Development of a low-metal adhesive tape to detect and localize metals in or on the stratum corneum at parts per million levels.

Tape-stripping is a well-established method for sampling the stratum corneum (SC). We have developed a tape with low-metal content suitable for use with particle-induced X-ray emission (PIXE), an analytical technique based on X-ray spectrometry. PIXE analysis of tape-stripped samples of SC is a reliable and minimally invasive means of identifying and quantifying metals present at parts per million levels. Assay feasibility and reproducibility was demonstrated using human volunteers. This new tape-strip technique has potential applications in exposure and decontamination assessment, diagnosis of metal dermatitis, forensics, and in environmental research.

Attomole quantitation of protein separations with accelerator mass spectrometry.

Quantification of specific proteins depends on separation by chromatography or electrophoresis followed by chemical detection schemes such as staining and fluorophore adhesion. Chemical exchange of short-lived isotopes, particularly sulfur, is also prevalent despite the inconveniences of counting radioactivity. Physical methods based on isotopic and elemental analyses offer highly sensitive protein quantitation that has linear response over wide dynamic ranges and is independent of protein conformation. Accelerator mass spectrometry quantifies long-lived isotopes such as 14C to subattomole sensitivity. We quantified protein interactions with small molecules such as toxins, vitamins, and natural biochemicals at precisions of 1-5%. Micro-proton-induced X-ray emission quantifies elemental abundances in separated metalloprotein samples to nanogram amounts and is capable of quantifying phopsphorylated loci in gels. Accelerator-based quantitation is a possible tool for quantifying the genome translation into proteome.

Olfactory bulb uptake and determination of biotransfer factors in the California ground squirrel (Spermophilus beecheyi) exposed to manganese and cadmium in environmental habitats.

Manganese (Mn) and cadmium (Cd) profiles in olfactory bulbs of California ground squirrels (Spermophilus beecheyi) trapped at Lawrence Livermore National Laboratory's Site 300 facility in California were determined with proton induced X-ray emission (PIXE). As a reference, Mn profiles in olfactory bulbs from laboratory rats exposed via nose-only inhalation to 0.53 mg/m3 Mn in the form of MnCl2 were also determined with PIXE. Atomic absorption spectrophotometry was used to measure soil Mn and Cd contents from the trapping sites and Mn and Cd contents in ground squirrel liver and leg muscle tissues. The data from laboratory rats revealed that Mn uptake into the olfactory bulb occurs via inhalation exposure. Data from ground squirrels and knowledge of the collection sites indicate that although several routes of exposure may occur, fossorial rodent olfactory uptake affords a significant exposure route to Mn and Cd in soils. Measured biotransfer factors (ratio of leg muscle tissue metal content to soil metal content) for Cd in ground squirrels were 10(3)-fold greater than exposure modeling estimates based on oral Cd uptake data from livestock. The measurements for ground squirrel tissues show that when conducting ecological risk assessments for natural habitats considerable care should be taken in selecting transfer factors. Specifically, transfer factors derived from data pertaining to comparable exposure pathways and ecological setting should be used wherever possible.

Characterization of mycobiont adaptations in the foliose lichen Xanthoparmelia chlorochroa (Parmeliaceae).

A cross section of the vagrant soil lichen Xanthoparmelia chlorochroa was analyzed using proton microprobe PIXE. Data were used to generate quantitative, two-dimensional element distribution maps for Al, Si, P, S, Cl, K, Ca, Ti, Cr, Mn, Fe, Ni, Cu, Zn, As, and Sr. Element maps show differential element partitioning between the stratified layers of the thallus. These data document transfer of inorganic nutrients across the thallus to the algal layer. Inorganic particle entrapment was also evident in the element maps. Dense accumulations of calcium oxalate at the junction of the medulla and the algal layer on the order of 10% by dry mass were discovered. Scanning electron microscopy and thermogravimetric analyses were used to characterize the calcium oxalate region. These data provide evidence for possible functional roles of the calcium oxalate layer, including regulation of water and light. Data also provide support for a mutualistic interpretation of the lichen association.

