Lysosomal pathways to cell death and their therapeutic applications

Lysosomes are the major cell digestive organelles that were discovered over 50 years ago. They contain a number of hydrolases that help them to degrade intracellular and extracellular material delivered. Among the hydrolases, the cathepsins, a group of proteases enclosed in the lysosomes, have a major role. About a decade ago, the cathepsins were found to participate in apoptosis. Following their release into the cytosol, they cleave Bid and degrade antiapoptotic Bcl-2 proteins, thereby triggering the mitochondrial pathway of apoptosis, with the lysosomal membrane permeabilization being the critical step in this pathway. Lysosomal dysfunction is linked with several diseases, including cancer and neurodegenerative disorders, thereby providing a potential for therapeutic applications. In this review lysosomes and lysosomal proteases involvement in apoptosis and their possible pharmaceutical targeting are discussed.     

Electric-field controlled liposome formation with embedded superparamagnetic iron oxide nanoparticles

Liposomes are one of the most promising biomaterial carriers to deliver DNA,(1) proteins, drugs and medicine in human bodies. However, artificially formed liposomes have to satisfy some crucial functions such as: (i) to efficiently carry drugs to targeted systems, (ii) to be biologically stable until they are removed from human body, (iii) to be biodegradable, and (iv) to be sufficiently small in size for effective drug delivery. Here, we report an efficient and novel method to simultaneously manufacture and incorporate super-paramagnetic iron-oxide nanoparticles (efficient target finder in the presence of external magnetic field) into the liposome's interior and its bilayer. In this technique, we use electric field to control the formation of liposomes and the incorporation of iron oxide nanoparticles. Our preparation procedure does not require any chemical or ultrasound treatments. Apart from that, we also provide further experimental investigations on the role of electric fields on the formation of liposomes using XPS(2) and the magnetic-optical microscope.     

Plant rDNA database: ribosomal DNA loci information goes online

Number, position and structure of the 5S and 18S-5.8S-26S ribosomal DNA (rDNA) loci are important species characteristics. In recent decades, we have witnessed accumulation of rDNA data, and there is a need to compile, store and analyse this information, and to make it accessible to a broader scientific community. An online resource, accessible at www.plantrdnadatabase.com , has been developed to accomplish these goals. Current knowledge regarding chromosomal rDNA sites is provided for more than 1,000 plant species (including more than 1,400 different accessions). The data comes from fluorescent in situ hybridisation experiments (FISH) from more than 300 publications. Additional information is also displayed, such as ploidy level, mutual arrangement of rRNA genes, genome size and life cycle. The webpage is intuitive and user-friendly, including different search options, and currently holds information published (or in press) up until January 2011; frequent updates are planned. We expect this database to be used for data-mining, analysing rDNAs from different angles, unit organisation, distribution, evolution and linkage of rDNA patterns with phylogenetic relationships.     

Influence of Temperature and Light Conditions on Germination,Growth and Conidiation of Oidium neolycopersici

The effect of temperature and light conditions (spectral quality, intensity and photoperiod) on germination, development and conidiation of tomato powdery mildew (Oidium neolycopersici) on the highly susceptible tomato cv. Amateur were studied. Conidia germinated across the whole range of tested temperatures (10–35°C); however, at the end‐point temperatures, germination was strongly limited. At temperatures slightly lower than optimum (20–25°C), mycelial development and time of appearance of the first conidiophores was delayed. Conidiation occurred within the range of 15–25°C, however was most intense between 20–25°C. Pathogen development was also markedly influenced by the light conditions. Conidiation and mycelium development was greatest at light intensities of approximately 60 μmol/m² per second. At lower intensities, pathogen development was delayed, and in the dark, conidiation was completely inhibited. A dark period of 24 h after inoculation had no stimulatory effect on later mycelium development. However, 12 h of light after inoculation, followed by continuous dark, resulted in delayed mycelium development and total restriction of pathogen conidiation (evaluated 8 days postinoculation). When a longer dark period (4 days) was followed by normal photoperiod (12 h/12 h light/dark), mycelium development accelerated and the pathogen sporulated normally. When only inoculated leaf was covered with aluminium foil while whole plant was placed in photoperiod 12 h/12 h, the intensive mycelium development and slight subsequent sporulation on covered leaf was recorded.     

Exercise-Induced Changes in Iron Status and Hepcidin Response in Female Runners

Background and Aims

Exercise-induced iron deficiency is a common finding in endurance athletes. It has been suggested recently that hepcidin may be an important mediator in this process.

Objective

To determine hepcidin levels and markers of iron status during long-term exercise training in female runners with depleted and normal iron stores.

Methods

Fourteen runners were divided into two groups according to iron status. Blood samples were taken during a period of eight weeks at baseline, after training and after ten days’ recovery phase.

