Surface modification of ZnO using triethoxysilane-based molecules.

Zinc oxide (ZnO) is an important material for hybrid inorganic-organic devices in which the characteristics of the interface can dominate both the structural and electronic properties of the system. These characteristics can be modified through chemical functionalization of the ZnO surface. One of the possible strategies involves covalent bonding of the modifier using silane chemistry. Whereas a significant body of work has been published regarding silane attachments to glass and SiO2, there is less information about the efficacy of this method for controlling the surface of metal oxides. Here we report our investigation of molecular layers attached to polycrystalline ZnO through silane bonding, controlled by an amine catalyst. The catalyst enables us to use triethoxysilane precursors and thereby avoid undesirable multilayer formation. The polycrystalline surface is a practical material, grown by sol-gel processing, that is under active exploration for device applications. Our study included terminations with alkyl and phenyl groups. We used water contact angles, infrared spectroscopy, and X-ray photoemission spectroscopy to evaluate the modified surfaces. Alkyltriethoxysilane functionalization of ZnO produced molecular layers with submonolayer coverage and evidence of disorder. Nevertheless, a very stable hydrophobic surface with contact angles approaching 106 degrees resulted. Phenyltriethoxysilane was found to deposit in a similar manner. The resulting surface, however, exhibited significantly different wetting as a result of the nature of the end group. Molecular layers of this type, with a variety of surface terminations that use the same molecular attachment scheme, should enable interface engineering that optimizes the chemical selectivity of ZnO biosensors or the charge-transfer properties of ZnO-polymer interfaces found in oxide-organic electronics.

Enhanced systemic transgene expression after nonviral salivary gland transfection using a novel endonuclease inhibitor/DNA formulation.

Gene transfer to the major salivary glands is an attractive method for the systemic delivery of therapeutic proteins. To date, nonviral gene transfer to these glands has resulted in inadequate systemic protein concentrations. We believe that identification of the barriers responsible for this inefficient transfection will enable the development of enhanced nonviral gene transfer in salivary glands and other tissues. One potential barrier is the degradation of plasmid DNA by endonucleases. To test this hypothesis, we coadministered two endonuclease inhibitors ((zinc and aurintricarboxylic acid (ATA)) with plasmid DNA, containing the secreted alkaline phosphatase gene (SEAP), to the submandibular glands of rats. The effect of zinc and ATA on SEAP expression, tissue accumulation of plasmid DNA, and plasmid DNA stability was then characterized. We observed that mixtures containing zinc/DNA, ATA/DNA, and zinc/ATA/DNA significantly enhanced both systemic transgene expression and the amount of plasmid DNA associated with treated tissues. The relative endonuclease inhibitory activity of zinc, ATA, and zinc/ATA correlated with the observed effects on transfection efficacy. The use of zinc/ATA enhanced the efficacy of salivary gland transfection by at least 1000-fold versus DNA alone. Importantly, this improved performance resulted in robust systemic secretion of an exogenous protein (SEAP), thus demonstrating the potential this nonviral gene transfer technology has as a method to treat systemic protein deficiencies.

Gastroprotection of DNA with a synthetic cholic acid analog.

The oral delivery of functional DNA to the gastrointestinal system would constitute a desirable, noninvasive method for potentially treating a variety of diseases. The digestive process, however, remains a formidable barrier. This dilemma may be addressed by using targeted liposomes both to protect the polynucleotide and to deliver the therapeutic DNA with high tissue specificity. The present study represents the initial steps toward developing a novel gene delivery system designed to interact with the enterohepatic receptors of the small intestine. Two cholic acid esters were synthetically modified at position C(3) to incorporate a DNA-binding domain. These novel compounds were evaluated for their ability to protect DNA from the nucleases found in gastrointestinal segments. Additionally, the compounds were screened as a component of a gene delivery vector. Formulations containing the new bile salt derivatives protected DNA from degradation for more than 2 h and were capable of transfecting cultured NIH 3T3 cells.

