C6 glioma cells express low levels of the gap junction protein connexin 43 and its mRNA and display very weak dye coupling. When implanted into the rat cerebrum, these cells quickly give rise to a large glioma. To investigate the role of gap junctions in the tumor characteristics of these cells, we have used Lipofectin-mediated transfection to introduce a full-length cDNA encoding connexin 43. Several transfected clones were obtained that exhibited various amounts of connexin 43 mRNA transcribed from the inserted cDNA. Immunocytochemical analysis revealed an increase in the amount of connexin 43 immunoreactivity in the transfected cells, being localized at areas of intercellular contact as well as in the cytoplasm. The level of dye coupling was also assessed and found to correlate with the amount of connexin 43 mRNA. When cell proliferation was followed over several days, cells expressing the transfected cDNA grew more slowly than nontransfected cells. These transfected cells will be useful in examining the role of gap junctions in tumorigenesis.Intercellular communication by gap junctions has been implicated in the control of cell growth and differentiation (1, 2). These intramembranous channels, or connexons, are composed of hexameric assemblies of the gap junction proteins connexins. Isolation ofconnexins and cloning oftheir cDNAs indicate that they are encoded by a family of genes that exhibit some degree of cellular and tissue specificity (3, 4).C6 glioma cells exhibit a low level of intercellular communication by gap junctions, as shown by morphological and functional studies (5-7). Consistent with these observations, these cells contain low levels of the same gapjunction mRNA and protein that is expressed in astrocytes, namely connexin 43 (6, 8). Since tumorigenicity has been correlated with a low level or absence of intercellular communication by gap junctions (1, 9), the low level of connexin 43 mRNA and protein in C6 glioma cells may relate to the invasive growth of these cells in vivo (10).As an initial step to understand the role of gap junctions in normal and abberant cellular growth, we sought to alter the expression of connexin 43 in C6 glioma cells. To this end, we transfected these cells with a full-length cDNA for connexin 43 (11). We have obtained several clones that transcribe the transfected cDNA at moderate to high levels. Whereas connexin 43-like immunoreactivity is not readily detectable in nontransfected cells, such reactivity is prevalent in these transfected cells. The level of intercellular dye coupling correlates with the relative level of connexin 43 mRNA. Furthermore, the growth rate of the glioma cells expressing the transfected connexin 43 cDNA was significantly lower than that of nontransfected C6 cells. MATERIALS AND METHODSExpression Vector. The plasmid used for expression of the connexin 43 cDNA in rat C6 glioma cells was derived from pLTR ( Fig. 1), a simian virus 40 (SV40)-based vector, which includes the enhancer portion of the Harvey sarcoma virus long terminal re...

Summary 1. A review is given of the optical and architectural analogies between cholesteric liquid crystals and certain insect cuticles (Coleoptera: Scarabaeidae). Earlier observations on the optical properties (reflexion of circularly polarized light and high form optical rotation) are confirmed and extended. Both cholesteric liquid crystals and lamellate cuticle have helicoidal structure (Fig. i). Even though their chemistry and physical states are very different, we are justified in making the analogy, since their optical properties depend primarily on the pitch of their helicoidal architecture. 2. The unusual optical properties were located for the first time in the outer 5 to 20 μ of the exocuticle. This layer is transparent and has regular spacings in the range required for interference colours according to Bragg's law. Among Scarabaeid beetles which show interference colours, we distinguish two types of outer exocuticle. (i) Optically active cuticles which reflect circularly polarized interference colours; show high angles of form optical rotation in transmitted light; and anomalous form birefringence perpendicular to the cuticle surface (reversible by deproteinization). (2) Optically inactive cuticles which show none of the above properties and in which the form birefringence is parallel to the cuticle surface. In the electron microscope the ultrastructure of these two types of outer exocuticle is clearly different. 3. All of the optically active species reflect left hand circularly polarized light, irrespective of the wavelength of the reflected colour. They therefore appear dark when viewed through a right hand circular analyser. The sense of reflected circularly polarized light does not reverse at higher wavelengths as recorded by previous workers. (A simple treatment is given for combinations of various wavelengths with retardation plates of varying values, as used in circular analysers.) We confirm earlier reports that the sense of reflected circularly polarized light is of the opposite sense to the transmitted light. 4. Using monochromatic light we have measured the anomalous dispersion with wavelength of the magnitude of optical rotation for various optically active cuticles. The dispersion curves change from negative values at lower wavelengths to positive values at higher wavelengths, and cross the zero optical rotation axis at a wavelength (AQ) corresponding to the interference colour of each sample. There is reasonable agreement between A0 and the interference colour calculated from ultrastructural evidence and by comparison with interference filters of known wavelength. A dispersion curve measured for a combined sample of two cuticles with different dispersion curves showed that the resultant is an algebraic summation of the two component curves. 5. We present the first experimental verification of existing mathematical treatments of anomalous form optical rotatory dispersion curves. Although these treatments were derived for cholesteric liquid crystals, they give a reasonable fit to our measu...

