Search PubMed⌕ Search

Biomedical subjects

R J Schmidt

Publications and source records attributed to R J Schmidt.

At least 73 records · Page 4Linked to original sources
PubMed1992

Biochemical responses of skin to allergenic and non-allergenic nitrohalobenzenes. Evidence that an NADPH-dependent reductase in skin may act as a prohapten-activating enzyme.

Using a selection of 'classic' haptens (dinitrohalobenzenes and picryl chloride) and related non-sensitizing analogous, we examined changes in levels of glutathione (GSH) and glutathione disulphide (GSSG) in mouse skin 12 h after their epicutaneous application. We observed that elevation of GSSG levels and/or depletion of GSH levels correlated well with contact allergenic potential. Non-sensitizing analogous failed to perturb GSH/GSSG status. In vitro assays using mouse skin and rat liver microsomal preparations indicated that only the allergenic nitrohalobenzenes initiated NADPH-dependent oxygen utilization, with the activity falling off in the order picryl chloride >> DNIB > DNBB > DNCB > DNFB. In addition, an examination of the colour of mouse skin homogenates ex vivo after application of the dinitrohalobenzenes showed significant yellowing (consistent with aromatic nucleophilic substitution) only with DNFB. Our results indicate that, while an aromatic nucleophilic substitution reaction with skin protein can possibly account for the allergenicity of DNFB, it does not seem to occur with DNCB, DNBB or DNIB. These may instead behave mainly as prohaptens which are activated enzymically by NADPH-dependent reductase(s) within the skin, with the concomitant generation of superoxide and hydrogen peroxide, to form potentially protein-reactive free radical and other metabolites. Picryl chloride appears capable of both conjugating directly with proteins by aromatic nucleophilic substitution and undergoing NADPH-dependent metabolism to other potentially protein-reactive metabolites.

Animals↗
PubMed1992

Nuclear localization signal(s) required for nuclear targeting of the maize regulatory protein Opaque-2.

The maize regulatory protein Opaque-2 (O2) localizes to the nucleus in both maize and tobacco cells. Here we show that in-frame carboxy- and amino-terminal fusions of O2 to reporter protein beta-glucuronidase (GUS) were sufficient to direct GUS to the nucleus in transgenic tobacco plants and in transiently transformed onion cells. Two independent regions of O2 containing 135 and 149 amino acids were identified that were able to redirect GUS to the nucleus in both systems. A quantitative biochemical analysis of GUS in nuclei isolated from transgenic tobacco plants revealed that the second region was more efficient than the first one. The precise location of nuclear localization signals (NLSs) was determined using an onion transformation system. The first NLS was located between residues 101 and 135 and had the structure of a simian virus 40 NLS. The second NLS was located in the basic, DNA binding domain (between residues 223 and 254) and had a bipartite structure. The presence of one of the O2 NLSs in the basic domain is in complete agreement with similar findings of NLSs in the basic domain of three other basic/leucine zipper proteins, suggesting that this domain may be bifunctional. The effect of amino- versus carboxy-terminal GUS fusions is discussed.

Allium↗
PubMed1992

Opaque-2 is a transcriptional activator that recognizes a specific target site in 22-kD zein genes.

opaque-2 (o2) is a regulatory locus in maize that plays an essential role in controlling the expression of genes encoding the 22-kD zein proteins. Through DNase I footprinting and DNA binding analyses, we have identified the binding site for the O2 protein (O2) in the promoter of 22-kD zein genes. The sequence in the 22-kD zein gene promoter that is recognized by O2 is similar to the target site recognized by other "basic/leucine zipper" (bZIP) proteins in that it contains an ACGT core that is necessary for DNA binding. The site is located in the -300 region relative to the translation start and lies about 20 bp downstream of the highly conserved zein gene sequence motif known as the "prolamin box." Employing gel mobility shift assays, we used O2 antibodies and nuclear extracts from an o2 null mutant to demonstrate that the O2 protein in maize endosperm nuclei recognizes the target site in the zein gene promoter. Mobility shift assays using nuclear proteins from an o2 null mutant indicated that other endosperm proteins in addition to O2 can bind the O2 target site and that O2 may be associated with one of these proteins. We also demonstrated that in yeast cells the O2 protein can activate expression of a lacZ gene containing a multimer of the O2 target sequence as part of its promoter, thus confirming its role as a transcriptional activator. A computer-assisted search indicated that the O2 target site is not present in the promoters of zein genes other than those of the 22-kD class. These data suggest a likely explanation at the molecular level for the differential effect of o2 mutations on expression of certain members of the zein gene family.