Optical fiber coupled light emitting diode based absorbance detector with a reflective flow cell.

We present a versatile, optical fiber coupled light emitting diode (LED) light source based flow-through optical absorbance detector. The LED source is readily changeable. Optical fibers are used to carry light from the electronics/display unit to a reflective flow-through cell and back. The cell can thus be located remotely from the electronics unit and the umbilical connection is not susceptible to electrical noise. The noise level of this detector with LEDs of different emission maxima were observed to be in the range of 3-20 muAU under actual use conditions, with a maximum short term drift of 4 muAU/min after the initial warm-up period. When the analyte absorbance is well matched with the source emission characteristics, the detector response is linear with concentration over at least two orders of magnitude. The liquid flow path through the cell is linear with a large exit aperture such that bubbles are not trapped in the optical path. The optical arrangement is such that the incident light crosses the liquid flow orthogonally and is reflected back by a rear mirror to the receiver fiber. This arrangement reduces the refractive index sensitivity by an order of magnitude relative to conventional Z-path flow cells.

Fibroblast growth factor receptor 4, implicated in progression of islet cell carcinogenesis by its expression profile, does not contribute functionally.

Fibroblast growth factor receptor 4 (FGFR4) gene expression is activated in late-stage beta-cell tumors that develop in transgenic mice harboring SV40 large T antigen (Tag) gene that is under the transcriptional control of the insulin promoter (RIP-Tag). The FGFR4 gene was active in cell lines derived from tumors but not in cells derived from hyperplastic islets. We used both gain-of-function and loss-of-function FGFR4 transgenic mice to determine whether FGFR4 modulates islet cell tumorigenesis and, if so, to identify the nature of the effect. Both types of FGFR4 transgenic mice were viable and fertile and developed islet tumors when crossed with RIP-Tag mice. Remarkably, there was no significant perturbation in the tumorigenesis pathway resulting from either chronic up-regulation or absence of FGFR4 gene expression. Analyses included the incidence and size of tumors, rate of cell proliferation, cell density, and life span. We conclude that FGFR4 gene activation is a marker of but is not causal for beta-cell transformation.

Pancreatic gene expression in rare cells of thymic medulla: evidence for functional contribution to T cell tolerance.

We report the initial characterization of rare cells within the thymus that express 'peripheral' self-antigens and are capable of inducing partial tolerance to a model protein. Mice from two transgenic families that express SV40 T antigen (Tag) in pancreatic islet beta cells under control of a rat insulin promoter (RIP) develop T cell tolerance toward this neo-self antigen. These mice express low levels of Tag mRNA in the thymus. Transplantation of thymus from tolerant RIP-Tag mice into athymic hosts is sufficient to confer tolerance by CD4+ Th cells and elicits variable tolerance by CD8+ cytotoxic T cells. Thymic medulla is shown to contain rare cells that express the endogenous insulin and somatostatin genes, and in the transgenic animals, Tag. These cells are referred to as 'peripheral antigen-expressing' (PAE) cells. Thymic cell fractionation reveals the PAE cells expressing insulin and Tag to be present in a fraction enriched for non-lymphoid, MHC class II+ cells. Notably, absence of thymic expression of the RIP-Tag gene in another transgenic family correlates with failure to establish self-tolerance and susceptibility to autoimmunity. Thus, expression of tissue-restricted genes such as insulin in PAE cells of thymic medulla may serve to limit development of potentially autoimmune T cells.

Differential induction of seven 1-aminocyclopropane-1-carboxylate synthase genes by elicitor in suspension cultures of tomato (Lycopersicon esculentum).