Zinc is sufficiently abundant within mammalian sperm nuclei to bind stoichiometrically with protamine 2.

Although studies have demonstrated that zinc can bind to sperm nuclear proteins, specifically protamine 2, it has not been shown that the metal is sufficiently abundant inside the sperm nucleus to interact stoichiometrically with these proteins. In this study proton-induced X-ray emission (PIXE) has been used to measure the amount of sulfur and zinc within the nuclei of individual sperm cells to infer the stoichiometry of zinc binding to protamine 2 in six species of mammal: bull, chinchilla, stallion, hamster, human, and mouse (protamine 2 comprises from 0% (bull) to 67% (mouse) of the protamine present in the sperm of these animals). Using the sulfur mass and electrophoretic data on the relative proportion of protamine 1 and protamine 2 in the sperm chromatin of these species, the protamine 1, protamine 2, and total protamine contents within each species sperm nuclei have been determined. The PIXE measurements reveal that the zinc content of the sperm nucleus varies proportionately with the protamine 2 content of sperm chromatin. PIXE analyses of hamster protamines extracted under conditions that appear to at least partially preserve zinc binding also confirm that the majority of the metal is bound to protamine. In five of the species examined, sufficient zinc is present for each protamine 2 molecule to bind one zinc. The results obtained for chinchilla sperm, conversely, indicate the chinchilla protamine 2 molecule may interact differently with zinc. Chinchilla sperm only contain enough zinc for one atom to be bound to two protamine 2 molecules.

A quantitative minimally invasive assay for the detection of metals in the stratum corneum.

A quantitative, minimally invasive tape-stripping assay for the detection of metals on and in skin that also has application to the detection of metallic elements on dry surfaces (where human contact could occur) has been developed. This development included construction, using commercial products, of an approximately 25 microm thick, low-metal content tape suitable both for tape-stripping and elemental analysis. Individual tapes were sequentially applied to the skin surface and then removed, taking with them a sample of the dead outer layer of the skin (stratum corneum). Analysis of such tape strip samples by particle induced X-ray emission (PIXE)--a well-characterized, sensitive, analytical technique based on X-ray spectrometry--identified and accurately quantified the metals in the sample. The assay had elemental sensitivities of approximately 1 ng/cm2 for many metals and analysis of elemental contents could be performed in as little as 5 min. The feasibility of the assay for measuring metals in the stratum corneum was demonstrated on the forearms of healthy human volunteers. Samples from approximately half the subjects were found to contain zirconium, possibly arising from the use of roll-on antiperspirants. The assay has potential as a tool: (1) for risk assessment, (2) to identify exposure levels following possible contact with a hazardous metal, and (3) to determine the effectiveness of cleanup or removal measures.

Quantitative particle-induced X-ray emission imaging of rat olfactory epithelium applied to the permeability of rat epithelium to inhaled aluminum.

Neurotoxicity from chronic metal inhalation has been suggested as an underlying contributor to late-developing neurodegenerative diseases that have symptoms similar to Alzheimer's and Parkinson's syndromes. If inhaled metals contribute to pathogenesis of these diseases, identifying, localizing, and quantitating metal deposition(s) within specific target regions of the central nervous system will be critical to our understanding of the mechanisms. Standard analytical techniques used to date require exposure to extremely high concentrations of metals to meet analytical detection limits in small tissue areas. The relevance to lower-dose environmentally relevant exposures and potential protective barriers is therefore questionable. The feasibility of microbeam particle-induced X-ray emission is investigated as a method for rapidly scanning tissues to study the inhalation of metals, nasal permeability, and central nervous system deposition. The optimal beam spot and analysis time used to image the rat olfactory epithelium to facilitate the rapid detection of aluminum localizations were determined. Measurements of aluminum localizations in rat olfactory bulb and brain sections are also presented.

