Although photosensitizers, molecules that produce active oxygen species upon activation by visible light, are being extensively used in photodynamic therapy to treat cancer and other clinical conditions, problems include normal cell and tissue damage and associated side effects, which are attributable in part to the fact that cytotoxic effects are largely restricted to the plasma membrane. We have previously shown that the photosensitizer chlorin e 6 has significantly higher photosensitizing activity when present in conjugates containing specific ligands and thus able to be internalized by receptor-expressing cells. In this study we use insulincontaining conjugates to which variants of the simian virus SV40 large tumor antigen nuclear localization signal (NLS) were linked to target chlorin e 6 to the nucleus, a hypersensitive site for active oxygen species-induced damage. NLSs were either included as peptides crosslinked to the carrier bovine serum albumin or encoded within the sequence of a ␤-galactosidase fusion protein carrier. The results for photosensitization demonstrate clearly for the first time that NLSs increase the photosensitizing activity of chlorin e 6 , maximally reducing the EC 50 by a factor of over 2000-fold. This has widereaching implications for achieving efficient cell typespecific photodynamic therapy.Photosensitizers such as porphyrins are molecules that produce active oxygen species upon activation by visible light and are currently being extensively used in photodynamic therapy to treat cancer and other clinical conditions (1-3). Because normal cells are able to accumulate porphyrins, however, and porphyrins are only excreted slowly from the body, prolonged skin photosensitization as well as other effects can be a problem (4, 5), leading to normal cell and tissue damage (6). A high priority with respect to photodynamic therapy is accordingly to increase the specificity of the uptake of photosensitizers in particular target cells, thereby enabling the active dose of porphyrins administered to patients to be reduced. We have previously shown that the photosensitizer chlorin e 6 has significantly higher photosensitizing activity when present in conjugates containing specific ligands such as insulin or concanavalin A and thus is able to be internalized by receptorexpressing cells (7-9). Photosensitization could be competed by incubating cells in the presence of an excess of unconjugated ligand (7-9), indicating that cellular uptake was receptor-dependent. Because only cells expressing specific receptors are targeted in this approach (see Refs. 7-9), it is clear that it enables selectivity in terms of the cell types targeted for photosensitization.Due to the fact that injury induced by singlet oxygen comprising 80% of all of the active oxygen species generated upon porphyrin activation is localized within less than 0.1 m of the site of its production, the effect of photosensitizers is integrally dependent on their site of cellular accumulation (1). Although most porphyrins such as chlorin e 6...

Background:Aflatoxin B1 (AFB1), produced by Aspergillus flavus, is one of the most life threatening food contaminants causing significant economic losses worldwide. Biological AFB1 degradation by microorganisms, or preferably microbial enzymes, is considered as one of the most promising approaches.Objectives:The current work aimed to study the AFB1-degrading metabolites, produced by Phoma glomerata PG41, sharing a natural substrate with aflatoxigenic A. flavus, and the preliminary determination of the nature of these metabolites.Materials and Methods:The AFB1-degrading potential of PG41 metabolites was determined by a quantitative high performance liquid chromatography (HPLC) of residual AFB1 after 72 hours incubation at 27ºC. The effects of pH, heat, and protease treatment on the AFB1-destroying activity of extracellular metabolites were examined.Results:The AFB1-degrading activity of protein-enriched fractions, isolated from culture liquid filtrate and cell-free extract, is associated with high-molecular-weight components, is time- and pH-dependent, thermolabile, and is significantly reduced by proteinase K treatment. The AFB1 degradation efficiency of these fractions reaches 78% and 66%, respectively.Conclusions:Phoma glomerata PG41 strain sharing natural substrate with toxigenic A. flavus secretes metabolites possessing a significant aflatoxin-degrading activity. The activity is associated mainly with a protein-enriched high-molecular-weight fraction of extracellular metabolites and appears to be of enzymatic origin.

