Molecular Charge-Transfer Cross Sections and Their Correlation with Reactant Ion Structures

Charge-transfer cross sections have been obtained by using time-of-flight techniques, and results correlated with reaction energetics and theoretical structures computed by self-consistent field-molecular orbital methods. Ion recombination energies, structures, heats of formation, reaction energy defects, and 3.0-keV charge-transfer cross sections are presented for reactions of molecular and fragment ions produced by electron bombardment ionization of CH30CH, and CH$l molecules. Relationships between experimental cross sections and reaction energetics involving different ion structures are discussed.

Sensitivity of charge transfer reactions to hydrocarbon ion structures

Charge transfer reactivities of hydrocarbon ions have been measured with time-of-flight techniques, and results correlated with theoretical structures computed by self-consistent field molecular orbital methods. Recombination energies, ion structures, heats of formation, reaction energetics and relative charge transfer cross-sections are presented for molecular and fragment ions produced by electron bombardment ionization of CH4, C2H4, C2H6, C3H8 and C4H10 molecules. Even-electron bridged cations have low ion recombination energies and relatively low charge transfer cross-sections as compared with odd-electron hydrocarbon cations.

Predicting spike timing of neocortical pyramidal neurons by simple threshold models

Neurons generate spikes reliably with millisecond precision if driven by a fluctuating current--is it then possible to predict the spike timing knowing the input? We determined parameters of an adapting threshold model using data recorded in vitro from 24 layer 5 pyramidal neurons from rat somatosensory cortex, stimulated intracellularly by a fluctuating current simulating synaptic bombardment in vivo. The model generates output spikes whenever the membrane voltage (a filtered version of the input current) reaches a dynamic threshold. We find that for input currents with large fluctuation amplitude, up to 75% of the spike times can be predicted with a precision of +/-2 ms. Some of the intrinsic neuronal unreliability can be accounted for by a noisy threshold mechanism. Our results suggest that, under random current injection into the soma, (i) neuronal behavior in the subthreshold regime can be well approximated by a simple linear filter; and (ii) most of the nonlinearities are captured by a simple threshold process.

Relative sensitivity factors of inorganic cations in frozen-hydrated standards in secondary ion MS analysis

We describe the measurement, at 100 K, of the SIMS relative sensitivity factors (RSFs) of the main physiological cations Na+, K+, Mg2+, and Ca2+ in frozen-hydrated (F-H) ionic solutions. Freezing was performed by either plunge freezing or high-pressure freezing. We also report the measurement of the RSFs in flax fibers, which are a model for ions in the plant cell wall, and in F-H ionic samples, which are a model for ions in the vacuole. RSFs were determined under bombardment with neutral oxygen (FAB) for both the fibers and the F-H samples. We show that referencing to ice-characteristic secondary ions is of little value in determining RSFs and that referencing to K is preferable. The RSFs of Na relative to K and of Ca relative to Mg in F-H samples are similar to their respective values in fiber samples, whereas the RSFs of both Ca and Mg relative to K are lower in fibers than in F-H samples. Our data show that the physical factors important for the determination of the RSFs are not the same in F-H samples and in homogeneous matrixes. Our data show that it is possible to perform a SIMS relative quantification of the cations in frozen-hydrated samples with an accuracy on the order of 15%. Referencing to K permits the quantification of the ionic ratios, even when the absolute concentration of the referencing ion is unknown. This is essential for physiological studies of F-H biological samples.

THE ISOLATION AND IDENTIFICATION OF O-ACETYLATED SIALIC ACIDS ON HUMAN CELLS (NEURAMINIC ACIDS, PAPER CHROMATOGRAPHY, HUMAN MALIGNANCY, BIOSYNTHETIC LABELING)

