Nuclear Magnetic Resonance Chemical Shift as a Probe for Single-Molecule Charge Transport.

Existing modelling tools, developed to aid the design of efficient molecular wires and to better understand their charge-transport behaviour and mechanism, have limitations in accuracy and computational cost. Further research is required to develop faster and more precise methods that can yield information on how charge transport properties are impacted by changes in the chemical structure of a molecular wire. In this study, we report a clear semilogarithmic correlation between charge transport efficiency and nuclear magnetic resonance chemical shifts in multiple series of molecular wires, also accounting for the presence of chemical substituents. The NMR data was used to inform a simple tight-binding model that accurately captures the experimental single-molecule conductance values, especially useful in this case as more sophisticated density functional theory calculations fail due to inherent limitations. Our study demonstrates the potential of NMR spectroscopy as a valuable tool for characterising, rationalising, and gaining additional insights on the charge transport properties of single-molecule junctions.

Enhanced charge transport across molecule-nanoparticle-molecule sandwiches.

The electrical properties of large area molecular devices consisting of gold nanoparticles (GNPs) sandwiched between a double layer of alkanedithiol linkers have been examined. These devices have been fabricated by a facile bottom-up assembly in which an alkanedithiol monolayer is first self-assembled on an underlying gold substrate followed by nanoparticle adsorption and then finally assembly of the top alkanedithiol layer. These devices are then sandwiched between the bottom gold substrates and a top eGaIn probe contact and current-voltage (I-V) curves recorded. Devices have been fabricated with 1,5-pentanedithiol, 1,6-hexanedithiol, 1,8-octanedithiol and 1,10-decanedithiol linkers. In all cases the electrical conductance of the double SAM junctions with GNPs is higher than the corresponding and much thinner single alkanedithiol SAM. Competing models for this enhanced conductance are discussed and it is suggested to have a topological origin arising from how the devices assemble or structure during the fabrication, which gives more efficient cross device electron transport pathways without the GNPs producing short circuits.

Single-molecule solvation-shell sensing.

We present a new route to single-molecule sensing via solvation shells surrounding a current-carrying backbone molecule. As an example, we show that the presence of a water solvation shell "gates" the conductance of a family of oligothiophene-containing molecular wires, and that the longer the oligothiophene, the larger is the effect. For the longest example studied, the molecular conductance is over 2 orders of magnitude larger in the presence of a shell comprising just 10 water molecules. A first principles theoretical investigation of electron transport through the molecules, using the nonequilibrium Green's function method, shows that water molecules interact directly with the thiophene rings, significantly shifting transport resonances and greatly increasing the conductance. This reversible effect is confirmed experimentally through conductance measurements performed in the presence of moist air and dry argon.

Single-molecule conductance of redox molecules in electrochemical scanning tunneling microscopy.

Experimental data and theoretical notions are presented for 6-[1'-(6-mercapto-hexyl)-[4,4']bipyridinium]-hexane-1-thiol iodide (6V6) "wired" between a gold electrode surface and tip in an in situ scanning tunneling microscopy configuration. The viologen group can be used to "gate" charge transport across the molecular bridge through control of the electrochemical potential and consequently the redox state of the viologen moiety. This gating is theoretically considered within the framework of superexchange and coherent two-step notions for charge transport. It is shown here that the absence of a maximum in the Itunneling versus electrode potential relationship can be fitted by a "soft" gating concept. This arises from large configurational fluctuations of the molecular bridge linked to the gold contacts by flexible chains. This view is incorporated in a formalism that is well-suited for data analysis and reproduces in all important respects the 6V6 data for physically sound values of the appropriate parameters. This study demonstrates that fluctuations of isolated configurationally "soft" molecules can dominate charge transport patterns and that theoretical frameworks for compact monolayers may not be directly applied under such circumstances.

Characterization and immunolocalization of rat liver annexin VI.

Annexin VI has been purified to homogeneity from rat liver and monospecific antibodies have been produced. The antibodies have been used for immunoblot analysis of rat tissues. Annexin VI is present in most tissues, with particularly high concentrations in liver, spleen, muscle, and intestine. In liver, annexin VI constitutes approximately 0.25% of total cellular protein. Immunohistochemical studies have located annexin VI on plasma membranes of hepatocytes with enhanced concentration on bile canaliculi. Annexin VI binds in a Ca(2+)-dependent manner to a sub-cellular fraction containing membranes. In the presence of physiological concentrations of ATP, the free Ca2+ concentration required for half-maximal binding of annexin VI to membranes is significantly reduced. While annexin VI binds in vitro to membranes in the presence of Ca2+, in rat liver about 31% of the annexin VI is associated with membranes in a Ca(2+)-independent manner and its solubilization requires the presence of Triton X-100. However, studies using Triton X-114 showed no increase in the hydrophobicity of this fraction of the protein compared to the purified EGTA-soluble annexin VI.