The iridescent cuticle of certain Rutelino scarab beetles, which is a form optically active and selectively reflects circularly polarized light, incorporates an NH 4 OH -extractable component The ultraviolet absorption spectrum of this component, together with its chromatographic and refractive properties, identify it as uric acid (2,6,8-trihydroxypurine). All species of Plusiotis examined have uric acid in their reflecting layers, as do several species of Anoplognathus. Plusiotis resplendens has a reflecting layer with a uric acid volume fraction of 0.7, P . optima a volume fraction of 0.6. The reflecting layer of P . resplenden s has an anticlockwise helicoidal architecture, the optical thickness of the helicoidal p itch being such that it constructively interferes with visible light wavelengths. An anticlockwise helicoid constructively interferes with only the left circularly polarized component of incident light, right circularly polarized light being transmitted without attenuation. P. resplendens has a 1.8 /xm thick unidirectional layer embedded within the helicoid which functions as a perfect halfwave retardation plate for wavelength 590 nm . This halfwave plate enables the helicoidal reflector in this species to reflect both left and right circularly polarized components of incident light. After passing through the halfwave plate, transmitted right circularly polarized light becomes left circularly polarized ; this light is now reflected and emerges from the cuticle right circularly polarized, after passing back through the halfwave plate. Consequently the total reflectivity of circularly polarized incident light is greater in P. resplendens than in any other species examined; the plate also reduces multiple internal reflexions. Interferometric analysis of the refractive properties of the helicoidal reflectors in species of Plusiotis showed that the ordered incorporation of uric acid increases the birefringence of the system by a factor of five times, e.g. the in tact birefringence of the unidirectional layer of P . resplendens is 0.166 at wavelength 560 nm ; after uric acid extraction the birefringence is reduced to 0.034. As the coefficient of reflexion of a helicoidal reflector is directly proportional to the birefringence of the individual planes comprising the helicoid, beetles incorporating uric acid into their reflecting surfaces reflect circularly polarized light far more efficiently than beetles lacking uric acid. Refractive index values for a single multicomponent plane of the helicoid have been summarized as a biaxial indicatrix, with the Z axis tilte dat 45° to the plane of the epicuticle. Beetle reflecting layers which incorporate uric acid have twenty times greater optical rotatory power compared with reflecting layers lacking this component. Mathematical treatments dealing with helicoidal reflectors predict the form optical rotatory power to be a function of the square of the birefringence, which is in agreement with the experimental observations. To enable uric acid to have the optical effects mentioned above, an epitaxial incorporation into the helicoidal framework is necessary. Although uric acid is a common cytoplasmic reflecting material in arthropods, this is the first record of its presence in an extracellular (cuticular) reflector.