Unlike most carbohydrates, sialic acids have a restricted distribution in nature, being present in higher animals and in certain bacteriae. Unfortunately, most studies have not taken into account the fact that the parent sialic acid molecules, N-acetyl(or N-glycolyl)-neuraminic acid can be O-substituted at the 4, 7, 8 and 9 positions, generating many compounds and isomers. The approach and results of this research study demonstrates that proportions of non-, mono-, di-, and tri-O-acetylated sialic acids can be identified and quantitated on normal and malignant human cells. This was accomplished using a paper chromatographic technique to isolate and resolve individual species of non and O-substituted sialic acids. The chemical nature of these O-substituents, as an acetyl ester, was determined on the basis of chemical degradation, enzymatic and fast atom bombardment-mass spectrometry analysis.^ The working hypothesis of this study, that O-acetylated sialic acids are expressed in a restricted manner on normal and malignant cells, was confirmed using the above experimental approach; which identified mono-, di-, and tri-O-acetylated sialic acids on a variety of normal and malignant human cells. These O-acetylated sialic acids were expressed in restricted manner on subpopulations and subcellular fractions of PHL melanoma cells. Aberrant expression of O-acetylated sialic acids was associated with adenocarcinoma of the colon, leading to a nearly complete loss of di- and tri-O-acetylated sialic acids.^ Thus, the ability to isolate and identify biosynthetically radiolabeled O-acetylated sialic acids offers an efficient method of monitoring the expression of O-acetylated sialic acids in biochemical and cellular interactions. Furthermore, the ability to identify abnormal ratios of O-acetylated sialic acids in the human colon, represents a possible diagnostic tool to evaluate and identify patients who may be genetically or culturally predisposed to the development of adenocarcinoma of the colon. ^

On the Methyl-Transfer Reaction in Crystalline Methyl 2-(Methylthio)benzenesulfonate: a Thermally Induced Non-Topochemical Solid-State Reaction

The rearrangement of methyl 2-(methylthio)benzenesulfonate (1) to the zwitterionic 2-(dimethyl-sulfonium)benzenesulfonate (2) is known to proceed in solution by intermolecular Me transfers. The same rearrangement has been observed to occur in crystalline 1, but the crystal structure shows that the molecular packing is not conducive to intermolecular Me transfer. The reaction has been carried out with mixed crystals composed of 1 and deuteriomethylated (D6)-l. By fast-atom-bombardment mass spectroscopy, it has been shown that the product consists of a 1:2:1 mixture of the non-, tri-, and hexadeuterated species, the mixture expected, if the solid-state reaction proceeds by intermolecular Me transfers. From this result, together with the slower rates of conversion in the single crystal compared with the melt, it can be concluded that the reaction must occur not topochemically but rather at defects such as microcavities, surfaces, and other irregularities in the ordered crystal arrangement.

Survivability analysis of tape-tether against multiple impact with tiny debris

We show that for a tether at 800 km altitude, which is 5 km long, 2 cm wide and 0.05 mm thick, the risk of substantial damage during a 3 month period due to multiple impacts with debris or micrometeoroids is low, of about 1.4%. By substantial damage we mean that if the tape is divided in 2 cm2 cm squares, then in some square the damaged area by bombardment with debris or micrometeoroids exceeds 11% of the area of the square. Furthermore, we show that the danger posed by the micrometeoroids is negligible compared to the risk posed by the debris.

Transgene organization in rice engineered through direct DNA transfer supports a two-phase integration mechanism mediated by the establishment of integration hot spots

Organization of transgenes in rice transformed through direct DNA transfer strongly suggests a two-phase integration mechanism. In the “preintegration” phase, transforming plasmid molecules (either intact or partial) are spliced together. This gives rise to rearranged transgenic sequences, which upon integration do not contain any interspersed plant genomic sequences. Subsequently, integration of transgenic DNA into the host genome is initiated. Our experiments suggest that the original site of integration acts as a hot spot, facilitating subsequent integration of successive transgenic molecules at the same locus. The resulting transgenic locus may have plant DNA separating the transgenic sequences. Our data indicate that transformation through direct DNA transfer, specifically particle bombardment, generally results in a single transgenic locus as a result of this two-phase integration mechanism. Transgenic plants generated through such processes may, therefore, be more amenable to breeding programs as the single transgenic locus will be easier to characterize genetically. Results from direct DNA transfer experiments suggest that in the absence of protein factors involved in exogenous DNA transfer through Agrobacterium, the qualitative and/or quantitative efficiency of transformation events is not compromised. Our results cast doubt on the role of Agrobacterium vir genes in the integration process.

A protein residing at the subunit interface of the bacterial ribosome

Surface labeling of Escherichia coli ribosomes with the use of the tritium bombardment technique has revealed a minor unidentified ribosome-bound protein (spot Y) that is hidden in the 70S ribosome and becomes highly labeled on dissociation of the 70S ribosome into subunits. In the present work, the N-terminal sequence of the protein Y was determined and its gene was identified as yfia, an ORF located upstream the phe operon of E. coli. This 12.7-kDa protein was isolated and characterized. An affinity of the purified protein Y for the 30S subunit, but not for the 50S ribosomal subunit, was shown. The protein proved to be exposed on the surface of the 30S subunit. The attachment of the 50S subunit resulted in hiding the protein Y, thus suggesting the protein location at the subunit interface in the 70S ribosome. The protein was shown to stabilize ribosomes against dissociation. The possible role of the protein Y as ribosome association factor in translation is discussed.