Effects of androgens on the transcription of secretory protein genes in rat seminal vesicle.

Run-on transcription in isolated nuclei has been used to study the effects of testosterone on gene expression in rat seminal vesicles. General transcriptional rates were increased by about 6-fold with an additional 2- to 3-fold differential stimulation of the genes for secretory proteins IV and V. These transcriptional changes are insufficient to explain overall changes in cellular mRNA levels, indicating that androgens must also have major effects on post-transcriptional processing of RNA transcripts or on mRNA stability. Analysis of nuclear RNA by Northern blotting with intron probes suggests substantial androgen effects on primary transcript processing.

The establishment and validation of the mobile immunization team concept at a clinic level.

Faced with unacceptably low and declining overall immunization compliance, as well as specific flu immunization compliance, the Executive Committee of a 32-person Air National Guard clinic asked the nursing service to devise a method of correcting these deficiencies as rapidly as possible and maintaining immunization compliance at a rate of 90% or better of the total patient population of approximately 1,100 full-time and Guard personnel for whom the clinic was responsible. The concept of a mobile immunization team was devised and validated over a 2-year interval. In order to successfully develop and implement this concept, command emphasis and a high level of cooperation from both clinic personnel and the Unit Commanders involved was requested and received. The make-up of the team, risk management, the timing and place of team visits, and record keeping were among the problems addressed and resolved. The mobile immunization team concept was a success, achieving excellent overall immunization compliance and outstanding compliance with the flu immunization program over a 2-year period.

Neonatal seminal vesicle mesenchyme induces a new morphological and functional phenotype in the epithelia of adult ureter and ductus deferens.

Mesenchyme from neonatal mouse and rat seminal vesicles (SVM) was grown in association with postnatal (adult) epithelial cells from the ureter (URE) and ductus deferens (DDE) in chimeric tissue recombinants composed of mouse mesenchyme and rat epithelium or vice versa. Functional cytodifferentiation was examined in these SVM + URE and SVM + DDE tissue recombinants with antibodies against major androgen-dependent seminal-vesicle-specific secretory proteins. Adult DDE and URE were induced to express seminal cytodifferentiation and produced the complete spectrum of major seminal vesicle secretory (SVS) proteins. The SVS proteins produced were specific for the species that provided the epithelium. In the case of SVM + URE recombinants, the URE, which normally lacks androgen receptors (AR), expressed AR. These results demonstrate that adult epithelial cells retain a developmental plasticity equivalent to their undifferentiated fetal counterparts and are capable of being reprogrammed to express a completely new morphological, biochemical and functional phenotype.

Characterization of antibodies to androgen-dependent secretory proteins of the mouse dorsolateral prostate.

The mouse prostate is an attractive model for studying the relationship between epithelial-mesenchymal interactions and the mechanism of androgen action because of the volume of information on tissue interactions in the development of the prostate of this species and the existence of a mutant mouse lacking functional androgen receptors (Tfm mouse). In this paper the major proteins of the mouse dorsolateral prostate (DLP) have been described, and antibodies to these proteins have been characterized. The two most abundant secreted proteins were of 110,000-115,000 (Mj1) and 55,000-62,000 (Mj2) mol wt. They were glycosylated, androgen dependent, and appeared to exist in an oligomeric complex. Antibodies raised against mouse DLP secretion reacted mainly with Mj1, Mj2, and a minor protein of 140,000 mol wt (Mn1). The antibodies were of a high titer and recognized these three mouse DLP proteins by Western blotting, immunoprecipitation, and immunocytochemical techniques. Mj1 and Mj2 were antigenically similar to proteins in the mouse coagulating gland and in the rat DLP, but were not found in other organs. Immunocytochemical staining of the DLP from intact mice revealed many ducts that were lined by a tall columnar epithelium whose cells stained intensely. However, ducts that were distended with luminal secretion had a low columnar epithelium that rarely showed intracellular staining. These marker proteins and the antibodies to them will be useful for detecting androgen-dependent functional activity in tissue recombinant studies with a variety of experimental tissues.

The contribution made by cells from a single somite to tissues within a body segment and assessment of their integration with similar cells from adjacent segments.

