Sorting out of normal and virus-transformed cells in cellular aggregates

The sorting-out behavior (self-segregation of two cell types from mixtures of the two) of five different established cell lines was studied. Eight of the ten possible binary combinations of these lines, cultured as cellular aggregates, were examined. Mouse BALB/c 3T3 cells sorted out internally to the corresponding malignant SV40 virus-transformed 3T3 cells. The transformed 3T3 line (SVT-2) did not sort out from a revertant line selected from SVT-2 cells by resistance to concanavalin A (con A). The revertant cells sorted out externally to the parent BALB/c 3T3 cells, although segregation was generally incomplete. BALB/c 3T3 cells did not sort out from another contact-inhibited line of 3T3 cells derived from Swiss albino mice (Swiss 3T3). Both BALB/c 3T3 and Swiss 3T3 cells sorted out from cells of the contact-inhibited hamster line, NIL B. Instead of a two-layered sphere, however, a three-layered structure was observed with most of the NIL B cells external to the 3T3 cells, and a few NIL B cells comprising the center of the sphere. On the other hand, NIL B cells did not consistently sort out from either the SVT-2 or con A cells. In general, sorting out between pairs of these five lines are slower and less complete than is generally observed between the more extensively studied chick embryonic tissue cells, suggesting that the cultured cells may be more closely related in their adhesive properties. The internal segregation of BALB/c 3T3 cells relative to SVT-2 cells is consistent with the hypothesis that transformed cells are less adhesive than their nontransformed counterparts.     

Phytochrome-mediated induction of ascorbate oxidase in different organs of a dicotyledonous seedling (Sinapis alba L.)

Summary The time courses of the level of ascorbate oxidase (AO; EC 1.10.3.3.) were followed in the different organs (cotyledons, hypocotyl, taproot) of the developing mustard seedling. Phytochrome (operationally, far-red light, cf. [20]) rapidly and strongly enhances the rate of apparent ascorbate oxidase (AO)¹ synthesis in cotyledons and hypocotyl, while in the taproot the detectable amount of AO is only small. However, the relative increase of AO as mediated by continuous far-red light is the same in all organs. Far-red dark kinetics indicate that the phytochrome-induced enzyme is much less stable in the hypocotyl than in the cotyledons, at least during the experimental period. It is concluded that the effect of phytochrome on enzyme induction is precisely the same in cotyledons and hypocotyl, while the processes of enzyme degradation are specific for the organ. Thus the time courses of enzyme levels can be determined by the nature of the particular organ, even if no isoenzymes are involved and the mechanism of the inductive process is the same in the different organs.     

Induction of amylase in mustard seedlings by phytochrome

Summary In the cotyledons of mustard seedlings (Sinapis alba L.) amylase activity can be induced by phytochrome. In the dark amylase activity remains low. Gibberellic acid (GA₃) does not stimulate an increase of amylase activity in this system. Inhibitors of RNA and protein synthesis strongly inhibit the increase of amylase activity mediated by phytochrome. In gel electrophoresis amylase from mustard seedlings reveals 3 bands. The electrophoretic pattern is the same for extracts from dark-grown and from irradiated seedlings. When mustard amylases were incubated with starch the pattern of products was similar to that produced by commercially available barley -amylase and not similar to that produced by Bacillus subtilis -amylase.     

Investigations on the role of ethylene in phytochrome-mediated photomorphogenesis

The etiolating, intact mustard (Sinapis alba L.) seedling exhibits a distinct temporal pattern of ethylene production. Light, operating through phytochrome, increases the rate of ethylene production without changing the pattern. Ethylene production of the isolated plant parts (segments), added together, exceed the production of the intact system even if the wound effect is taken into account. There is no significant light effect on ethylene production of the segments. Phytochrome-mediated anthocyanin synthesis in the cotyledons is inhibited by ethylene. The responsiveness towards ethylene of the anthocyanin producing metabolic chain is decreased by phytochrome. As anthocyanin synthesis is only partly inhibited under saturating ethylene concentrations in the atmosphere around the seedlings (100 l l^–1), a twofactor analysis becomes feasible. This analysis leads to the result that phytochrome and ethylene show multiplicative behavior, meaning that phytochrome and ethylene act on the same metabolic sequence (leading to anthocyanin) but independently of each other, and at different sites. Therefore, the hypothesis that ethylene mediates the action of phytochrome in anthocyanin synthesis and photomorphogenesis in general appears to be inapplicable.Abbreviations P_fr far-red absorbing form of phytochrome - P_r red absorbing form of phytochrome - P_tot total phytochrome, i.e. [P_r]+[P_fr]     

Myocardial gene transfer and long-term expression following intracoronary delivery of adeno-associated virus

Adeno-associated viral vectors (AAV) can direct long-term gene expression in post-mitotic cells. Previous studies have established that long-term cardiac gene transfer results from intramuscular injection into the heart. Cardiac gene transfer after direct intracoronary delivery of AAV in vivo, however, has been minimal in degree, and indirect intracoronary delivery, an approach used in an increasing number of studies, appears to be receiving more attention. To determine the utility of indirect intracoronary gene transfer of AAV, we used aortic and pulmonary artery cross clamping followed by proximal aortic injection of AAV encoding enhanced green fluorescent protein (AAV.EGFP) at 10(11) DNase resistant particles (drp; high-performance liquid chromatography (HPLC)-purified) per rat. Gene expression was quantified by fluorescent microscopy at four time points up to 1 year after vector delivery, revealing 20-32% transmural gene expression in the left ventricle at each time point. Histological analysis revealed little or no inflammatory response and levels of transgene expression were low in liver and undetectable in lung. In subsequent studies in pigs, direct intracoronary delivery into the left circumflex coronary artery of AAV.EGFP (2.64-5.28 x 10(13) drp; HPLC-purified) resulted in gene expression in 3 of 4 pigs 8 weeks following injection with no inflammatory response in the heart. PCR analysis confirmed AAV vector presence in the left circumflex perfusion bed. These data indicate that intracoronary delivery of AAV vector is associated with transgene expression in the heart, providing a means to obtain long-term expression of therapeutic genes.     

