Hemolysis of rabbit erythrocytes in the presence of copper ions

The hemolysis of red blood cells (RBC) induced by Cu(II) is modified by ceruloplasmin (Cp) and albumin. The time course of hemolysis for rabbit RBC by Cu(II) consisted of two parts, an induction period followed by a catastrophic lysis period. The induction period decreased and the lysis rate increased with increasing Cu(II) concentration. Cp or albumin, modified Cu(II) induced hemolysis, by increasing the duration of the induction period and decreasing the overall rate of hemolysis of RBC. The catastrophic lysis period coincided with a sharp increase in the formation of metHb within the cell and in a rapid uptake of Cu(II). The presence of Cp led to an increase in the induction period prior to the rapid increase in metHb formation and in Cu(II) uptake. Porcine Cp was prepared with either two or three nonprosthetic copper binding sites (sites where Cu(II) is easily removed by passing over Chelex-100). Cp with three nonprosthetic binding sites gave more protection than Cp with two. Likewise, albumin can be prepared with three and five nonprosthetic copper binding sites. The albumin with five sites gave more protection than the albumin with three sites.     

Development of Anncaliia algerae (Microsporidia) in Drosophila melanogaster

Representatives of the genus Anncaliia are known as natural parasites of dipteran and coleopteran insects, amphipod crustaceans, but also humans, primarily with immunodeficiency. Anncaliia algerae‐caused fatal myositis is considered as an emergent infectious disease in humans. A. (=Nosema, Brachiola) algerae, the best studied species of the genus, demonstrates the broadest among microsporidia range of natural and experimental hosts, but it has never been propagated in Drosophila. We present ultrastructural analysis of development of A. algerae in visceral muscles and adipocytes of Drosophila melanogaster 2 wk after per oral experimental infection. We observed typical to Anncaliia spp. features of ultrastructure and cell pathology including spore morphology, characteristic extensions of the plasma membrane, and presence of “ridges” and appendages of tubular material at proliferative stages. Anncaliia algerae development in D. melanogaster was particularly similar to one of A. algerae and A.(Brachiola) vesicularum in humans with acute myositis. Given D. melanogaster is currently the most established genetic model, with a fully sequenced genome and easily available transgenic forms and genomic markers, a novel host–parasite system might provide new genetic tools to investigate host–pathogen interactions of A. algerae, as well to test antimicrosporidia drugs.     

A multi-generation analysis of the stability of transgenic virus resistance in doubled-haploid tobacco lines

Plants can be genetically engineered for virus resistance by transformation with a viral gene. We transformed tobacco with the tomato spotted wilt virus (TSWV) nucleocapsid gene from the Hawaiian L isolate in order to obtain TSWV resistant breeding lines. Doubled-haploid lines were produced from primary transgenic plants that were selected for resistance to the virus. Several of these lines showed very high levels of resistance and were symptomless after inoculation with the Hawaiian L isolate of TSWV. The accumulation of only low levels of full-length transgene RNA and protein observed in these lines is consistent with an RNA-mediated mechanism of resistance. The lines that were highly resistant to the Hawaiian L isolate of TSWV were also found to be highly resistant to several other isolates of TSWV, while lines that were only moderately resistant to the Hawaiian L isolate were often susceptible to the other isolates. The highly resistant lines were advanced over several generations by self-pollination. Although these lines were fully homozygous, several lines lost resistance in later generations, indicating that the resistance was unstable. Selection for resistance in these unstable lines did not prevent the occurrence of susceptible progeny in subsequent generations. Therefore, testing over several generations is required to determine the stability of resistance when breeding crops with transgenic virus resistance.     

Adenine nucleotide translocase-dependent anion transport in pea chloroplasts

Pea chloroplasts were found to take up actively ATP and ADP and exchange the external nucleotides for internal ones. Using carrier-free [¹⁴C]ATP, the rate of nucleotide transport in chloroplasts prepared from 12–14-day-old plants was calculated to be 330 μmol ATP/g chlorophyll/min, and the transport was not affected by light or temperature between 4 and 22°C. Adenine nucleotide uptake was inhibited only slightly by carboxyatractylate, whereas bongkrekic acid was nearly as effective an inhibitor of the translocator in pea chloroplasts as it was in mammalian mitochondria. There was no counter-transport of adenine nucleotides with substrates carried on the phosphate translocator including inorganic phosphate, 3-phosphoglycerate and dihydroxyacetone phosphate. However, internal or external phosphoenolpyruvate, normally considered to be transported on the phosphate carrier in chloroplasts, was able to exchange readily with adenine nucleotides. Furthermore, inorganic pyrophosphate which is not transported by the phosphate carrier initiated efflux of phosphoenolpyruvate as well as ATP from the chloroplast. These findings illustrate some interesting similarities as well as differences between the various plant phosphate and nucleotide transport systems which may relate to their role in photosynthesis.     