BAC-VAC, a novel generation of (DNA) vaccines: A bacterial artificial chromosome (BAC) containing a replication-competent, packaging-defective virus genome induces protective immunity against herpes simplex virus 1

This study aimed to exploit bacterial artificial chromosomes (BAC) as large antigen-capacity DNA vaccines (BAC-VAC) against complex pathogens, such as herpes simplex virus 1 (HSV-1). The 152-kbp HSV-1 genome recently has been cloned as an F-plasmid-based BAC in Escherichia coli (fHSV), which can efficiently produce infectious virus progeny upon transfection into mammalian cells. A safe modification of fHSV, fHSVΔpac, does not give rise to progeny virus because the signals necessary to package DNA into virions have been excluded. However, in mammalian cells fHSVΔpac DNA can still replicate, express the HSV-1 genes, cause cytotoxic effects, and produce virus-like particles. Because these functions mimic the lytic cycle of the HSV-1 infection, fHSVΔpac was expected to stimulate the immune system as efficiently as a modified live virus vaccine. To test this hypothesis, mice were immunized with fHSVΔpac DNA applied intradermally by gold-particle bombardment, and the immune responses were compared with those induced by infection with disabled infectious single cycle HSV-1. Immunization with either fHSVΔpac or disabled infectious single cycle HSV-1 induced the priming of HSV-1-specific cytotoxic T cells and the production of virus-specific antibodies and conferred protection against intracerebral injection of wild-type HSV-1 at a dose of 200 LD50. Protection probably was cell-mediated, as transfer of serum from immunized mice did not protect naive animals. We conclude that BAC-VACs per se, or in combination with genetic elements that support replicative amplification of the DNA in the cell nucleus, represent a useful new generation of DNA-based vaccination strategies for many viral and nonviral antigens.

enod40 induces dedifferentiation and division of root cortical cells in legumes

Under nitrogen-limiting conditions Rhizobium meliloti can establish symbiosis with Medicago plants to form nitrogen-fixing root nodules. Nodule organogenesis starts with the dedifferentiation and division of root cortical cells. In these cells the early nodulin gene enod40, which encodes an unusually small peptide (12 or 13 amino acids), is induced from the beginning of this process. Herein we show that enod40 expression evokes root nodule initiation. (i) Nitrogen-deprived transgenic Medicago truncatula plants overexpressing enod40 exhibit extensive cortical cell division in their roots in the absence of Rhizobium. (ii) Bombardment of Medicago roots with an enod40-expressing DNA cassette induces dedifferentiation and division of cortical cells and the expression of another early nodulin gene, Msenod12A. Moreover, transient expression of either the enod40 region spanning the oligopeptide sequence or only the downstream region without this sequence induces these responses. Our results suggest that the cell-specific growth response elicited by enod40 is involved in the initiation of root nodule organogenesis.

Gene replacement by homologous recombination in the multicellular green alga Volvox carteri

With only two different cell types, the haploid green alga Volvox represents the simplest multicellular model system. To facilitate genetic investigations in this organism, the occurrence of homologous recombination events was investigated with the intent of developing methods for gene replacement and gene disruption. First, homologous recombination between two plasmids was demonstrated by using overlapping nonfunctional fragments of a recombinant arylsulfatase gene (tubulin promoter/arylsulfatase gene). After bombardment of Volvox reproductive cells with DNA-coated gold microprojectiles, transformants expressing arylsulfatase constitutively were recovered, indicating the presence of the machinery for homologous recombination in Volvox. Second, a well characterized loss-of-function mutation in the nuclear nitrate reductase gene (nitA) with a single G → A nucleotide exchange in a 5′-splice site was chosen as a target for gene replacement. Gene replacement by homologous recombination was observed with a reasonably high frequency only if the replacement vector containing parts of the functional nitrate reductase gene contained only a few nucleotide exchanges. The ratio of homologous to random integration events ranged between 1:10 and 1:50, i.e., homologous recombination occurs frequently enough in Volvox to apply the powerful tool of gene disruption for functional studies of novel genes.