Some ATP-binding cassette (ABC) transporters of subfamilies B, C and G confer resistance to xenobiotics including insecticides. We identified genes of these subfamilies expressed by the lepidopterans Trichoplusia ni and Bombyx mori. The B. mori genome includes eight, six and 13 ABC-B, -C and -G genes, respectively, which encode P-glycoprotein, multidrug resistance protein, MRP, and breast cancer resistance protein, BCRP, homologues. Among the ABC-C and -G subfamilies, gene duplication contributes to protein diversity. We have identified three ABC-B and two ABC-C T. ni genes. Analyses of the T. ni MRP (TrnMRP) revealed unique features, including the potential for TrnMRP4 hyperglycosylation and the alternative splicing of TrnMRP1. Taken together, these attributes of moth multidrug resistance-associated ABCs may confer distinct functional capacities to xenobiotic efflux.

For several millennia, stem extracts of Ephedra (Ephedraceae, Gnetales) have been used as folk medicines in both the Old and New World. Some species were used in treatments of questionable efficacy for venereal disease in North America during the last century. Many Eurasian species produce phenylethylamine alkaloids, mostly ephedrine and pseudoephedrine, that interact with adrenergic receptors in the mammalian sympathetic nervous system. Asian Ephedra have been used recently in the clandestine manufacture of a street drug, methamphetamine. Although ephedrine alkaloids are not detectable in New World species of Ephedra, together with Asian species they contain other nitrogen-containing secondary metabolites with known neuropharmacological activity. Many mesic and particularly xeric species worldwide accumulate substantial amounts of quinoline-2-carboxylic acids, or kynurenates, in their aerial parts. Many species of Ephedra accumulate cyclopropyl amino acid analogues of glutamate and proline in their stems and roots, and particularly in the seed endosperm. Mesic species synthesize substantial amounts of three L-2-(carboxycyclopropyl)glycine stereomers rarely seen in nature. A cyclopropyl analogue of proline with known antimicrobial activity, cis-3,4-methanoproline, is found in large amounts in the stems and seeds of many Ephedra species. The ability to synthesize cyclopropyl amino acids may be an ancestral feature in the taxon. The natural function in the taxon of these three groups of secondary compounds remains to be established.

To examine the role of pp-junctional inter

No abstract

SUMMARY Selective Na+-dependent re-uptake of biogenic monoamines at mammalian nerve synapses is accomplished by three types of solute-linked carrier family 6 (SLC6) membrane transporter with high affinity for serotonin(SERTs), dopamine (DATs) and norepinephrine (NETs). An additional SLC6 monoamine transporter (OAT), is responsible for the selective uptake of the phenolamines octopamine and tyramine by insect neurons. We have characterized a similar high-affinity phenoloamine transporter expressed in the CNS of the earthworm Lumbricus terrestris. Phylogenetic analysis of its protein sequence clusters it with both arthropod phenolamine and chordate catecholamine transporters. To clarify the relationships among metazoan monoamine transporters we identified representatives in the major branches of metazoan evolution by polymerase chain reaction (PCR)-amplifying conserved cDNA fragments from isolated nervous tissue and by analyzing available genomic data. Analysis of conserved motifs in the sequence data suggest that the presumed common ancestor of modern-day Bilateria expressed at least three functionally distinct monoamine transporters in its nervous system: a SERT currently found throughout bilaterian phyla, a DAT now restricted in distribution to protostome invertebrates and echinoderms and a third monoamine transporter (MAT), widely represented in contemporary Bilateria, that is selective for catecholamines and/or phenolamines. Chordate DATs, NETs,epinephrine transporters (ETs) and arthropod and annelid OATs all belong to the MAT clade. Contemporary invertebrate and chordate DATs belong to different SLC6 clades. Furthermore, the genes for dopamine and norepinephrine transporters of vertebrates are paralogous, apparently having arisen through duplication of an invertebrate MAT gene after the loss of an invertebrate-type DAT gene in a basal protochordate.