The key enzyme of ethylene biosynthesis, ACC synthase, is encoded by a multigene family. We describe three new DNA sequences encoding members of the ACC synthase family of the tomato. One of these sequences encodes a novel ACC synthase, LE-ACS6, which is phylogenetically related to the ACC synthases LE-ACS1A and LE-ACS1B. Gene-specific probes for seven tomato ACC synthase genes were prepared. They were used for RNase protection assays to study the accumulation of ACC synthase transcripts in suspension-cultured tomato cells after the addition of an elicitor. The ACC synthase genes LE-ACS2, LE-ACS5 and LE-ACS6 were strongly induced by the elicitor. In contrast, the genes LE-ACS1B, LE-ACS3 and LE-ACS4 were constitutively expressed and LE-ACS1B was present at all times at a particularly high level. Thus, there are two groups of ACC synthase transcripts expressed in these cells, either elicitor-induced or constitutive. A transcript of LE-ACS1A was not detected. Despite the presence of LE-ACS1B, LE-ACS2, LE-ACS3, LE-ACS4 and LE-ACS5, there was only little ethylene produced in the absence of the elicitor. Increased ethylene production is usually correlated with the accumulation of ACC synthase transcripts, indicating that ethylene production is controlled via the transcriptional activation of ACC synthase genes. However, the abundance of several ACC synthase mRNAs studied was not strictly correlated with the rate of elicitor-induced ethylene production. Our data provide evidence that the activity of these ACC synthases may not solely be controlled by the transcriptional activation of ACC synthase genes.

The endocrine secretion of human insulin and growth hormone by exocrine glands of the gastrointestinal tract.

The exocrine pancreas, liver, and submandibular glands of the rat were used to express and secrete two exogenous, human protein hormones (growth hormone and insulin) into blood at physiological concentrations. Transfection, expression, and secretion were achieved by the in vivo retrograde injection of plasmid DNA into the secretory ducts of these glands. Pancreatic acinar cells secreted physiological concentrations of growth hormone into the circulation, and its secretion was enhanced by cholinergic stimulation. A human insulin gene was engineered to allow normal processing of insulin in non-beta cells. With this gene, the secretion of human insulin by the exocrine pancreas normalized elevated blood glucose levels in diabetic rats. These in vivo observations demonstrate the utility of retrograde ductal administration of naked DNA into exocrine organs as a novel method for the regulated systemic delivery of protein-based pharmaceuticals.

Upregulation of fibroblast growth factors and their receptors during multi-stage epidermal carcinogenesis in K14-HPV16 transgenic mice.

Upregulation of acidic and basic fibroblast growth factors (FGF-1 and -2), and their cognate receptors FGFR-1 and -2, has been demonstrated in a variety of epithelial malignancies. However, the patterns of FGF/FGFR expression at specific stages of epithelial carcinogenesis have not been extensively characterized. In this report, the levels of FGF-1, FGF-2, FGF-7 mRNA and their receptors FGFR-1 and FGFR-2, were investigated during epidermal carcinogenesis in transgenic mice expressing the early region of the 'high risk' papillomavirus type 16 (HPV16) under control of the human keratin-14 enhancer/promoter (K14-HPV16 transgenic mice). FGF-1 was first upregulated in dysplasias, while FGF-2 was constitutively expressed in non-transgenic, neoplastic, and malignant keratinocytes throughout carcinogenesis. Expression of FGF-7 was undetectable in non-transgenic epidermis, and remained at threshold levels at all stages of progression. In well differentiated squamous cancers, FGFR-1 was upregulated and co-localized with angiogenic capillaries in the dermis underlying dysplastic lesions and within papillary fronds of invasive cancers. In contrast, FGFR-1 was upregulated specifically within the malignant squamous cells of moderate-poorly differentiated squamous cancers. The expression of FGFR-2 was essentially constitutive in both non-transgenic and neoplastic epidermis. Collectively the data suggest that the FGF/FGFR signaling pathways may potentially contribute to several facets of multi-stage epithelial carcinogenesis, including auto- or paracrine growth stimulation, upregulation of angiogenesis, and stromal remodeling.

Analysis of LE-ACS3, a 1-aminocyclopropane-1-carboxylic acid synthase gene expressed during flooding in the roots of tomato plants.

