Subcellular distribution of aluminum, bismuth, cadmium, chromium, copper, iron, manganese, nickel, and lead in cultivated mushrooms (Agaricus bisporus and Pleurotus ostreatus)

In this work, the distribution of nine metals in two types of cultivated mushroom had been investigated. For Agaricus bisporus, the biomass was separated into caps and stalks, and for Pleurotus ostreatus, the entire mushrooms were taken for analysis. Electrothermal atomic absorption spectrometry was used for total element determination in acid digests. For accuracy checking, the certified reference material (NIST 1571, citrus leaves) was analyzed. The results obtained for the two fungi species were within the ranges of concentration reported previously by other authors. Subcellular fractionation was accomplished by centrifugation of cell homogenates, which has been suspended in Tris-HCl buffer. In the first centrifugation (7300g, 4°C, 10 min), cell walls were separated (pellet I), and the second centrifugation (147,000g, 4°C, 60 min) yielded mixed membrane fraction (pellet II) and cytosol (supernatant II). Recoveries of the fractionation procedure were in the range 70–100% (with the exception of Fe). For all elements studied, the highest relative contributions were found in cytosol fractions of the fruiting bodies (63–72%, 49–76%, 44–93%, 26–87pc, 55–85%, 50–68%, 41–78%, 39–78%, 54–67% respectively for Al, Bi, Cd, Cr, Cu, Fe, Mn, Ni, and Pb. Lower contributions were found in cell walls (respectively 22–32%, 24–44%, 6.1–47%, 12–52%, 7.3–37%, 7.9–32%, 19–52%, 20–42%, and 25–38%) and only minute amounts in the mixed membrane fraction (3.0–5.8%, 0.7–7.0%, 0.7–8.3%, 1.0–22%, 7.5–14%, 16–24%, 1.1–19%, and 5.1–7.7%). The results obtained indicate that small water-soluble molecules were the primary forms of nine elements in two mushroom species studied. On the other hand, the evidence has been provided on elements binding to larger, water-insoluble molecules contained in the structures of cell wall and membranes. The relative distribution was both element and fungi dependent. Thus, in P. ostreatus, total element levels were higher than in A. bisporus, with the preference for their accumulation in cytosol. On the contrary, total element content in the latter fungi was lower; however, a clear tendency toward more efficient element incorporation to the water-insoluble structures was observed (no apparent differences between stalks and caps). These findings might contribute in a better understanding of the accumulation of metals in mushrooms.     

Life history and systematics of Albusambia elaphoglossumae (Lepidoptera: Crambidae): a new genus and species of musotimine with leaf-mining biology from Costa Rica

Albusambia elaphoglossumae Solis & Davis, a new genus and species, is described. It was discovered mining the fronds of the fern Elaphoglossum conspersum in Costa Rica (San José and Cartago Provinces, at elevations of 2300-3100 m). The type series was obtained by rearing of the immature stages in laboratory. The adult is defined by unique genital characters, and the pupa with a medial depression on the vertex and with two anterolateral horn-like structures on the prothorax. The larva is a gregarious leaf miner with its body flattened dorsoventrally and head prognathous; morphological adaptations to its leaf-mining habit are new to the Musotiminae. Fern-feeding musotimines are important to the discovery of new biological control agents for invasive ferns.     

Identification of acetylation and methylation sites of histone H3 from chicken erythrocytes by high-accuracy matrix-assisted laser desorption ionization-time-of-flight,matrix-assisted laser desorption ionization-postsource decay,and nanoelectrospray ionization tandem mass spectrometry

A new strategy has been employed for the identification of the covalent modification sites (mainly acetylation and methylation) of histone H3 from chicken erythrocytes using low enzyme/substrate ratios and short digestion times (trypsin used as the protease) with analysis by HPLC separation, matrix-assisted laser desorption ionization-time-of-flight (MALDI-TOF), matrix-assisted laser desorption ionization-postsource decay, and tandem mass spectrometric techniques. High-accuracy MALDI-TOF mass measurements with representative immonium ions (126 for acetylated lysine, 98 for monomethylated lysine, and 84 for di-, tri-, and unmethylated lysine) have been effectively used for differentiating methylated peptides from acetylated peptides. Our results demonstrate that lysines 4, 9, 14, 27, and 36 of the N-terminal of H3 are methylated, while lysines 14, 18, and 23 are acetylated. Surprisingly, a non-N-terminal residue, lysine 79, in the loop region hooking up to the bound DNA, was newly found to be methylated (un-, mono-, and dimethylated isoforms coexist). The reported mass spectrometric method has the advantages of speed, directness, sensitivity, and ease over protein sequencing and Western-blotting methods and holds the promise of an improved method for determining the status of histone modifications in the field of chromosome research.     

