Mapping of trichodermin resistance in Saccharomyces cerevisiae: a genetic locus for a component of the 60S ribsomal subunit.

Resistance to the protein synthesis inhibitor trichodermin in Saccharomyces cerevisiae has been studied. A single recessive nuclear gene was responsible for resistance. The resistance locus, tcm1 was found to be closely linked (1 centi-morgan) to the locus pet 17 on the right arm of chromosome XV. The mutation to trichodermin resistance conferred resistance to other 12,13-epoxytrichothecenes and to the structurally unrelated antibiotic anisomycin.

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.

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.

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.

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.

The effect of position on the migration of muscle.

The tibial insertion of the M. semitendinosus of young rabbits was moved to a new location on the shaft of the tibia, either 5 or 10 mm proximal, or 0, 10, 20, 30, 40, or 50 mm distal to its original position. The animals were killed 8.5 months later. The results show that the greatest amount of proximal migration of the experimental muscles was 11.3 mm and this occurred in the muscles moved 10 mm distally. This compared with a migration of 12.8 mm for control muscles. The amount of proximal migration decreased progressively when the insertion was moved more or less than 10 mm distally. There was a small amount of distal migration in the 10 mm proximal, and the 40 and 50 mm distal groups. These results confirm experiments reported earlier in which muscles were not moved more than 20 mm distally. They support the hypothesis that migration is controlled by the position on the growing bone rather than by the tension in the muscle.

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.

The skin primates. XLIV. Numerical taxonomy of primate skin.

Data on 84 characteristics of the skin of 36 species of primates were extracted from a series of articles describing the histological and histochemical properties of the skin of primates. The data were subjected to a cluster analysis. The results were in reasonably good agreement with orthodox primate taxonomies although some exceptions were apparent. The species clustered into four main groups approximately comparable to Prosimii, Cercopithecoidea, pithecoidea, and Hominoidea are commensurate with standard taxonomic practice. Within the Ceboidea, however, the Atelinae and Alouattinae tend to group with the Hominoidea, Aotus and Saimiri show variable placements, and Callimico groups with the Callithricidae.

Experimental modification of muscle migration in the rabbit.

The insertion of the M. semimenbranosus was translocated distally through distances of 0, 5, 10, and 20 mm in 8 weeks old rabbits. Markers were placed in the bone to allow measurements of the distance that each muslce migrated during growth. Rabbits were sacrificed after 3 months. The results showed that the further distally a muscle is moved, the further it will migrate proximally during this time. This may indicate that there are factors other than, or in addition to, the growth of the bone that control migration.

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.

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.

Positional relationships of structures attached to long bones during growth. Cross-sectional studies.

The various soft structures attached to bones maintain relatively constant relationships during growth. The exact number of these relationships has, however, never been studied. As part of our ongoing research into the factors controlling muscle migration, we have determined these relationships for some structures. We studied the positions of 37 muscles from 24 New Zealand white rabbits ranging in age from birth to maturity. The muscles were selected to illustrate different kinds of attachments: fleshy and tendonous, restricted and extensive, and at both ends of a bone. The proximal and distal edges of attachments were carefully exposed and measured in a device that allowed us to measure the position relative to the ends of the bones without parallax distortion. We used the data to compute correlation coefficients and regression equations for position vs. length of bone. Results show that the correlation coefficient was above 0.9 for most cases and was significant at the 0.5 level for all but 4 cases. Slopes of the regression equations varied considerably, but in all cases they indicated that the closer to the end of a bone, the greater the distance migrated. The significance of these results is discussed.

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.

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.

Antibiotic effects on the photoinduced affinity labeling of Escherichia coli ribosomes by puromycin.

The effect of ribosomal antibiotics on the photoinduced affinity labeling of Escherichia coli ribosomes by puromycin [Cooperman, B.S., Jaynes, E.N., Brunswick, D.J., & Luddy, M.A. (1975) Proc. Natl. Acad. Sci. U.S.A. 72, 1974; Jaynes, E.N. Jr., Grant, P.G., Giangrande, G., Wieder, R., & Cooperman, B.S. (1978) Biochemistry 17, 561] has been studied. Although blasticidin S, sparsomycin, lincomycin, and erythromycin are essentially without effect, major changes are seen on addition of either chloramphenicol or tetracycline. The products of photoincorporation have been characterized by one- and two-dimensional gel electrophoresis and by specific immunoprecipitation with antibodies to ribosomal proteins. In the presence of chloramphenicol, protein S14 becomes the major labeled protein. In the presence of tetracycline, L23 remains the major labeled protein, but the yield of labeled ribosomes is enormously increased, and the labeling is more specific for L23. These results are discussed in terms of the known modes of action of these antibiotics and the photoreactivity of tetracycline.