The plant hormone ethylene is produced in response to a variety of environmental stresses. Previous work has shown that flooding or anaerobic stress in the roots of tomato plants caused an increase in the production of the ethylene precursor 1-aminocyclopropane-1-carboxylate (ACC) in the roots, due to flooding-induced activity of ACC synthase (EC 4.4.1.14). RNA was extracted from roots and leaves of tomato plants flooded over a period of 48 h. Blot analysis of these RNAs hybridized with probes for four different ACC synthases revealed that the ACC synthase gene LE-ACS3 is rapidly induced in roots. LE-ACS2 is also induced, but at later times. The genomic clone for LE-ACS3 was isolated and sequenced. At all time points, the probe from the LE-ACS3 coding region hybridized to two bands in the RNA blots. Hybridization using the first and third introns of LE-ACS3 separately as probes indicate that flooding may inhibit processing of the LE-ACS3 transcript. Sequence homology analysis identified three putative cis-acting response elements in the promoter region, corresponding to the anaerobic response element from the maize adh1 promoter, the root-specific expression element from the cauliflower mosaic virus 35S promoter and a recognition element for chloroplast DNA binding factor I from the maize chloroplast ATP synthase promoter.

An apparatus for the determination of volatile analytes by stopped-flow injection analysis using an integrated fiber optic detector.

A new method for the analysis of volatile analytes using a stopped-flow injection system originating from either a gas or liquid phase has been developed. It uses an integrated fiber optic detector which also serves as a reactor. This system combines the advantages of gas diffusion and stopped-flow, making the overall assay very sensitive. Both gas streams and aqueous solutions containing ammonia were analyzed. The limits of detection are 40 ppb for gas phase analysis and 1.0 ppm for aqueous phase analysis.

The properties of p53 proteins selected for the loss of suppression of transformation.

The wild-type p53 protein can act as a suppressor of transformation in that it will block or reduce the formation of adenovirus E1A plus ras mediated transformants of primary rat embryo fibroblasts (C. A. Finlay et al., Cell, 57: 1083-1093, 1989). In those experiments, all of the transformed cell lines that arose selected for mutations in the transfected p53 gene, and many of these cell lines now express mutant p53 proteins. These mutant p53 genes are unusual because they were selected only for their inability to act as a transformation suppressor of other oncogenes as opposed to mutant p53 genes that arise spontaneously in tumors or transformed cell lines. p53 mutants that arise in tumors may be selected for several properties, and these mutants do have a number of phenotypes in common; for example, (a) they no longer block the cell division of transformed cells in culture (growth suppressor); (b) they cooperate with ras to transform rat embryo cells; (c) they enhance the plating efficiency of rat embryo cells; (d) some mutants have an altered protein conformation; (e) most mutants have a much longer half-life and greater concentration in the cells; and (f) mutants have lost or have a reduced ability to act as a transcription factor. Experiments were carried out to test whether the selection for p53 mutants that fail to block oncogene mediated transformation would also have some or all of the other properties of p53 mutants that arise in spontaneous tumors. Two mutants selected for their lost ability to block transformation were cloned, sequenced, and tested for all of the phenotypes listed above. The properties that these mutants had in common were (a) cooperation with ras to transform cells, (b) enhanced plating efficiency of cells, (c) elevated steady-state expression levels, and (d) a lost or reduced ability to act as a transcription factor.

Identification and characterization of multiple mdm-2 proteins and mdm-2-p53 protein complexes.