Polymorphic distribution of the ovine prion protein (PrP) gene in scrapie-infected sheep flocks in which embryo transfer was used to circumvent the transmissions of scrapie

The genetic sequence of the ovine prion protein (PrP) gene between codons 102 and 175 with emphasis on ovine PrP gene codons 136 and 171 was determined, and the polymorphic distribution of the ovine PrP gene in the scrapie-exposed Suffolk embryo donors and offspring from these donors that were transferred to scrapie-free recipient ewes was investigated in this study. The most common genotype was AA(136)QQ(171) (70% and 63% in the donor and offspring flocks, respectively), which is considered a high risk genotype in US Suffolk sheep. Although embryos were collected from scrapie-positive donors and many embryos had the high risk genotype, no scrapie occurred in the resulting offspring. Based upon the results of this study, we conclude that vertical transmission of scrapie can be circumvented using embryo transfer procedures even when the offspring have the high risk genotype.     

Differential proteome analysis of replicative senescence in rat embryo fibroblasts

Normal somatic cells undergo a finite number of divisions and then cease dividing whereas cancer cells are able to proliferate indefinitely. To identify the underlying mechanisms that limit the mitotic potential, a two-dimensional differential proteome analysis of replicative senescence in serially passaged rat embryo fibroblasts was undertaken. Triplicate independent two-dimensional gels containing over 1200 spots each were run, curated, and analyzed. This revealed 49 spots whose expression was altered more than 2-fold. Of these, 42 spots yielded positive protein identification by mass spectrometry comprising a variety of cytoskeletal, heat shock, and metabolic proteins, as well as proteins involved in trafficking, differentiation, and protein synthesis, turnover, and modification. These included gelsolin, a candidate tumor suppressor for breast cancer, and alpha-glucosidase II, a member of the family of glucosidases that includes klotho; a defect in klotho expression in mice results in a syndrome that resembles human aging. Changes in expression of TUC-1, -2, -4, and -4 beta, members of the TUC family critical for neuronal differentiation, were also identified. Some of the identified changes were also shown to occur in two other models of senescence, premature senescence of REF52 cells and replicative senescence of mouse embryo fibroblasts. The majority of these candidate proteins were unrecognized previously in replicative senescence. They are now implicated in a new role.     

Bacterial stimulation upregulates the surface expression of the stress protein gp96 on B cells in the frog Xenopus

The presence of the soluble intracellular heat shock protein gp96 (an endoplasmic reticulum resident protein) at the surface of certain cell types is an intriguing phenomenon whose physiological significance has been unclear. We have shown that the active surface expression of gp96 by some immune cells is found throughout the vertebrate phylum including the Agnatha, the only vertebrate taxon whose members (lamprey, hagfish) lack an adaptive immune system. To determine whether gp96 surface expression can be modulated by pathogens, we investigated the effects of in vitro stimulation by purified lipopolysaccharide (LPS) and the heat-killed gram-negative bacteria, Escherichia coli and Aeromonas hydrophilia. Purified Xenopus B cells are readily activated and markedly proliferate in vitro in response to the heat-killed bacteria but not to purified LPS. Furthermore, messenger ribonucleic acid, and intracellular and surface protein expressions of both gp96 and immunoglobulin were upregulated only after activation of B cells by heat-killed bacteria. These data are consistent with an ancestral immunological role of gp96 as an antigen-presenting or danger-signaling molecule, or both, interacting directly with antigen-presenting cells, T cells, or natural killer cells, (or all), to trigger or amplify immune responses.     

Topology of constitutional reciprocal translocations in metaphase

We studied in 39 carriers of 26 reciprocal translocations (including five de novo and seven of indeterminate occurrence) the metaphase localization of the derivative chromosomes, their normal non-homologous counterparts (here called A and B), and two control pairs (C and D). In eight familial translocations, we analysed two to five carriers. We digitally captured 10 G-banded lymphocyte metaphases per individual and measured in microns the largest diameter (d) of the metaphase and six intercentromeric distances: (1) der A<-->der B (problem distance 1, pd1), (2) der A<-->B (pd2), (3) der B<-->A (pd3), (4) A<-->B (control distance 1, cd1), (5) the smaller distance between C and D (cd2) and (6) the largest distance between C and D (cd3); in addition, the average between C and D (cd4) was calculated. We used the formula Delta = 100(cd - pd)/d 12 times per metaphase, compared each pd vs. each cd, and tested the differences by the Wilcoxon matched-pair test. Although, in the whole sample there were not significant differences respect to cd1, this distance emerged as the proper control. In the eight familial translocations, the three pd vs. cd1 comparisons revealed that in 19/24 times the pd was smaller but only once reached significance (cd1 vs. pd2 in t[3;4]). In the analysis per individual the pd was smaller than cd1 in 19 (pd1), 22 (pd2) and 22 (pd3) cases although only twice reached significance. We conclude that in some translocations, the derivative chromosomes actually lie close from each other or from a normal non-homologous counterpart.