Base Sequence↗
PubMed1992

Mutations of the 22- and 27-kD zein promoters affect transactivation by the Opaque-2 protein.

By utilizing a homologous transient expression system, we have demonstrated that the Opaque-2 (O2) gene product O2 confers positive trans-regulation on a 22-kD zein promoter. This trans-acting function of the O2 protein is mediated by its sequence-specific binding to a cis element (the O2 target site) present in the 22-kD zein promoter. A multimer of a 32-bp promoter fragment containing this O2 target site confers transactivation by O2. A single nucleotide substitution in the O2 target sequence not only abolishes O2 binding in vitro, but also its response to transactivation by O2 in vivo. We have also demonstrated that an amino acid domain including the contiguous basic region and the heptameric leucine repeat is essential for the trans-acting function of the O2 protein. Similar but not identical O2 target sequence motifs can be found in the promoters of zein genes of different molecular weight classes. Conversion of such a motif in the 27-kD zein promoter to an exact O2 target sequence by site-directed mutagenesis was sufficient to increase the binding affinity of the O2 protein in vitro and to confer transactivation by O2 in vivo.

Base Sequence↗
PubMed1992

Paraphenylenediamine, a contact allergen, induces oxidative stress and ICAM-1 expression in human keratinocytes.

In an investigation of the role of keratinocytes in the pre-immunological phase of contact allergy, we have studied the effect of paraphenylenediamine (PPD) on cell proliferation, membrane lipid peroxidation and the expression of the intercellular adhesion molecule 1 (ICAM-1). Because PPD undergoes rapid autoxidation in the culture medium, the effect of PPD-modified medium on keratinocyte proliferation and ICAM-1 expression was also examined. PPD at low concentrations (up to 10 micrograms/ml) and with low exposure times (0.5 h) enhanced keratinocyte proliferation, but at high concentrations and with longer exposure times resulted in cell stasis and toxicity. These effects and the enhanced membrane lipid peroxidation that was also observed can be ascribed to the production of superoxide and hydrogen peroxide by the autoxidation of PPD in the medium. At non-cytotoxic concentrations, PPD induced ICAM-1 expression on the keratinocytes. PPD-modified medium was also cytotoxic to the keratinocytes and induced ICAM-1 expression in non-cytotoxic concentrations. It appeared that superoxide and hydrogen peroxide were not responsible for the cytotoxicity. These results are consistent with the view that oxidative stress may be an essential part of the pre-immunological phase in the induction of allergic contact dermatitis.

Allergens↗
PubMed1992

Single center success with a high risk peritoneal dialysis population.

A large end stage renal failure population treated by chronic ambulatory peritoneal dialysis (CAPD) was examined for rates of infection, CAPD modality failure and patient survival (N = 347). Nearly half were considered high risk for survival for reasons of age (39% older than 60 years), diabetes mellitus (33%), hemodialysis access failure (10%), poor cardiopulmonary reserve (16%) or technical challenges (30% had morbid obesity, history of abdominal aortic aneurysm repair or multiple abdominal surgeries). Hence, CAPD was often initiated by default rather than choice in the 347 patients studied (mean age: 51 +/- 17 years). Infections greatly outnumbered technical failures as grounds for cessation of CAPD. Over 5521 patient-months, 51% of patients developed infection with peritonitis predominating (80%) when compared to exit site infections (20%). The frequency of infections was 1.9 mean episodes per patient; however, 55% of these patients had only one episode of peritonitis. A rate of 0.75 infections per patient per year was seen with an average interval of 16 months between infections. Technique and patient survival rates at 4 years were 50% and 61% respectively. High risk status does not preclude successful CAPD and should not preclude its implementation.

Adolescent↗
PubMed1991

An arginine to lysine substitution in the bZIP domain of an opaque-2 mutant in maize abolishes specific DNA binding.

The opaque-2 (o2) locus in maize encodes a transcription factor involved in the regulation of zein storage proteins. We have shown previously that the O2 protein contains a leucine zipper domain that binds to promoters of 22-kD zein genes. In this paper we characterize an EMS-induced o2 allele, o2-676, that causes a 50% reduction in zein. We have found that the o2-676 mutant protein does not show specific recognition of zein promoter fragments because of the substitution of a lysine residue for an arginine residue within the bZIP domain of o2-676. This particular arginine is conserved within the bZIP domains of all mammalian, fungal, and plant DNA binding proteins of this class. The correlation between this mutation in o2 and the altered pattern of zein expression strongly suggests that O2 regulates transcription of certain members of the zein multigene family through direct interaction with the zein promoters and not through the transcriptional activation of some other regulator of zein gene expression.