A protein product of the mdm-2 oncogene (p90) has been recently shown to associate with the protein encoded by the tumor-suppressor gene p53. The mdm-2 gene was originally identified as a gene amplified in a spontaneously transformed Balb/c 3T3 cell line (3T3DM). This report describes the characterization of mdm-2 gene products and their interactions with the p53 protein. Polyclonal and monoclonal antibodies were generated against murine and human mdm-2 protein. These antibodies detected the mdm-2 p90 protein and at least four additional polypeptides (p85, p76, p74, p58-p57) in cultured cells. These additional proteins may arise from different spliced mRNA forms of the mdm-2 gene or post-translational modifications of the mdm-2 protein. The monoclonal antibodies distinguished at least three sets of mdm-2 proteins with distinct combinations of epitopes (p90 and p85; p76 and p74; p58-57). One or two of these proteins forms a complex with the p53 protein (p90, p58). These mdm-2 proteins were found to be overexpressed in 3T3DM cells and a subset of these proteins were complexed with p53. In 3T3DM cells, p90, like p53, had a short half-life of approximately 20 min and was localized to the cell nucleus. In resting cells stimulated with serum p90 levels and p90/p53 complex levels increased in the late G1 phase of the cell cycle. The p90 mdm-2 protein could regulate p53 activity in the late G1 phase of the cell cycle.

The mdm-2 oncogene product forms a complex with the p53 protein and inhibits p53-mediated transactivation.

A cellular phosphoprotein with an apparent molecular mass of 90 kd (p90) that forms a complex with both mutant and wild-type p53 protein has been characterized, purified, and identified. The protein was identified as a product of the murine double minute 2 gene (mdm-2). The mdm-2 gene enhances the tumorigenic potential of cells when it is overexpressed and encodes a putative transcription factor. To determine if mdm-2 could modulate p53 transactivation, a p53-responsive element from the muscle creatine kinase gene was employed. A wild-type p53-expressing plasmid enhanced the expression of the p53-responsive element when cotransfected into cells that contain no endogenous p53. When a cosmid expressing mdm-2 was transfected with this p53-expressing plasmid, the transactivation of the p53-responsive element was inhibited. Thus, a product of the mdm-2 oncogene forms a tight complex with the p53 protein, and the mdm-2 oncogene can inhibit p53-mediated transactivation.

Differential expression of two genes for 1-aminocyclopropane-1-carboxylate synthase in tomato fruits.

1-Aminocyclopropane-1-carboxylate synthase (ACC synthase; S-adenosyl-L-methionine methylthioadenosine-lyase, EC 4.4.1.14) is the regulated enzyme in the biosynthetic pathway of the plant hormone ethylene. A full-length cDNA encoding this enzyme has been cloned from tomato fruits [Van Der Straeten, D., Van Wiemeersch, L., Goodman, H. M. & Van Montagu, M. Proc. Natl. Acad. Sci. USA (1990) 87, 4859-4863]. We report here the complete nucleotide and derived amino acid sequences of a cDNA encoding a second isoform of ACC synthase from tomato fruits. The cDNAs coding for both isoforms contain highly conserved regions that are surrounded by regions of low homology, especially at the 5' and 3' ends. Gene-specific probes were constructed to examine the expression of transcripts encoding the two ACC synthase isoforms under two conditions of enhanced ethylene formation--namely, during fruit ripening and in response to mechanical stress (wounding). The level of mRNA encoding both isoforms, ACC synthase 1 and 2, increased during ripening. In contrast, wounding caused an increase in only the level of mRNA coding for ACC synthase 1. Blot analysis of genomic DNA digested with restriction enzymes confirmed that ACC synthase 1 and 2 are encoded by different genes.

Determination of phenylalanine in serum using reversed-phase liquid chromatography and fluorescence detection.

A liquid chromatography procedure is reported for determining phenylalanine in small volumes of serum. A 10-microliter volume of serum was deproteinized with ethanol and an aliquot was derivatized with dansyl chloride reagent. The dansylated phenylalanine and the norleucine internal standard were separated using reversed-phase chromatography and measured with a fluorescence detector. Linearity was excellent over the range 50-800 mg/l. Within-run precision was better than 4%. Total analysis time including chromatography was approximately 40 min. As little as 300 pg of dansylated phenylalanine was detected.