Somites represent the first visual evidence of segmentation in the developing vertebrate embryo and it is becoming clear that this segmental pattern of the somites is used in the initial stages of development of other segmented systems such as the peripheral nervous system. However, it is not known whether the somites continue to contribute to the maintenance of the segmental pattern after the dispersal of the somitic cells. In particular, the extent to which cells from a single somite contribute to all of the tissues of a single body segment and the extent to which they mix with cells from adjacent segments during their migration is not known. In this study, we have replaced single somites in the future cervical region of 2-day-old chick embryos with equivalent, similarly staged quail somites. The chimerae were then allowed to develop for a further 6 days when they were killed. The cervical region was dissected and serially sectioned. The sections were stained with the Feulgen reaction for DNA to differentiate between the chick and quail cells. The results showed that the cells from a single somite remained as a clearly delimited group throughout their migration. Furthermore, the sclerotome, dermatome and myotome portions from the single somites could always be recognised as being separate from similar cells from other somites. The somitic cells formed all of the tissues within a body segment excluding the epidermis, notochord and neural tissue. There was very little mixing of the somitic cells between adjacent segments. The segmental pattern of the somites is therefore maintained during the migration of the somitic cells and this might be fundamental to a mechanism whereby the segmentation of structures, such as the peripheral nervous system, is also maintained during development.

Induction of functional cytodifferentiation in the epithelium of tissue recombinants. I. Homotypic seminal vesicle recombinants.

Functional cytodifferentiation of seminal vesicle epithelium was investigated in tissue recombinants. Neonatal rat and mouse seminal vesicles were separated into epithelium and mesenchyme using trypsin. Epithelium and mesenchyme were then recombined in vitro to form interspecific rat/mouse homotypic recombinants. Growth as renal grafts in adult male athymic mice resulted in seminal vesicle morphogenesis in 70% of the recombinants (the remaining 30% failed to grow). Functional cytodifferentiation was judged by the expression of the major androgen-dependent secretory proteins characteristic of the seminal vesicles of adult rats and mice. Antibodies specific for each of these proteins were used to screen tissue sections by immunocytochemistry and to probe protein extracts by immunoblotting techniques. The heterospecific recombinants synthesized the full range of seminal vesicle secretory proteins that typifies the species providing the epithelium of the recombinant, not the mesenchyme. There was little functional variation between individual recombinants. The time course of development corresponded to that of intact neonatal seminal vesicles grown under the same conditions. Morphogenesis and functional cytodifferentiation were not evident after one week, but were well advanced after two weeks. Seminal vesicle recombinants grown for three weeks were indistinguishable morphologically and functionally from normal adult seminal vesicles. In addition, the ability of adult seminal vesicle epithelium to be induced to proliferate was examined. In association with neonatal seminal vesicle mesenchyme, the epithelium of the adult seminal vesicle proliferated and retained its normal functional activity. Thus, seminal vesicle functional cytodifferentiation can be faithfully reproduced in homotypic tissue recombinants. The methods used in this study will be used to investigate seminal vesicle development in instructive inductions of heterotypic epithelia.

Induction of functional cytodifferentiation in the epithelium of tissue recombinants. II. Instructive induction of Wolffian duct epithelia by neonatal seminal vesicle mesenchyme.

When grown as renal grafts in adult male hosts, the upper (cranial), middle and lower (caudal) portions of fetal mouse and rat Wolffian ducts developed into epididymis, epididymis plus ductus deferens, and seminal vesicle, respectively. In heterotypic tissue recombinants, the epithelia from upper and middle Wolffian ducts were instructively induced to undergo seminal vesicle morphogenesis by neonatal seminal vesicle mesenchyme. Functional cytodifferentiation was examined in these recombinants using antibodies against major androgen-dependent, seminal vesicle-specific secretory proteins. The instructively induced Wolffian duct epithelia synthesized normal amounts of all of the secretory proteins characteristic of mature seminal vesicles, as judged by immunocytochemistry on tissue sections and gel electrophoresis plus immunoblotting of secretions extracted from the recombinants. In heterospecific recombinants composed of rat and mouse tissues, the seminal vesicle proteins induced were specific for the species that had provided the epithelium. This showed that the seminal vesicle epithelium in the recombinants was derived from instructively induced Wolffian duct epithelium and not from epithelial contamination of the mesenchymal inductor. Upper Wolffian duct epithelium, instructively induced to undergo seminal vesicle morphogenesis, did not express epididymis-specific secretory proteins, showing that its normal development had been simultaneously repressed.

The contribution made by a single somite to the vertebral column: experimental evidence in support of resegmentation using the chick-quail chimaera model.

The somitic involvement in the formation of the vertebral column was examined using the chick-quail chimaera model. Single cervical somites from quail donor embryos were transplanted into similarly staged chick host embryos. Following further incubation, serial sections of variously staged embryos were stained with the Feulgen reaction to distinguish the two cell populations. Quail cells were generally located within a delimited region in one half of each of the two adjacent vertebrae, as well as in the intervening disc. The horizontal plane of division through each vertebra passed approximately through the centre of the body and divided the neural arch into rostral and caudal halves through the rostral border of the caudal notch. These results give support to the controversial theory of resegmentation, in which it was suggested that there is an apparent realignment of segmentation between the somite stage and the subsequent vertebral stage of development.

The effects of somite removal on vertebral formation in the chick.