In vitro studies on the use of clay, clay minerals and charcoal to adsorb bovine rotavirus and bovine coronavirus.

Rotaviruses are the leading cause and coronaviruses are the major contributors of acute gastroenteritis in the young of various mammalian and avian species. Despite numerous trials and decades of research, vaccines have limited efficacy particularly for calves. As an alternative method of controlling infection, we have investigated broad spectrum antiviral agents that are not discriminatory among various viruses. This report involves testing a variety of adsorbent agents including charcoal, clay, and clay minerals to adsorb rotavirus and coronavirus in vitro. Results revealed that all the adsorbent agents had good to excellent capability of adsorbing rotavirus and excellent capability of adsorbing coronavirus. Percent adsorptions ranged from 78.74% to 99.89% for rotavirus and 99.99% for coronavirus; while sand (negative control) was < 0.01%. A high affinity binding was present as determined by a low percent desorption (0.06-3.09%). However, the adsorbent bound virus complex retained, and may have actually enhanced, infectivity.

An in vitro study of theaflavins extracted from black tea to neutralize bovine rotavirus and bovine coronavirus infections.

Crude theaflavin was extracted from black tea and then fractionated by HPLC into five components (initial peaks (IP), TF1, TF2A, TF2B, and TF3). The crude extract and the various fractions of theaflavin were collected and tested, individually and in combination, for antirotaviral activity. The mean effective concentration (EC50) was calculated and compared. Activity varied from the most active being the uncharacterized theaflavin-like initial peaks (IP) with an EC50 of 0.125 microgram/ml to the least active being theaflavin-3 monogallate (TF2A) with an EC50 of 251.39 micrograms/ ml. The combination of TF1 + TF2A + TF2B + TF3 was more active than the sum of the activities of these four fractions individually, indicating synergism among the peaks. Only the crude extract was assayed for activity against coronavirus; the EC50 was 34.7 micrograms/ml.

Selective chemisorption and detoxification of aflatoxins by phyllosilicate clay.

Practical and effective strategies for the detoxification of aflatoxins are critically needed. We have shown that a phyllosilicate clay (HSCAS): i) tightly binds aflatoxins in aqueous solutions, including milk; ii) markedly decreases the bioavailability of radiolabeled aflatoxins; iii) greatly diminishes aflatoxicosis in young animals, i.e., rats, chickens, turkeys, lambs, and pigs; and iv) reduces the level of aflatoxin M1 in the milk from lactating dairy cattle and goats. In further studies, ligands with one or more of the functional groups in common with aflatoxin were reacted with HSCAS in vitro in an attempt to elucidate the specificity and mechanism of tight binding (or chemisorption). A chemisorption index (C alpha) was developed, allowing for direct comparison of various clay and zeolitic minerals with HSCAS. Chemisorption indices were determined by HPLC analysis of extracts of the supernatants and sorbed pellets (exhaustively extracted with methanol and chloroform). The beta-dicarbonyl system of aflatoxin was found to be essential for tight binding by HSCAS. Comparison of the chemisorption indices from various classes of compounds with spectral data (DRIFTS) indicated that the molecular mechanism of aflatoxin binding may involve the chelation of metal ions in HSCAS with the beta-dicarbonyl moiety in aflatoxin. Computer modeling was utilized to provide additional information. Preliminary evidence suggests that aflatoxin B1 may react at surfaces and within the interlayers of HSCAS particles. With knowledge of the mechanism involved, it has been possible to further enhance the propensity of HSCAS for aflatoxins.

Purification and characterization of a cyclic GMP-stimulated cyclic nucleotide phosphodiesterase from the cytosol of human platelets.

