Study of the influence of plasmids on the arbitrary primer polymerase chain reaction fingerprint of Escherichia coli strains

Abstract To study the effect of plasmids on the arbitrary primer-polymerase chain reaction fingerprint of bacterial strains, the Escherichia coli strains DH5, Top10, and W3110 were transformed with plasmids of different sizes: respectively, pUC19, pCEP and two clinically important plasmids carrying resistance to several antibiotics. Total DNA, i.e. both chromosomal and plasmid DNA, was prepared from transformed cells by boiling the cell suspensions and by phenol-chloroform extraction; chromosomal DNA was prepared by the same methods from the non-transformed, plasmid-free strains; plasmid DNA of pUC19 was purchased; plasmid DNA of pCEP was purified from the transformed strains by caesium chloride density gradient centrifugation. Arbitrarily primed polymerase chain reaction was carried out for all of these preparations. Amplification carried out independently with three different primers resulted in similar patterns for the chromosomal preparations whether or not plasmid was present. Amplification of plasmid DNA gave different patterns, characterized by fragments larger than those obtained when total or chromosomal DNA were used as the target. These data illustrate that the plasmids studied here do not influence the chromosomal arbitrarily primed PCR fingerprint, although plasmids alone are amplified in the absence of chromosomal DNA. Experiments comparing different relative concentrations of plasmid and chromosomal DNA indicate that under natural conditions the amount of chromosomal DNA per cell is sufficient to inhibit observable amplification of the plasmid(s) present.     

Local accumulations of B-50/GAP-43 evoke excessive bleb formation in PC12 cells

B-50 (GAP-43) is an axonal, plasma membrane-associated protein involved in growth cone morphology and function. We have conducted immunocytochemical, electron microscopic, and time-lapse experiments to visualize morphological consequences of local accumulations of B-50 at the plasma membrane of B-50-transfected PC-B2 cells, a clonal PC12 cell line with very low expression of endogenous B-50. The distribution of the transfected B-50 within these cells was inhomogeneous. At sites where the B-50 concentration was locally increased up to twofold, numerous filopodia were present in growth cone-like, substrate-attached regions. When local B-50 concentrations were even higher (up to 6.2-fold), blebs were formed, often containing vesicular structures, heavily decorated with B-50 immunoreactivity. Double labeling with f-actin binding phalloidin revealed that local B-50 accumulations were accompanied by increased actin filament concentrations. Colocalization of B-50 with actin filaments was prominent in filopodia, but was virtually absent in blebs, suggesting a disconnection of the bleb plasma membrane from the actin cytoskeleton. We conclude that B-50 evokes distinct effects on cell-surface activity in PC12 cells depending on its local concentration.     

Dehydroascorbate influences the plant cell cycle through a glutathione-independent reduction mechanism

Glutathione is generally accepted as the principal electron donor for dehydroascorbate (DHA) reduction. Moreover, both glutathione and DHA affect cell cycle progression in plant cells. But other mechanisms for DHA reduction have been proposed. To investigate the connection between DHA and glutathione, we have evaluated cellular ascorbate and glutathione concentrations and their redox status after addition of dehydroascorbate to medium of tobacco (Nicotiana tabacum) L. cv Bright Yellow-2 (BY-2) cells. Addition of 1 mm DHA did not change the endogenous glutathione concentration. Total glutathione depletion of BY-2 cells was achieved after 24-h incubation with 1 mm of the glutathione biosynthesis inhibitor l-buthionine sulfoximine. Even in these cells devoid of glutathione, complete uptake and internal reduction of 1 mm DHA was observed within 6 h, although the initial reduction rate was slower. Addition of DHA to a synchronized BY-2 culture, or depleting its glutathione content, had a synergistic effect on cell cycle progression. Moreover, increased intracellular glutathione concentrations did not prevent exogenous DHA from inducing a cell cycle shift. It is therefore concluded that, together with a glutathione-driven DHA reduction, a glutathione-independent pathway for DHA reduction exists in vivo, and that both compounds act independently in growth control.     

Profiling of oligolignols reveals monolignol coupling conditions in lignifying poplar xylem

Lignin is an aromatic heteropolymer, abundantly present in the walls of secondary thickened cells. Although much research has been devoted to the structure and composition of the polymer to obtain insight into lignin polymerization, the low-molecular weight oligolignol fraction has escaped a detailed characterization. This fraction, in contrast to the rather inaccessible polymer, is a simple and accessible model that reveals details about the coupling of monolignols, an issue that has raised considerable controversy over the past years. We have profiled the methanol-soluble oligolignol fraction of poplar (Populus spp.) xylem, a tissue with extensive lignification. Using liquid chromatography-mass spectrometry, chemical synthesis, and nuclear magnetic resonance, we have elucidated the structures of 38 compounds, most of which were dimers, trimers, and tetramers derived from coniferyl alcohol, sinapyl alcohol, their aldehyde analogs, or vanillin. All structures support the recently challenged random chemical coupling hypothesis for lignin polymerization. Importantly, the structures of two oligomers, each containing a gamma-p-hydroxybenzoylated syringyl unit, strongly suggest that sinapyl p-hydroxybenzoate is an authentic precursor for lignin polymerization in poplar.     