Amino Acid Sequence↗
PubMed1991

Monocot regulatory protein Opaque-2 is localized in the nucleus of maize endosperm and transformed tobacco plants.

Protein targeting to the nucleus has been studied extensively in animal and yeast systems; however, nothing is known about nuclear targeting in plants. The Opaque-2 (O2) gene produces a regulatory protein that is responsible for inducing transcription of the alpha-zein class of storage proteins in maize kernels. The cloned O2 gene encodes a protein that contains a leucine zipper DNA binding domain that can interact with zein gene promoters. We have used immunolocalization to show that the O2 protein is present in nuclei in the maize endosperm tissues known to produce alpha-zeins. In addition, neither embryo tissue from wild-type kernels nor endosperm from kernels harboring a null o2 allele contain the O2 protein. Analysis of a transposable, element-induced o2 allele, o2-m20, revealed that sectors of endosperm cells contained the nuclear-localized O2 protein, indicating excision of the transposable element. To study further the nuclear transport of the O2 protein, we have transformed this gene, under the control of a constitutive promoter, into tobacco. Plants were shown to have detectable levels of steady-state O2 mRNA and O2 protein. Immunolocalization of O2 protein in transformed tobacco plants indicated that the O2 protein was transported into tobacco nuclei. Therefore, we have developed a system to study nuclear targeting in plants and have established that the nuclear transport machinery is similar in monocots and dicots.

Cell Nucleus↗
PubMed1990

Are free radicals and not quinones the haptenic species derived from urushiols and other contact allergenic mono- and dihydric alkylbenzenes? The significance of NADH, glutathione, and redox cycling in the skin.

The induction of allergic contact dermatitis to urushiols from poison ivy and related plants is generally believed to involve an initial oxidation event by which a protein-reactive quinone is formed. However, this does not readily account for the contact allergenicity of closely related mono- and dihydric alkylbenzenes such as the alkylphenols and alkylresorcinols which are not so easily oxidised to quinones in vitro. When the redox processes known to occur in living tissues are taken into consideration, a more plausible unifying mechanism involving the formation of protein-reactive radical species becomes apparent. Experiments described here examine the autoxidation of p-benzoquinone and various mono- and dihydric benzenes and alkylbenzenes, and their reactions with the diphenylpicrylhydrazyl radical, cysteine, glutathione, and NADH. We have also demonstrated that administration to mice of 2-oxo-4-thiazolidine carboxylate, a compound known to elevate intracellular glutathione levels, inhibits the irritancy and sensitising activity of 3-pentadecylphenol. This work suggests that redox cycling in the skin following penetration of allergenic mono- and dihydric alkylbenzenes initially depletes local levels of endogenous reducing equivalents such as glutathione and NADH; once depleted, further cycling results in the uncontrolled generation of radical species which may reasonably be expected to exhibit protein reactivity.

Animals↗
PubMed1990

Maize regulatory gene opaque-2 encodes a protein with a "leucine-zipper" motif that binds to zein DNA.

The opaque-2 locus (o2) in maize regulates the expression of many members of the zein multigene family of storage proteins. cDNA clones for a wild-type allele of the (o2) locus (O2) were isolated from a maize endosperm cDNA library and sequenced. We found a 258-nucleotide 5' leader sequence containing three short open reading frames followed by a sequence specifying a protein of 437 amino acids. The presumptive amino acid sequence of the protein (O2) specified by the O2 cDNA contains a "leucine-zipper" domain characteristic of some mammalian and fungal transcription activation factors. lacZ-O2 fusion constructs, using nearly the entire coding region of O2 or only a fragment specifying the leucine-zipper domain, were expressed in Escherichia coli. In an in vitro binding assay, the beta-galactosidase-O2 fusion proteins bound to two specific regions on the 5' side of the coding sequence in a zein genomic clone. This suggests that the O2 protein affects zein transcription through direct interaction with one or more zein promoter elements.

Amino Acid Sequence↗
PubMed1989

Biocompatibility of wound management products: standardization of and determination of cell growth rate in L929 fibroblast cultures.

To facilitate the development of a bioassay procedure by which the biocompatibilities of materials used in wound management may be assessed and compared, those environmental factors affecting cell growth in mouse L929 fibroblast cultures have been identified. Standardization of the initial cell number and frequency of change of medium resulted in the virtual elimination of variation of growth curves of L929 cells cultured in flasks of specified surface area. In addition, three methods for assessing fibroblast growth rate in the presence of alginate products used in wound management were evaluated. These were the haemacytometer counting chamber method, the Coulter counting method, and a liquid scintillation counting method. The first two methods determine the number of cells in a given volume of a cell suspension, whereas the third method determines the rate of synthesis of deoxyribonucleic acid (DNA), and hence cell growth, by measuring the incorporation of [3H]thymidine. The haemacytometer method had significant advantages over the other two procedures in providing both qualitative and quantitative data on culture morphology and cell growth response.