Metabolic pathway engineering in cotton: Biosynthesis of polyhydroxybutyrate in fiber cells

Alcaligenes eutrophus genes encoding the enzymes, β-ketothiolase (phaA), acetoacetyl-CoA reductase (phaB), and polyhydroxyalkanoate synthase (phaC) catalyze the production of aliphatic polyester poly-d-(−)-3-hydroxybutyrate (PHB) from acetyl-CoA. PHB is a thermoplastic polymer that may modify fiber properties when synthesized in cotton. Endogenous β-ketothiolase activity is present in cotton fibers. Hence cotton was transformed with engineered phaB and phaC genes by particle bombardment, and transgenic plants were selected based on marker gene, β-glucuronidase (GUS), expression. Fibers of 10 transgenic plants expressed phaB gene, while eight plants expressed both phaB and phaC genes. Electron microscopy examination of fibers expressing both genes indicated the presence of electron-lucent granules in the cytoplasm. High pressure liquid chromatography, gas chromatography, and mass spectrometry evidence suggested that the new polymer produced in transgenic fibers is PHB. Sixty-six percent of the PHB in fibers is in the molecular mass range of 0.6 × 106 to 1.8 × 106 Da. The presence of PHB granules in transgenic fibers resulted in measurable changes of thermal properties. The fibers exhibited better insulating characteristics. The rate of heat uptake and cooling was slower in transgenic fibers, resulting in higher heat capacity. These data show that metabolic pathway engineering in cotton may enhance fiber properties by incorporating new traits from other genetic sources. This is an important step toward producing new generation fibers for the textile industry.

A link between DNA methylation and epigenetic silencing in transgenic Volvox carteri

Epigenetic silencing of foreign genes introduced into plants poses an unsolved problem for transgenic technology. Here we have used the simple multicellular green alga Volvox carteri as a model to analyse the relation of DNA methylation to transgenic silencing. Volvox DNA contains on average 1.1% 5-methylcytosine and 0.3% N6-methyladenine, as revealed by electrospray mass spectrometry and phosphoimaging of chromatographically separated 32P-labelled nucleotides. In two nuclear transformants of V.carteri, produced in 1993 by biolistic bombardment with a foreign arylsulphatase gene (C-ars), the transgene is still expressed in one (Hill 181), but not in the other (Hill 183), after an estimated 500–1000 generations. Each transformant clone contains multiple intact copies of C-ars, most of them integrated into the genome as tandem repeats. When the bisulphite genomic sequencing protocol was applied to examine two select regions of transgenic C-ars, we found that the inactivated copies (Hill 183) exhibited a high-level methylation (40%) of CpG dinucleotides, whereas the active copies (Hill 181) displayed low-level (7%) CpG methylation. These are average values from 40 PCR clones sequenced from each DNA strand in the two portions of C-ars. The observed correlation of CpG methylation and transgene inactivation in a green alga will be discussed in the light of transcriptional silencing.

Developmental Regulation of Intercellular Protein Trafficking through Plasmodesmata in Tobacco Leaf Epidermis1

Plasmodesmata mediate direct cell-to-cell communication in plants. One of their significant features is that primary plasmodesmata formed at the time of cytokinesis often undergo structural modifications, by the de novo addition of cytoplasmic strands across cell walls, to become complex secondary plasmodesmata during plant development. Whether such modifications allow plasmodesmata to gain special transport functions has been an outstanding issue in plant biology. Here we present data showing that the cucumber mosaic virus 3a movement protein (MP):green fluorescent protein (GFP) fusion was not targeted to primary plasmodesmata in the epidermis of young or mature leaves in transgenic tobacco (Nicotiana tabacum) plants constitutively expressing the 3a:GFP fusion gene. Furthermore, the cucumber mosaic virus 3a MP:GFP fusion protein produced in planta by biolistic bombardment of the 3a:GFP fusion gene did not traffic between cells interconnected by primary plasmodesmata in the epidermis of a young leaf. In contrast, the 3a MP:GFP was targeted to complex secondary plasmodesmata and trafficked from cell to cell when a leaf reached a certain developmental stage. These data provide the first experimental evidence, to our knowledge, that primary and complex secondary plasmodesmata have different protein-trafficking functions and suggest that complex secondary plasmodesmata may be formed to traffic specific macromolecules that are important for certain stages of leaf development.