trichoplusia ni, revealed a high sequence homology (70%) with Manduca sexta apolipophorin-III. A polyclonal antibody developed against VPF, however, was not immunoreactive with either purified M. sexta or T. ni apolipophorin-III. Immunoblots of tissue homogenates of T. ni indicated that VPF was present in imaginal wing discs, central nervous system (CNS), silk glands, midgut and hemocytes from fifth instar larvae, and also in the IAL-TND1 cell line which can grow as either fluid-filled multicellular vesicles or multicellular aggregates. VPF was also detected immunologically in the hemolymph of adults of T. ni, and in hemolymph of adults and larvae of Galleria mellonella and Heliothis virescens. Testes, midgut, hemocytes, and wing discs, but not Malpighian tubules, of T. ni released VPF into tissue culture medium during a 3 h incubation period. © 1995 Wiley-Liss, Inc.

1 This article is a US Government work and, as such, is in the public domain in the United States of America.

     

Mating-induced loss of sex pheromone and sexual receptivity in insects with emphasis on Helicoverpa zea and Lymantria dispar

Mating in most species of insects leads to a transient or permanent loss in sexual receptivity of the females. Among moths, this loss of receptivity is often accompanied with a loss of the sex pheromone in the absence of calling, which also could be temporary or permanent. Most of the earlier work on changes in reproductive behavior after mating was done with Diptera in which sperm and/or male accessory gland secretions were shown to be responsible for termination of receptivity. In the corn earworm moth, Helicoverpa zea, mated females become depleted of pheromone and become nonreceptive to further mating attempts, but only for the remainder of the night of mating. A pheromonostatic peptide isolated from the accessory glands of males may be responsible for the depletion of pheromone, while the termination of receptivity is independently controlled. In the gypsy moth, Lymantria dispar, the changes in behavior following mating are permanent. In this species, the switch from virgin to mated behavior involves three steps: a physical stimulation associated with mating, transfer of viable sperm to the spermatheca, and commencement of oviposition. Signals generated by these factors operate through neural pathways and, unlike in H. zea, accessory gland factors seem not to be involved. © 1994 Wiley-Liss, Inc.

1 This article is a US Government work and, as such, is in the public domain in the United States of America.

     

Transgenic Zebrafish for Studying Nervous System Development and Regeneration

1 tubulin gene expression is induced in the developing and regenerating CNS of vertebrates. Therefore, 1 tubulin gene expression may serve as a good probe for mechanisms underlying CNS development and regeneration. One approach to identify these mechanisms is to work backwards from the genome. This requires identification of 1 tubulin DNA sequences that mediate its developmental and regeneration-dependent expression pattern. Therefore, we generated transgenic zebrafish harboring a fragment of the 1 tubulin gene driving green fluorescent protein expression (GFP). In these fish, and similar to the endogenous gene, transgene expression was dramatically induced in the developing and regenerating nervous system. Although transgene expression generally declined during maturation of the nervous system, robust GFP expression was maintained in progenitor cells in the retinal periphery, lining brain ventricles and surrounding the central canal of the spinal cord. When these cells were cultured in vitro they divided and gave rise to new neurons. We also show that optic nerve crush in adult fish re-induced transgene expression in retinal ganglion cells. These studies identified a relatively small region of the 1 tubulin promoter that mediates its regulated expression pattern in developing and adult fish. This promoter will be extremely useful to investigators interested in targeting gene expression to the developing or regenerating nervous system. As adult transgenic fish maintain transgene expression in neural progenitors, these fish also provide a valuable resource of labeled adult neural progenitor cells that can be studied in vivo or in vitro. Finally, these fish should provide a unique in vivo system for investigating mechanisms mediating CNS development and regeneration.     

Effect of aging on EGF-stimulated replication of specific genes in rat hepatocytes

EGF-stimulated replication of specific genes was examined in primary hepatocyte cultures from mature (6 months) and senescent (24 months) rats. Basal and EGF-stimulated [³H]thymidine incorporation and DNA polymerase α activities, as well as total cellular DNA, were also assessed. The genes examined were dihydrofolate reductase (DHFR) and c-myc, as well as total mitochondrial DNA (mt DNA). Although [³H]thymidine incorporation, DNA polymerase α activity, total cellular DNA, DHFR, and c-myc gene specific DNA replication stimulated by EGF are reduced with age, mt DNA replication is not affected by either EGF or age. Chromosomal DNA replication is mediated mainly by DNA polymerase α while mt DNA replication is mediated by its own DNA polymerase γ. Thus, the age-related decline in stimulated DNA replication appears to be associated mainly with the DNA polymerase α activation pathway. J. Cell. Physiol. 176:32–39, 1998. Published 1998 Wiley-Liss, Inc.

1 This article is a US Government work and, as such, is in the public domain in the United States of America.