Ecological genetics of adaptive color polymorphism in pocket mice: geographic variation in selected and neutral genes

Patterns of geographic variation in phenotype or genotype may provide evidence for natural selection. Here, we compare phenotypic variation in color, allele frequencies of a pigmentation gene (the melanocortin-1 receptor, Mc1r), and patterns of neutral mitochondrial DNA (mtDNA) variation in rock pocket mice (Chaetodipus intermedius) across a habitat gradient in southern Arizona. Pocket mice inhabiting volcanic lava have dark coats with unbanded, uniformly melanic hairs, whereas mice from nearby light-colored granitic rocks have light coats with banded hairs. This color polymorphism is a presumed adaptation to avoid predation. Previous work has demonstrated that two Mc1r alleles, D and d, differ by four amino acids, and are responsible for the color polymorphism: DD and Dd genotypes are melanic whereas dd genotypes are light colored. To determine the frequency of the two Mc1r allelic classes across the dark-colored lava and neighboring light-colored granite, we sequenced the Mc1r gene in 175 individuals from a 35-km transect in the Pinacate lava region. We also sequenced two neutral mtDNA genes, COIII and ND3, in the same individuals. We found a strong correlation between Mc1r allele frequency and habitat color and no correlation between mtDNA markers and habitat color. Using estimates of migration from mtDNA haplotypes between dark- and light-colored sampling sites and Mc1r allele frequencies at each site, we estimated selection coefficients against mismatched Mc1r alleles, assuming a simple model of migration-selection balance. Habitat-dependent selection appears strong but asymmetric: selection is stronger against light mice on dark rock than against melanic mice on light rock. Together these results suggest that natural selection acts to match pocket mouse coat color to substrate color, despite high levels of gene flow between light and melanic populations.     

Functional organization of the sortilin Vps10p domain

A Vps10p domain makes up the entire luminal part of Sortilin, and this type of domain is the hallmark of a new family of neuronal receptors that target a variety of ligands, including neurotrophins and neuropeptides. We have shown that two structural features of the Vps10p domain, the N-terminal propeptide and the C-terminal segment of ten conserved cysteines (10CC), are key elements in the function of Sortilin. The propeptide has two functions. (i) It binds the mature part of Sortilin and prevents ligands in the biosynthetic pathway from binding to the uncleaved proreceptor, and (ii) it facilitates receptor transport in early Golgi compartments by a mechanism that does not depend on its ability to prevent ligand binding. In contrast, other Vps10p domain receptors, such as SorLA and SorCS3, do not need their propeptide for normal and swift processing. The 10CC segment constitutes an exchangeable module containing five conserved disulfide bridges, and using module-shuffling and truncations, we have shown that the 10CC segment is a major ligand-binding region in Sortilin.     

The narrow-spectrum anthelmintic oxantel is a potent agonist of a novel acetylcholine receptor subtype in whipworms

In the absence of efficient alternative strategies, the control of parasitic nematodes, impacting human and animal health, mainly relies on the use of broad-spectrum anthelmintic compounds. Unfortunately, most of these drugs have a limited single-dose efficacy against infections caused by the whipworm, Trichuris. These infections are of both human and veterinary importance. However, in contrast to a wide range of parasitic nematode species, the narrow-spectrum anthelmintic oxantel has a high efficacy on Trichuris spp. Despite this knowledge, the molecular target(s) of oxantel within Trichuris is still unknown. In the distantly related pig roundworm, Ascaris suum, oxantel has a small, but significant effect on the recombinant homomeric Nicotine-sensitive ionotropic acetylcholine receptor (N-AChR) made up of five ACR-16 subunits. Therefore, we hypothesized that in whipworms, a putative homolog of an ACR-16 subunit, can form a functional oxantel-sensitive receptor. Using the pig whipworm T. suis as a model, we identified and cloned a novel ACR-16-like subunit and successfully expressed the corresponding homomeric channel in Xenopus laevis oocytes. Electrophysiological experiments revealed this receptor to have distinctive pharmacological properties with oxantel acting as a full agonist, hence we refer to the receptor as an O-AChR subtype. Pyrantel activated this novel O-AChR subtype moderately, whereas classic nicotinic agonists surprisingly resulted in only minor responses. We observed that the expression of the ACR-16-like subunit in the free-living nematode Caenorhabditis elegans conferred an increased sensitivity to oxantel of recombinant worms. We demonstrated that the novel Tsu-ACR-16-like receptor is indeed a target for oxantel, although other receptors may be involved. These finding brings new insight into the understanding of the high sensitivity of whipworms to oxantel, and highlights the importance of the discovery of additional distinct receptor subunit types within Trichuris that can be used as screening tools to evaluate the effect of new synthetic or natural anthelmintic compounds.