Chemotactic assay for biological effects of silver nanoparticles

A method is proposed for assessing the biocidal efficacy of water-dispersed nanoparticles of silver. It is based on negative chemotaxis of the plasmodia of the slime mold Physarum polycephalum. Biocidal and repellent effects were compared for silver nanoparticles, Ag⁺ ions, and AOT in solution and in the agar gel. In such characteristics as increasing the period of auto-oscillations of contractile activity, decreasing the area of spreading on substrate, and substrate preference in spatial tests, silver nanoparticles proved to be substantially more effective than Ag⁺ and AOT. The lethal concentrations of the nanoparticles were close to those found earlier for bacteria and viruses. The chemotactic tests allow quantitative assessment of the biological reaction and monitoring its dynamics; in resolution, they are superior to the tests based on the lethal action of biocidal agents.     

A silver stain for the detection of nanogram amounts of tRNA following two-dimensional electrophoresis

Three ultrasensitive protein silver-staining methods have been compared with respect to the detection of tRNA in polyacrylamide gels. The method of Sammons (D. W. Sammons, L.D. Adams, and E.E. Nishizawa (1981) Electrophoresis 2, 135-141) has been shown to have remarkable sensitivity, with a detection limit of 0.3 ng tRNA/mm2, allowing the two-dimensional fractionation of submicrogram amounts of bulk tRNA. The application of this technique to developmental and differentiation problems and other areas where the amounts of nonradioactive tRNA available are limited is anticipated.     

Silver ions inhibit the ethylene-stimulated production of ripening-related mRNAs in tomato

Abstract. Silver ions effectively inhibited both the initiation and the continuation of tomato ( Lyeopersicon esculentum Mill) ripening. Studies of protein synthesis in vivo showed that application of 2 mol m⁻³ silver thiosulphate to mature green fruit prevented the appearance of several novel proteins associated with ripening, including the softening enzyme polygalacturonase. However, total protein synthesis, as judged by the incorporation of [³⁵S] methionine into proteins, continued unabated after silver treatment. Ripening was also arrested when silver was supplied after ripening had begun. The accumulation of several ripening-related mRNAs, including that for polygalacturonase, was studied by translation in vitro and using cDNA clones as hybridization probes. Silver was shown to prevent the appearance of polygalaturonase mRNA when supplied to mature green fruit and to cause a rapid reduction in the concentration of mRNA for polygalacturonase and other ripening-related proteins when supplied after ripening had begun. It is proposed that silver exerts its effects due to interaction with the ethylene perception mechanism. The results suggest that perception of ethylene is vital not only for the initiation of ripening but also for the continued expression of genes required for ripening.     

Keratinolysis by Absidia cylindrospora and Rhizomucor pusillus: biochemical proof

Absidia cylindrospora and Rhizomucor pusillus, causal agents of phycomycoses, were cultured on sterile natural keratins in a mineral solution and the keratin degradation products analyzed. The excess of sulphur was removed by oxidation to inorganic sulphate and thiosulphate, which were the main products of sulphitolysis of keratin. The proteolytic activity of the two fungi depended on the nature of the keratin substrate. Human scalp hair was the most favoured keratin substrate by both the fungi.     

Temporal fluctuations of silver,copper and zinc in the bivalve Macoma balthica at five stations in South San Francisco Bay

Concentrations of Cu, Ag and Zn were measured in the soft tissues of the estuarine bivalve Macoma balthica in South San Francisco Bay at near-monthly intervals for periods of two to three years at four stations, and eight years at a metal-enriched station. The amplitude and frequency of fluctuations differed among stations and among metals. Fluctuations were greatest at stations with the greatest metal enrichment and with the least dilution and flushing of wastes. A consistent seasonal pattern of fluctuation in Cu and Ag concentrations was evident in M. balthica at the metal-enriched station. These seasonal changes in tissue metal concentrations appeared to be affected by metal inputs, hydrologic processes that may affect both metal concentrations and bioavailability, and seasonal changes in the weight of the bivalve. The contributions of each of these interacting factors could not be determined quantitatively. At the metal-enriched station significant variation in the amplitude of seasonal fluctuations was also evident from year to year. Interpretation of metal concentrations in bivalves from estuaries will require careful consideration of the processes which affect metal dynamics in these complex environments.     

Ammoniacal silver staining of proteins: mechanism of glutaraldehyde enhancement

When the conditions for detecting proteins by ammoniacal silver staining (B. R. Oakley, D. R. Kirsch, and N. R. Morris (1980) Anal. Biochem. 105, 361-363.) following gel electrophoresis were varied, it was noted that glutaraldehyde pretreatment was necessary for maximal staining, which could not be explained simply as the result of "fixation." Further studies indicated that glutaraldehyde enhancement of protein staining with this silver reagent was probably due to oxidation of the aldehyde groups by silver ions, resulting in metallic silver depositions within the gel which act as nucleation sites for additional metallic silver localization in the protein bands upon the addition of formaldehyde developer. This proposed mechanism is consistent with the Tollen's reaction, as well as some aspects of the photographic process. Consistent with this notion, silver-staining intensities are directly related to mole percentage lysine of various standard proteins.     