Alginates↗
PubMed1989

Biocompatibility of wound management products: the effect of various monosaccharides on L929 and 2002 fibroblast cells in culture.

The effects of various monosaccharides on the growth of human 2002 and mouse L929 fibroblast cultures have been investigated. Eleven monosaccharides having acidic, neutral, and basic characteristics were evaluated in a bioassay procedure developed for the investigation of biocompatibility of wound management materials. Rate of growth in both cell lines was inhibited by D-galacturonic acid and by D-glucuronic acid. Although most neutral sugars produced no significant change in the growth rate or in the morphology of the cells, galactose produced a significant increase in the growth rate of both cell lines whilst L-fucose caused a significant decrease in growth of the L929 cells but did not significantly affect the growth of 2002 cells; xylose increased the growth rate of L929 but not 2002 cells. D-Glucosamine, a basic sugar, produced inhibition of growth which followed a different pattern from that produced by the acidic sugars; N-acetylglucosamine produced a species specific increase in cell growth of L929 cells. The results show that the effects produced by the monosaccharides on the cultured fibroblasts are related to their chemical structure and to cell line, and suggest that the use of galactose as a possible aid to wound healing should be investigated.

Animals↗
PubMed1988

Advantages and limitations of using Spm as a transposon tag.

Transposon tagging has become the method of choice for isolating genes whose products are in low abundance. We have recently used the transposable element Spm to tag and clone maize regulatory loci. Our choice of Spm was dictated by several factors: The frequency of transposition of Spm is high enough to obtain detectable transposition events, into loci affecting kernel traits, in populations of less than 10(6) seed. Although the copy number of Spm is high in the maize genome, insertions into the gene of interest can be distinguished from other Spm copies by digesting DNAs from segregating populations with methyl-sensitive restriction enzymes, and hybridizing with Spm-specific probes. Since all members of the Spm family thus far examined share DNA homology, hybridization with appropriate probes allows detection of insertions of both autonomous and defective elements. Thus, if a mutable allele can be shown to be under Spm control, one can be reasonably confident of successfully cloning that allele.

Cloning, Molecular↗
PubMed1988

An assay procedure to compare sorptive capacities of activated carbon dressings: the detection of impregnation with silver.

A semiquantitative assay procedure for the estimation of uptake of diethylamine by activated carbon dressings has been developed. The procedure involves the use of Nessler's reagent as a comparative indicator. The use of diethylamine allows the concomitant detection of silver with which the dressings may be impregnated. The implications of silver/amine complex formation during dressing use are discussed. Quantification of the silver content of one such fabric was carried out by atomic absorption spectrophotometry. A silver content of 2770 ppm was found. It is suggested that this method might be suitable for adoption as the basis for a pharmacopoeial assay procedure for activated carbon dressings.

Adsorption↗
PubMed1987

Transposon tagging and molecular analysis of the maize regulatory locus opaque-2.

Genetic analyses suggested that the opaque-2 (o2) locus in maize acts as a positive, transacting, transcriptional activator of the zein seed storage-protein genes. Because isolation of the gene is requisite to understanding the molecular details of this regulation, transposon mutagenesis with the transposable element suppressor-mutator (Spm) was carried out, and three mutable o2 alleles were obtained. One of these alleles contained an 8.3-kilobase autonomous Spm, another a 6.8-kilobase nonautonomous Spm, and the third an unidentified transposon that is unrelated to Spm. A DNA sequence flanking the autonomous Spm insertion was verified to be o2-specific and provided a probe to clone a wild-type allele. Northern blots indicated that the gene is expressed in wild-type endosperm but not in leaf tissues or in endosperms homozygous for a mutant allele of the O2 gene. A transcript was detected in endosperms homozygous for mutations at opaque-7 and floury-2, an indication that O2 expression is independent of these two other putative regulators of zein synthesis.

Alleles↗
PubMed1987

Allergic contact dermatitis from proprietary topical analgesic sprays containing 3-(aminomethyl)-pyridyl salicylate.

Eleven cases of allergic contact dermatitis following the use of proprietary topical analgesic sprays (Algiospray and Pangesic) are described. Patch testing revealed that the adverse reactions were attributable to 3-(aminomethyl)-pyridine, which is present in these products as an ion pair with salicylic acid. The similarity between the two products investigated is not immediately apparent from the chemical nomenclature used by the manufacturers to describe the ingredients of their sprays.

Administration, Topical↗