Internalization of Human 5-HT<Subscript>4a</Subscript> and 5-HT<Subscript>4b</Subscript> Receptors is Splice Variant Dependent

The family of 5-HT₄ receptors comprises 16 putative splice variants. We have previously shown that there are differences in signal transduction of the h5-HT_4a and h5-HT_4b receptors. In the present study, the internalization of these two splice variants following receptor stimulation was investigated with confocal microscopy on living cells. Chimeric receptors, h5-HT_4a-GFP and h5-HT_4b-GFP were generated by fusing the coding sequence of the 5-HT₄ receptor with the coding sequence of the GFP. The agonist stimulation of fluorescent receptors resulted in a time-dependent internalization of the h5-HT_4b-GFP receptor, but not of the h5-HT_4a-GFP receptor. The h5-HT_4b receptor displays a dual coupling to Gαi,o and Gαs proteins, in contrast to the h5-HT_4a receptor, which couples to Gαs proteins only. We investigated whether the difference in internalization of the two splice variant receptors was related to their differential coupling. Therefore, we performed agonist-stimulation of the receptor following inhibition of the Gαi,o protein coupling using PTX. The h5-HT_4b receptor internalization is PTX insensitive. We co-transfected the fluorescent chimeric receptors with other wild-type variants, which did not produce an alteration of the receptor trafficking. These findings provide the first evidence of differential internalization between the two splice variants, 5-HT_4a and 5-HT_4b receptors.     

Molecular mechanisms underlying specificity of excitotoxic signaling in neurons

The central role of glutamate receptors in mediating excitotoxic neuronal death in stroke, epilepsy and trauma has been well established. Glutamate is the major excitatory amino acid transmitter within the CNS and it's signaling is mediated by a number of postsynaptic ionotropic and metabotropic receptors. Although calcium ions are considered key regulators of excitotoxicity, new evidence suggests that specific second messenger pathways rather than total Ca(2+) load, are responsible for mediating neuronal degeneration. Glutamate receptors are found localized at the synapse within electron dense structures known as the postsynaptic density (PSD). Localization at the PSD is mediated by binding of glutamate receptors to submembrane proteins such as actin and PDZ containing proteins. PDZ domains are conserved motifs that mediate protein-protein interactions and self-association. In addition to glutamate receptors PDZ-containing proteins bind a multitude of intracellular signal molecules including nitric oxide synthase. In this way PDZ proteins provide a mechanism for clustering glutamate receptors at the synapse together with their corresponding signal transduction proteins. PSD organization may thus facilitate the individual neurotoxic signal mechanisms downstream of receptors during glutamate overactivity. Evidence exists showing that inhibiting signals downstream of glutamate receptors, such as nitric oxide and PARP-1 can reduce excitotoxic insult. Furthermore we have shown that uncoupling the interaction between specific glutamate receptors from their PDZ proteins protects neurons against glutamate-mediated excitotoxicity. These findings have significant implications for the treatment of neurodegenerative diseases using therapeutics that specifically target intracellular protein-protein interactions.     

Combined subtractive cDNA cloning and array CGH: an efficient approach for identification of overexpressed genes in DNA amplicons

Background  

Activation of proto-oncogenes by DNA amplification is an important mechanism in the development and maintenance of cancer cells. Until recently, identification of the targeted genes relied on labour intensive and time consuming positional cloning methods. In this study, we outline a straightforward and efficient strategy for fast and comprehensive cloning of amplified and overexpressed genes.     

Rapid population decline in red knots: fitness consequences of decreased refuelling rates and late arrival in Delaware Bay

Most populations of migrant shorebirds around the world are in serious decline, suggesting that vital condition-dependent rates such as fecundity and annual survival are being affected globally. A striking example is the red knot (Calidris canutus rufa) population wintering in Tierra del Fuego, which undertakes marathon 30,000 km hemispheric migrations annually. In spring, migrant birds forage voraciously on horseshoe crab eggs in Delaware Bay in the eastern USA before departing to breed in Arctic polar deserts. From 1997 to 2002 an increasing proportion of knots failed to reach threshold departure masses of 180-200 g, possibly because of later arrival in the Bay and food shortage from concurrent over-harvesting of crabs. Reduced nutrient storage, especially in late-arriving birds, possibly combined with reduced sizes of intestine and liver during refuelling, had severe fitness consequences for adult survival and recruitment of young in 2000-2002. From 1997 to 2002 known survivors in Delaware Bay were heavier at initial capture than birds never seen again, annual survival of adults decreased by 37% between May 2000 and May 2001, and the number of second-year birds in wintering flocks declined by 47%. Population size in Tierra del Fuego declined alarmingly from 51,000 to 27,000 in 2000-2002, seriously threatening the viability of this subspecies. Demographic modelling predicts imminent endangerment and an increased risk of extinction of the subspecies without urgent risk-averse management.     

A local influence approach applied to binary data from a psychiatric study

Recently, a lot of concern has been raised about assumptions needed in order to fit statistical models to incomplete multivariate and longitudinal data. In response, research efforts are being devoted to the development of tools that assess the sensitivity of such models to often strong but always, at least in part, unverifiable assumptions. Many efforts have been devoted to longitudinal data, primarily in the selection model context, although some researchers have expressed interest in the pattern-mixture setting as well. A promising tool, proposed by Verbeke et al. (2001, Biometrics 57, 43-50), is based on local influence (Cook, 1986, Journal of the Royal Statistical Society, Series B 48, 133-169). These authors considered the Diggle and Kenward (1994, Applied Statistics 43, 49-93) model, which is based on a selection model, integrating a linear mixed model for continuous outcomes with logistic regression for dropout. In this article, we show that a similar idea can be developed for multivariate and longitudinal binary data, subject to nonmonotone missingness. We focus on the model proposed by Baker, Rosenberger, and DerSimonian (1992, Statistics in Medicine 11, 643-657). The original model is first extended to allow for (possibly continuous) covariates, whereafter a local influence strategy is developed to support the model-building process. The model is able to deal with nonmonotone missingness but has some limitations as well, stemming from the conditional nature of the model parameters. Some analytical insight is provided into the behavior of the local influence graphs.