A cyclic GMP-stimulated cyclic nucleotide phosphodiesterase was purified to near homogeneity from the 150,000 g supernatant fraction of human platelets by a combination of DEAE-cellulose chromatography and cyclic GMP affinity chromatography. Overall purification was about 7400-fold with a 10% to 15% recovery of activity. On NaDodSO4-containing polyacrylamide gels, the purified enzyme migrates as a single band Mr = 105,000. Phosphodiesterase activity co-migrates with the protein band on native polyacrylamide gels. Both Mg2+ and Mn2+ support the activity of this phosphodiesterase. The enzyme hydrolyzes both cyclic AMP and cyclic GMP with similar maximal rates. The hydrolysis of both nucleotides exhibits positive homotropic cooperativity with S0.5 values of 50 +/- 12 microM for cyclic AMP and 35 +/- 15 microM for cyclic GMP and Hill coefficients of 1.2 to 1.5 for both nucleotides. Low levels of cyclic GMP stimulate the rate of cyclic AMP hydrolysis from 3- to 10-fold. The activity of this phosphodiesterase is not stimulated by the calcium binding protein, calmodulin. The cyclic GMP stimulation of cyclic AMP hydrolysis by this phosphodiesterase may provide a possible regulatory link between the metabolism of these two nucleotides in platelets.

Three new potential cAMP affinity labels. Inactivation of human platelet low Km cAMP phosphodiesterase by 8-[(4-bromo-2,3-dioxobutyl)thio]adenosine 3',5'-cyclic monophosphate.

Three new analogues of cAMP have been synthesized and characterized: 2-[(4-bromo-2,3-dioxobutyl)thio]adenosine 3',5'-cyclic monophosphate (2-BDB-TcAMP), 2-[(3-bromo-2-oxopropyl)thio]-adenosine 3',5'-cyclic monophosphate (2-BOP-tcAMP), and 8-[(4-bromo-2,3-dioxobutyl)thio]adenosine 3',5'-cyclic monophosphate (8-BDB-TcAMP). The bromoketo moiety has the ability to react with the nucleophilic side chains of several amino acids, while the dioxobutyl group can interact with arginine. These cAMP analogues were tested for their ability to inactivate the low Km (high affinity) cAMP phosphodiesterase from human platelets. The 2-BDB-TcAMP and 2-BOP-TcAMP were competitive inhibitors of cAMP hydrolysis by the phosphodiesterase with Ki values of 0.96 +/- 0.12 and 0.70 +/- 0.12 microM, respectively. However, 2-BDB-TcAMP and 2-BOP-TcAMP did not irreversibly inactivate the phosphodiesterase at pH values from 6.0 to 7.5 and at concentrations up to 10 mM. These results indicate that although the 2-substituted TcAMP analogues bind to the enzyme, there are no reactive amino acids in the vicinity of the 2-position of the cAMP binding site. In contrast, incubation of the platelet low Km cAMP phosphodiesterase with 8-BDB-TcAMP resulted in a time-dependent, irreversible inactivation of the enzyme with a second-order rate constant of 0.031 +/- 0.009 min-1 mM1. Addition of the substrates, cAMP and cGMP, and the product, AMP, to the reaction mixture resulted in marked decreases in the inactivation rate, suggesting that the inactivation was due to reaction at the active site of the phosphodiesterase.(ABSTRACT TRUNCATED AT 250 WORDS)

cAMP-mediated phosphorylation of the low-Km cAMP phosphodiesterase markedly stimulates its catalytic activity.