Characteristics of structural proteins of infectious flacherie virus from the silkworm, Bombyx mori

Structural proteins and the characteristics of infectious flacherie virus (IFV) purified from the silkworm, Bombyx mori, are described. The purified IFV had four major structural proteins, which were detected only in high concentration gels of sodium dodecyl sulfate-polyacrylamide gel electrophoresis, and a few minor ones. Molecular weights of the major proteins were 35,200 (VP 1), 33,000 (VP 2), 31,200 (VP 3), and 11,600 (VP 4), and numbers per virion were 62, 57, 54, and 31, respectively. Amino acid compositions of VP 1, VP 2, and VP 3 were similar to each other but that of VP 4 was somewhat different. By isoelectric focusing and two-dimensional electrophoresis, high resolution of the structural proteins was obtained with silver staining. The isoelectric points of the four major proteins were determined as 7.7(VP 1), 6.7(VP 2), 4.8(VP 3), and 5.5(VP 4). This work is the first report on insect picornaviruses that presents some discriminative properties of each viral protein that was compared to those of mammalian picornaviruses.     

Synergistic effect of 1-aminocyclopropane-1-carboxylic acid and ethylene during senescence of isolated carnation petals

The effects of ethylene (C₂H₄), (2-chloroethyl)phosphonic acid (ethefon) and 1-aminocyclopropane-1-carboxylic acid (ACC) on senescence of isolated intact petals and of upper petal parts of carnation flowers ( Dianthus caryophyllus L. cv. White Sim) were investigated.
Isolated upper petal parts did not respond to treatment with ethefon or ACC. These tissues did, however, show severe wilting in intact petals that were treated with ethefon or ACC. When isolated upper petal parts were simultaneously treated with ACC and ethefon or ACC and ethylene, a marked synergistic effect on senescence was found. Treatment of isolated petals with radiolabeled ACC led to the accumulation of radiolabeled ACC and N-malonyl-ACC (MACC) in the upper parts. The formation of ethylene and the malonylation of ACC were inhibited by pretreatment of the flower with the inhibitor of ethylene action, silver thiosulphate (STS), which indicates that both were induced by endogenously produced ethylene. Treatment of isolated upper parts with ACC slightly increased their ethylene production. However, when these petal parts were simultaneously treated with ethylene and ACC, the conversion of ACC to ethylene was markedly stimulated.
The results indicate that, in intact petals, ethylene may be translocated from the basal to the upper part where it stimulates the activity of the ethylene-forming enzyme (EFE), thereby making the tissue receptive to ACC.
In addition, it was found that upon incubation of petal portions in radiolabeled ACC, both the petal tissue and the incubation solutions produced radiolabeled carbon dioxide. This was shown to be due to microorganisms that were able to metabolize the carbon atoms in the 2 and 3 position of ACC into carbon dioxide.     

Chromosomal localization of ribosomal DNA sequences in an apple rootstock using a digoxygenin detection system

A 6kb rDNA probe comprising the 18S coding plus spacer sequences has been hybridized to the metaphase chromosomes of apple rootstock cultivar MM106 demonstrating the localization of ribosomal gene arrays in the vicinity of the telomeric regions of the short arms of chromosomes 6 and 14.The in situ results using digoxygenin labelling coupled to an alkaline phosphatase immunoassay were confirmed by silver staining for NORs and nucleoli.This study demonstrates the feasibility of molecular cytogenetic analysis of very small chromosomes(1.0-2.7μm） of apple.     

Selective Axonal Argentaffin Staining in Rat Central Nervous System after Protein Mercuration

The mercury-silver (Hg-Ag) argentaffin technique, known to stain specifically proteins in the lateral components of triads/diads in striated muscle cells, was applied to the central nervous system of adult rats. Following fixation in glutaraldehyde, axons in white and gray matter were selectively stained, but not perikarya or their proximal axon and dendrites. Neural tissues were postfixed 24 hr in 5% (w/v) mercuric acetate in 2% (v/v) acetic acid in distilled water, stained for 12-24 hr in darkness at 37-43 C with ammoniacal silver nitrate solution, freshly prepared by adding concentrated ammonia to 60% (w/v) silver nitrate solution until a small amount of silver oxide precipitate remained undissolved. Samples were then washed with freshly prepared 5% (w/v) sodium sulfite and distilled water. All steps were carried out using dark-colored glass flasks. Samples were dehydrated with ethanol and embedded in Paraplast or Poly Bed. Electron microscopy showed the silver-reducing protein inside the axons. Methylation abolished Hg-Ag axonal reactivity indicating that carboxyl groups were necessary for silver staining. Proteins with solubility properties characteristic of neurofilament proteins were involved in Hg-Ag staining. In the cerebellum the plexus of parallel fibers in the molecular layer were not stained, while basket cell axonal processes reacted intensely. The method appears to distinguish neuronal protein variants related to cytotypic differences in cytoskeletal neurofilaments.