We have examined the effects on vertebral development of various combinations of somite removal in two day old chick embryos as shown by vertebral formation after a further seven days of incubation. Each combination produced one of a variety of results ranging from completely normal vertebral formation, through fusion of various vertebral elements, to the absence of complete vertebral halves and the formation of hemivertebrae. Assessment of our operating ability showed that we were removing at least 90% of the somitic material and therefore these results suggest that there is a regulating mechanism available to the embryo, at least with regards to vertebral development. When two consecutive somites were removed, vertebrae frequently developed that were lacking certain elements. This suggests that the somitic cells are already determined with regards to formation of specific vertebral elements. Experiments involving the removal of a bilateral pair of somites (a repetitive unit) also provided evidence of a counting mechanism which ensures that the correct number of total vertebrae are present.

Growth of rat seminal vesicle epithelial cells in culture: neurotransmitters are required for androgen-regulated synthesis of tissue-specific secretory proteins.

Epithelial cells from enzymically dispersed seminal vesicle tissue of castrated adult rats were grown on collagen-coated surfaces in serum-containing medium for up to 6 weeks. Androgens were neither required for growth nor were they mitogenic. They also failed to induce the synthesis of tissue-specific secretory proteins that serve as markers of androgen action in vivo. A variety of potential growth factors and culture procedures (such as feeder layers, extracellular matrices, collagen gels, and growth from tissue explants) were examined for their ability to support androgen action. Testosterone and 5 alpha-dihydrotestosterone were only able to induce seminal vesicle secretory protein synthesis in the presence of neurotransmitters (acetylcholine, noradrenaline, or serotonin). The mechanism of this permissive effect of neurotransmitters on androgenic induction has been examined. It is not related to their mitogenic activity since other mitogens (epidermal growth factor and corticosterone) did not facilitate androgen action. Agents that raise intracellular cAMP were also ineffective so cAMP is probably not involved, but the phosphatidyl inositol pathway may be important.

Formation of rat copulatory plug: purified seminal vesicle secretory proteins serve as transglutaminase substrates.

An in vitro system has been used to study the role of purified rat seminal vesicle proteins in the formation of the copulatory vaginal plug. Proteins II, IV (or S) and V (or F) were each separately coagulated using the transglutaminase in coagulating gland extracts. In each case the coagulum required Ca2+ ions for its formation and was insoluble in denaturing solvents. In experiments with [3H]lysine, proteins II and S incorporated [3H]lysine into glu-lys dipeptide with similar kinetics. Both the N-terminal and C-terminal glutamine residues of protein S participated in the reaction.

Comparison of seminal vesicle secretory proteins of rodents using antibody and nucleotide probes.

The copulatory vaginal plug is a conspicuous feature of rodent reproduction. The five major seminal vesicle secretory proteins of Rattus norvegicus (proteins I-V), which form the copulatory plug, constitute a closely related androgen-regulated family that appears to share a common evolutionary origin. The relationships between these rat proteins and the major seminal vesicle proteins of other rodents were explored using antibodies specific for the individual rat proteins. Immunoblotting of proteins separated by SDS-PAGE showed that the vesicular proteins of R. rattus are identical to those of R. norvegicus except for an additional protein related to protein III. No differences were seen in inbred and outbred strains of R. norvegicus. Of the major proteins of Mus musculus, one showed strong homology with rat protein II and three others were weakly homologous to proteins I, IV (or S) and V (or F); none showed homology to rat protein III. The only homology between the vesicular proteins of Mesocricetus auratus (Syrian hamster) and Meriones ungulatus (Mongolian gerbil) was with rat protein II while those of Cavia porcellus (guinea pig) showed no homology at all with the rat proteins. In addition, cDNA probes for rat genes IV and V both detected weak homologues in seminal vesicle RNA from mice but not guinea pigs.(ABSTRACT TRUNCATED AT 250 WORDS)

Tissue distribution, developmental profile and hormonal regulation of androgen-responsive secretory proteins of rat seminal vesicles studied by immunocytochemistry.

The seminal vesicles of the rat synthesise large amounts of androgen-regulated secretory proteins. Indirect immunofluorescence cytochemistry and immunoblotting with monospecific polyclonal antibodies against three of the major secretory proteins (II, S and F) have been used to investigate the tissue distribution, subcellular localisation, androgen-regulation and developmental profile of secretory protein synthesis. There was no evidence for regional specialisation of the seminal vesicle epithelium; every epithelial cell synthesizes all three proteins via a classical secretory involving storage in secretory vesicles. Proteins S and II are contained within the same secretory vesicles. The time course of deinduction of proteins S and F after castration and their reinduction by testosterone closely followed that for their specific mRNAs described previously. During development, proteins S and F first appear between 10 and 15 days after birth. A protein immunologically related to seminal vesicle protein II is present in the lateral and dorsal lobes of the prostatic complex.