Hsp90α/β associates with the GSK3β/axin1/phospho-β-catenin complex in the human MCF-7 epithelial breast cancer model

Hsp90α/β, the signal transduction chaperone, maintains intracellular communication in normal, stem, and cancer cells. The well characterised association of Hsp90α/β with its client kinases form the framework of multiple signalling networks. GSK3β, a known Hsp90α/β client, mediates β-catenin phosphorylation as part of a cytoplasmic destruction complex which targets phospho-β-catenin to the 26S proteasome. The canonical Wnt/β-catenin pathway promotes stem cell self-renewal as well as oncogenesis. The degree of Hsp90α/β involvement in Wnt/β-catenin signalling needs clarification. Here, we describe the association of Hsp90α/β with GSK3β, β-catenin, phospho-β-catenin and the molecular scaffold, axin1, in the human MCF-7 epithelial breast cancer cell model using selective inhibition of Hsp90α/β, confocal laser scanning microscopy and immunoprecipitation. Our findings suggest that Hsp90α/β modulates the phosphorylation of β-catenin by interaction in common complex with GSK3β/axin1/β-catenin.     

Epitope mapping of snake venom phospholipases A2 with pseudexin monoclonal antibodies

Fifteen different monoclonal antibodies, developed against a pseudexin A, B, and C mixture, were screened for linear epitope recognition. Peptides (9-mers) spanning pseudexin B were synthesized on alanine-derivatized polyethylene pins and subsequently probed with antibody. Four antibodies recognized linear epitopes of pseudexin A, pseudexin B, and also nonidentical sequences found in other phospholipases A₂ (PLA₂s) as determined by enzyme-linked immunosorbent assays. Three antibodies recognized a highly conserved site important in calcium binding and the interlocking of dimeric forms of PLA₂. Antibodies neutralizing lethal or enzymatic effects of PLA₂ did not recognize linear epitopes.     

Metabolism of mevalonic acid to phosphorylated intermediates in a cell-free extract from Nepeta cataria leaves

A cell-free extract has been prepared from leaves of Nepeta cataria plants which converts mevalonic acid (MVA) to mevalonic acid phosphate (MVAP), mevalonic acid pyrophosphate (MVAPP) and isopentenylpyrophosphate (IPP). These enzymes are found in the 30 000 g supernatant. The activities are maximal at pH 7 and the formation of mevalonic acid pyrophosphate and isopentenyl-pyrophosphate reaches a maximal level after an incubation time of 180 min whereas the level of mevalonic acid phosphate begins to decrease after 90 min.     

Organically modified silica nanoparticles are biocompatible and can be targeted to neurons in vivo

The application of nanotechnology in biological research is beginning to have a major impact leading to the development of new types of tools for human health. One focus of nanobiotechnology is the development of nanoparticle-based formulations for use in drug or gene delivery systems. However most of the nano probes currently in use have varying levels of toxicity in cells or whole organisms and therefore are not suitable for in vivo application or long-term use. Here we test the potential of a novel silica based nanoparticle (organically modified silica, ORMOSIL) in living neurons within a whole organism. We show that feeding ORMOSIL nanoparticles to Drosophila has no effect on viability. ORMOSIL nanoparticles penetrate into living brains, neuronal cell bodies and axonal projections. In the neuronal cell body, nanoparticles are present in the cytoplasm, but not in the nucleus. Strikingly, incorporation of ORMOSIL nanoparticles into the brain did not induce aberrant neuronal death or interfered with normal neuronal processes. Our results in Drosophila indicate that these novel silica based nanoparticles are biocompatible and not toxic to whole organisms, and has potential for the development of long-term applications.