Photosensitizers, molecules that produce active oxygen species upon activation by visible light, are currently being used in photodynamic therapy (PDT) to treat cancer and other conditions, where limitations include normal cells and tissue damage and associated side effects, and the fact that cytotoxic effects are largely restricted to the plasma and other peripheral membranes. In this study, we used insulincontaining conjugates to which variants of the simian-virus-SV40 large-tumor antigen (T-ag) nuclear localization signal (NLS) were linked in order to target the photosensitizer chlorin e 6 to the nucleus. NLSs were included either as peptides coupled co-valently to the carrier bovine serum albumin, or within the coding sequence of ␤-galactosidase fusion proteins. The most potent photosensitizing conjugate was the NLS-containing T-ag ␤-galactosidase fusion protein (P10)-(chlorin e 6 )-insulin, exhibiting an EC 50 more than 2400-fold lower than the value for free chlorin e 6 , and more than 15-fold lower than that of an NLS-deficient ␤-galactosidase-(chlorin e 6 )-insulin construct, thus demonstrating that NLSs can increase the photosensitizing activity of chlorin e 6 . Attenuated adenoviruses were used to increase the nuclear delivery of conjugates through its endosomal-membrane-disrupting activity. In the case of the NLS-containing P10-conjugate, co-incubation with adenovirus increased the proportion of cells whose nuclear photosensitizing activity was higher than that in the cytoplasm by 2.5-fold. This use of adenoviruses in conjunction with photosensitizers has clear implications for achieving efficient cell-type-specific PDT. Int. J. Cancer 81:734-740, 1999.1999 Wiley-Liss, Inc.

Aim: Create a method for highly sensitive, selective, rapid and easy-to-use detection and identification of economically significant potato pathogens, including viruses, bacteria and oomycetes, be it single pathogen, or a range of various pathogens occurring simultaneously. Methods and Results: Test-systems for real-time PCR, operating in the unified amplification regime, have been developed for Phytophthora infestans, Pectobacterium atrosepticum, Dickeya dianthicola, Dickeya solani, Ralstonia solanacearum, Pectobacterium carotovorum, Clavibacter michiganensis subsp. sepedonicus, potato viruses Y (ordinary and necrotic forms as well as indiscriminative test system, detecting all forms), A, X, S, M, potato leaf roll virus, potato mop top virus and potato spindle tuber viroid. The test-systems (including polymerase and revertase) were immobilized and lyophilized in miniature microreactors (1Á2 ll) on silicon DNA/RNA microarrays (micromatrices) to be used with a mobile AriaDNA â amplifier. Conclusions: Preloaded 30-reaction micromatrices having shelf life of 3 and 6 months (for RNA-and DNA-based pathogens, respectively) at room temperature with no special conditions were successfully tested on both reference and field samples in comparison with traditional ELISA and microbiological methods, showing perfect performance and sensitivity (1 pg). Significance and Impact of the Study: The accurate, rapid and user-friendly diagnostic system in a micromatrix format may significantly contribute to pathogen screening and phytopathological studies.

Photosensitizers, molecules that produce active oxygen species upon activation by visible light, are currently being used in photodynamic therapy (PDT) to treat cancer and other conditions, where limitations include normal cells and tissue damage and associated side effects, and the fact that cytotoxic effects are largely restricted to the plasma and other peripheral membranes. In this study, we used insulin‐containing conjugates to which variants of the simian‐virus‐SV40 large‐tumor antigen (T‐ag) nuclear localization signal (NLS) were linked in order to target the photosensitizer chlorin e6 to the nucleus. NLSs were included either as peptides coupled co‐valently to the carrier bovine serum albumin, or within the coding sequence of β‐galactosidase fusion proteins. The most potent photosensitizing conjugate was the NLS‐containing T‐ag β‐galactosidase fusion protein (P10)‐(chlorin e6)‐insulin, exhibiting an EC50 more than 2400‐fold lower than the value for free chlorin e6, and more than 15‐fold lower than that of an NLS‐deficient β‐galactosidase‐(chlorin e6)‐insulin construct, thus demonstrating that NLSs can increase the photosensitizing activity of chlorin e6. Attenuated adenoviruses were used to increase the nuclear delivery of conjugates through its endosomal‐membrane‐disrupting activity. In the case of the NLS‐containing P10‐conjugate, co‐incubation with adenovirus increased the proportion of cells whose nuclear photosensitizing activity was higher than that in the cytoplasm by 2.5‐fold. This use of adenoviruses in conjunction with photosensitizers has clear implications for achieving efficient cell‐type‐specific PDT. Int. J. Cancer 81:734–740, 1999. © 1999 Wiley‐Liss, Inc.