Treatment of intact human platelets with the adenylate cyclase agonist forskolin (100 microM) resulted in an increase in cAMP phosphodiesterase activity in freeze-thaw lysates. When the low-Km (high affinity), cGMP-inhibited cAMP phosphodiesterase was isolated from such lysates by blue dextran-Sepharose chromatography, the specific activity of the enzyme was increased an average of 11-fold over similarly processed control platelets. The increase in the low-Km, cGMP-inhibited cAMP phosphodiesterase activity was inhibited when platelets were incubated with the protein kinase inhibitor H-8 prior to treatment with forskolin, suggesting that the stimulation of cAMP phosphodiesterase activity involved a cAMP-dependent phosphorylation. When intact platelets that had been prelabeled with 32Pi were treated with forskolin and the low-Km, cGMP-inhibited phosphodiesterase was isolated by blue dextran-Sepharose chromatography, a protein of 110,000 kDa was phosphorylated. By using a monospecific antiserum to the purified phosphodiesterase, this protein was shown to be the low-Km, cGMP-inhibited cAMP phosphodiesterase by electrophoretic transfer blot (Western blot) analysis and by immunoprecipitation. The stable prostacyclin analog iloprost also stimulated the low-Km cAMP phosphodiesterase activity about 2-fold and caused phosphorylation of the enzyme. These results suggest that phosphorylation of the low-Km, cGMP-inhibited phosphodiesterase may be an important regulatory mechanism for this enzyme in platelets.

Purification and characterization of a human platelet cyclic nucleotide phosphodiesterase.

A cyclic nucleotide phosphodiesterase was extensively purified from the 100000g supernatant fraction of human platelets. The purification was 2500-3000-fold with 30% recovery of activity. The enzyme was isolated by DEAE-cellulose chromatography followed by adsorption to blue dextran-Sepharose and elution with cAMP. The protein has a molecular weight of 140 000 as determined by gel filtration. On NaDodSO4-containing polyacrylamide gels the major band is at 61 000 daltons, suggesting that the enzyme may exist as a dimer in solution under nondenaturing conditions. The enzyme requires Mg2+ or Mn2+ for activity. The calcium binding protein calmodulin does not stimulate hydrolysis of cAMP by this enzyme. The purified enzyme hydrolyzes both cAMP and cGMP with normal Michaelis-Menten kinetics with Km values of 0.18 microM and 0.02 microM, respectively. The hydrolysis of cGMP, however, is only one-tenth as rapid as the hydrolysis of cAMP. Cyclic GMP does not stimulate cAMP hydrolysis but instead is a potent competitive inhibitor of cAMP hydrolysis. The enzyme is also competitively inhibited by the phosphodiesterase inhibitors papaverine, 3-isobutyl-l-methylxanthine, and dipyridamole. The enzyme did not cross-react with an antibody raised to a cAMP phosphodiesterase isolated from dog kidney, indicating that the enzymes are not immunologically related. The inhibition of cAMP hydrolysis by cGMP suggests a possible regulatory link between these two cyclic nucleotides. One of the roles of cGMP in platelets may be to potentiate increases in intracellular cAMP by inhibiting the hydrolysis of cAMP by this enzyme.

Puromycin binding to the small subunit of Escherichia coli ribosomes. Localization of the antibiotic in subunits reconstituted with puromycin-modified components.

Small (30 S) ribosomal subunits from Escherichia coli strain TPR 201 were photoaffinity-labeled with [3H]puromycin in the presence of chloramphenicol under conditions in which more than 1 mol of antibiotic was incorporated per mol of ribosomes. The subunits were than washed with 3 M NH4Cl to yield core particles and a split protein fraction; the split proteins were further fractionated with ammonium sulfate. Subunits were then reconstituted using one fraction (core, split proteins, or ammonium sulfate supernatant) from photoaffinity-modified subunits and other components from unmodified (control) subunits. The distribution of [3H]puromycin in ribosomal proteins was monitored by one-dimensional polyacrylamide gel electrophoresis, and the sites of puromycin binding were visualized by immunoelectron microscopy. Two areas of puromycin binding were identified. A high affinity puromycin site, found on the upper third of the subunit and distant from the platform, is identical to the primary site previously identified (Olson, H. M., Grant, P. G., Glitz, D. G., and Cooperman, B. S. (1980) Proc. Natl. Acad. Sci. U. S. A. 77, 890-894). Binding at this site is maximal in subunits reconstituted with high levels of puromycin-modified protein S14, and is decreased when unmodified S14 is incorporated. Because the percentage of antibody binding at the primary site always exceeds the percentage of puromycin label in protein S14, the primary site must include components other than S14. A secondary puromycin site of lower affinity is found on the subunit platform. This site is enriched in subunits reconstituted from puromycin-modified core particles and may include protein S7. Our results demonstrate the feasibility of localizing specifically modified components in reconstituted ribosomal subunits.