Results

Of 14 runners, 7 were iron deficient at baseline and 10 after training. Hepcidin was lower at recovery compared with baseline (p<0.05). The mean cell haemoglobin content, haemoglobin content per reticulocyte and total iron binding capacity all decreased, whereas soluble transferrin receptor and hypochromic red cells increased after training and recovery (p<0.05 for all).

Conclusion

The prevalence of depleted iron stores was 71% at the end of the training phase. Hepcidin and iron stores decreased during long-term running training and did not recover after ten days, regardless of baseline iron status.     

Expression of a hepatitis A virus antigen in Lactococcus lactis and Escherichia coli and evaluation of its immunogenicity

An epidemic shift in Hepatitis A virus (HAV) infection has been observed in recent years in rapidly developing countries, with increasing numbers of severe adult cases which has led to renewed interest in vaccination. Our approach in vaccine development uses recombinant expression of the highly immunogenic HAV antigen VP1-P2a in food-grade lactic acid bacterium Lactococcus lactis and in Escherichia coli. We used genetic constructs that enable nisin-controlled expression of the antigen in L. lactis in three different forms: (a) intracellularly, (b) on the bacterial surface and (c) on the bacterial surface fused with the fragment of the E. coli flagellin molecule that can act as a molecular adjuvant. Expression of the two surface forms of the antigen was achieved in L. lactis, and the resulting antigen-displaying bacteria were administered orally to mice. Half the animals in each of the two groups developed specific IgGs, with titers increasing over time and reaching 1:422 without flagellin and 1:320 with flagellin. A much higher titer 1:25,803 was observed with the parenterally administered antigen, which was purified from E. coli. With the latter, a significant mucosal IgA response was also observed. Despite significant titers, the IgGs elicited with oral or parenteral administration could not prevent HAV from infecting cells in a virus neutralization assay, suggesting that the antibodies cannot recognize viral surface epitopes. Nevertheless, orally administered HAV antigen expressed in L. lactis elicited significant systemic humoral immune response showing the feasibility for development of effective HAV vaccine for mucosal delivery.     

Comparative proteome profiling of host–pathogen interactions: insights into the adaptation mechanisms of Francisella tularensis in the host cell environment

The intracellular pathogens have the unique capacity to sense the host cell environment and to respond to it by alteration in gene expression and protein synthesis. Proteomic analysis of bacteria exposed directly to the host cell milieu might thus greatly contribute to the elucidation of processes leading to bacterial adaptation and proliferation inside the host cell. Here we have performed a global proteome analysis of a virulent Francisella tularensis subsp. holarctica strain during its intracellular cycle within the macrophage-like murine cell line J774.2 using the metabolic pulse-labeling of bacterial proteins with ³⁵S-methionine and ³⁵S-cysteine in various periods of infection. The two-dimensional gel analysis revealed macrophage-induced bacterial proteome changes in which 64 identified proteins were differentially expressed in comparison to controls grown in tissue culture medium. Nevertheless, activation of macrophages with interferon gamma before in vitro infection decreased the number of detected alterations in protein levels. Thus, these proteomic data indicate the F. tularensis ability to adapt to the intracellular hostile environment that is, however, diminished by prior interferon gamma treatment of host cells.     

Synthesis and characterization of a pH-sensitive conjugate of isoniazid with Fe3O4@SiO2 magnetic nanoparticles

The Letter describes the preparation and characterization of a conjugate of isoniazid (INH) with magnetic nanoparticles Fe₃O₄@SiO₂ 115 ± 60 nm in size. The INH molecules were attached to the surface of nanoparticles by a covalent pH-sensitive amidine bond. The conjugate was characterized by X-ray diffraction, SEM, dynamic light scattering, IR spectroscopy and microanalysis. The conjugate released isoniazid under in vitro conditions (pH = 4; 37 °C; t_1/2 ≈ 115 s). In addition, the cytotoxicity of the Fe₃O₄@SiO₂–INH conjugate was evaluated in SK-BR-3 cells using the xCELLigence system.     

Accumulation of DNA damage and cell death after fractionated irradiation

The purpose of this work was to determine how fractionated radiation used in the treatment of tumors affects the ability of cancer as well as normal cells to repair induced DNA double-strand breaks (DSBs) and how cells that have lost this ability die. Lymphocytic leukemia cells (MOLT4) were used as an experimental model, and the results were compared to those for normal cell types. The results show that cancer and normal cells were mostly unable to repair all DSBs before the next radiation dose induced new DNA damage. Accumulation of DSBs was observed in normal human fibroblasts and healthy lymphocytes irradiated in vitro after the second radiation dose. The lymphocytic leukemia cells irradiated with 4 × 1 Gy and a single dose of 4 Gy had very similar survival; however, there was a big difference between human fibroblasts irradiated with 4 × 1.5 Gy and a single dose of 6 Gy. These results suggest that exponentially growing lymphocytic leukemia cells, similar to rapidly proliferating tumors, are not very sensitive to fraction size, in contrast to the more slowly growing fibroblasts and most late-responding (radiation therapy dose-limiting) normal tissues, which have a low proliferation index.