The effect of a medicinal Chinese herb on platelet function.

We investigated the effect of the Chinese herb Injectio Salvia Miltiorrhizae (ISM) on human platelet function in vitro. ISM inhibited platelet aggregation and serotonin release induced by either ADP or epinephrine in a dose dependent manner. This effect of ISM was observed with both gel-filtered platelets (ID50 = 8-30 micrograms ISM/ml gel-filtered platelets) and platelets in plasma (ID50 = 400-900 micrograms ISM/ml of platelet-rich plasma). The active molecule(s) in ISM was heat stable, resistant to acid, base and proteolysis and fractionated on Sephadex 6-25 at MW approximately 280. ISM did not interact with the platelet alpha-adrenergic receptor, but increased cAMP in intact platelets. The results are consistent with the concept that ISM inhibition of platelet aggregation and release is mediated by an increase in platelet cAMP. The exact mechanism whereby ISM increases platelet cAMP appears to be that of inhibition of cyclic AMP phosphodiesterase. The effect of ISM on platelet function is one mechanism which might explain the therapeutic effect of ISM in experimental and clinical coronary artery disease.

Immunoelectron microscopic localization of puromycin binding on the large subunit of the Escherichia coli ribosome.

Ribosomes from Escherichia coli strain Q13 have been photoaffinity labeled with [3H]puromycin in the presence of tetracycline. Puromycin-modified 50 S subunits appear to be identical with untreated subunits in electron micrographs and are precipitated by antibodies to the N6,N6'dimethyladenosine moiety of puromycin. Electron micrographs of subunit-antibody complexes show ribosomal subunits to which an individual antibody molecule is bound and pairs of subunits linked by an IgG molecule. Two regions of puromycin binding have been identified. The primary area, seen in 76% of the ribosome monomer complexes and 93% of the antibody-linked dimers, is beside (or on) the small central protuberance and on the side opposite the L7/L12 arm. A secondary area, maximally distant from the central protuberance, is seen in 22% of the monomeric complexes but only 7% of the antibody-linked dimers. In conjunction with our earlier localization of puromycin binding on the 30 S subunit (Olson, H. M., Grant, P. G., Glitz, D. G., and Cooperman, B. S. (1980) Proc. Natl. Acad. Sci. U. S. A. 77, 890-894), we now define a puromycin-binding neighborhood of the 70 S ribosome. In addition to providing evidence for the localization of the peptidyl transferase center within the 50 S subunit, our results contribute to the formulation of a model for tRNA binding to both 30 S subunits and 70 S ribosomes.

The effect of changes in muscle function and bone growth on muscle migration.

The elastic sleeve model of the periosteum of a long bone presents the periosteum as a structure which, because it is attached to the epiphyses rather than the diaphysis, expands interstitially and equally at all points as the bone grows at its ends. Structures attached to the periosteum are seen as essentially passive hitchhikers on the expanding periosteum. Two corollaries of this model are tested here. First, that changes in the magnitude or direction of the force that an attached structure exerts on the periosteum do not affect the migration of the structure. Second, that changes in the proportion of growth that occur at each end of the bone do not affect the migration of attached structures. Experiments performed on rabbits to test these corollaries include muscle paralysis, muscle transection, changes in the direction pull of a muscle, and epiphysiodesis. The results are in agreement with the hypotheses. This model should have applicability to functional and comparative anatomy, since it postulates that differences in positions of attachment of muscles and ligaments to bones reflect underlying genetic differences (phylogeny) rather than the effects of differences in behavior of the animal (ontogeny).