Judith Hanania

New trends in photobiology bactericidal effects of photoactivated porphyrins — An alternative approach to antimicrobial drugs

Photoactivated porphyrins display a potent cytotoxic activity towards a variety of Gram positive bacteria, mycoplasma and yeasts, but not Gram negative cells. The prerequisite for photosensitization of a microbial cell is the binding of porphyrin to the cytoplasmic membrane in a pH-dependent manner. On illumination, the membrane bound, and possibly, cytoplasmic porphyrin molecules generate singlet oxygen and radicals which sensitize biomolecules and lead to cell death. The immediate inhibition of cell growth on photodynamic treatment is accompanied by alterations in cell wall and membrane synthesis, leading to the formation of large mesosomes adjacent to the unaccomplished septa. Hemin bound to microbial cells exerts cytotoxic activity by peroxidative and oxidative reactions independent of light. Future research in the field may enhance the possibility of using porphyrin photosensitization for treatment of microbial infections. Such clinical use will be unrelated to the antibiotic resistance of the pathogen. Resistance of Gram negative bacteria to porphyrin photosensitization is the main impediment to its use as a broad spectrum antibacterial method.

The effect of EDTA and serum on endogenous porphyrin accumulation and photodynamic sensitization of human K562 leukemic cells

The interrelationship between the effect of serum on the induction of porphyrin synthesis, intracellular porphyrin accumulation and photodynamic sensitization of human K562 cells is described. Endogenous porphyrins, synthesized from supplemented 5-amino levulinic acid (5-ALA), were shown to accumulate in the cells, while an addition of serum triggered porphyrin translocation from the cell to the serum. In order to enhance porphyrin accumulation in the cells themselves, they were further stimulated by EDTA, which in combination with 5-ALA reduces Fe++ cellular content. The higher porphyrin cellular content under EDTA and 5-ALA induction was exploited to photoinactivate the human leukemic cells by more then 3 orders of magnitude.

Ultrastructure, autofluorescence, callose deposition and lignification in susceptible and resistant muskmelon leaves infected with the powdery mildew fungus Sphaerotheca fuliginea

Abstract Leaves of the susceptible cv. Ananas-Yokneam (AY) and of resistant cvs PI 124111F, PI 124112, PMR-45, PMR-6 of muskmelon ( Cucumis melo ) were inoculated with either race 1 or race 2 of Sphaerotheca fuliginea and examined microscopically after staining with Calcofluor, basic aniline blue or phloroglucinol. At 20–24 h post inoculation of the susceptible AY at 23 °C, the fungal spores (both race 1 or race 2) developed one or two germtubes which penetrated into one or two epidermal cells. The penetration zones were surrounded with callose-like material but no autofluorescence nor lignin-like materials were observed in the penetrated epidermal cells. A similar response was observed in PMR-45 inoculated with race 2 (compatible). In contrast, the fungus developed a single germtube on the resistant PI 124111F, PI 124112, and PMR-6 inoculated with either race, as well as in PMR-45 inoculated with race 1, which induced autofluorescence, callose accumulation and lignification in the penetrated epidermal cells. Electron microscopical studies revealed that the rapid collapse of epidermal cells in the resistant cultivars was accompanied by the accumulation of callose-like deposits in cell walls and around haustoria, electron-opaque deposits in the plasma membrane and electron-opaque deposits between the cell wall and the plasma membrane. Occasionally, callose also appeared in epidermal and mesophyll cells adjacent to the penetrated cells. By 96–120 h post inoculation, abundant sporulation was observed in the compatible interactions whereas only 1–3 germtubes with no sporulation were seen in the incompatible interactions. Heat shock or chemical inhibitors (cycloheximide, blasticidin-S, cordycepin, 2-deoxy-D-glucose, α-aminooxyacetic acid, 2,4-dinitrophenol and sodium azide) failed to induce susceptibility in the resistant cultivars. The results suggest a similar structural response to powdery mildew in C. melo cultivars carrying the resistance genes Pm-1 (PMR-45), Pm-2 (PMR-6), Pm-3 (PI 124111F), Pm-4 (PI 124112), Pm-5 (PI 124112) and Pm-6 (PI 124111F).

Ultrastructure of Pseudoperonospora cubensis in muskmelon genotypes susceptible and resistant to downy mildew

Abstract The causal agent of muskmelon (Cucumis melo L.) downy mildew, Pseudoperonospora cubensis (Berk. et Curt.) Rost., produced 10–15-mm lesions in the susceptible cv. Ananas-Yokneam (AY) but 1–3-mm, water-soaked lesions in the resistant cv. PI 124111F (PI). Sporulation on cv. PI was extremely limited compared to the conspicuous sporulation on cv. AY. In both cvs the fungus produced intercellular hyphae, rich in β-1,3-glucans, from which digitate (cauliflower-like) haustoria grew into the host cells. The haustoria had induced only minor chemical and ultrastructural changes in the cytoplasm and/or walls of cv AY 144 h after inoculation, whereas in contrast major changes were observed in cells of cv. PI as early as 20 h after inoculation. Changes induced in cv. PI included a heavy deposition of paramural, layered, callose-like materials along the inner surfaces of the host cell walls; enrichment of host cell walls with lignin-like materials (staining with phloroglucinol and chlorine sulphite); and encasement of the haustoria with heavy deposits of callose-like materials staining with resorcinol blue and lacmoid, fluorescing with aniline blue, and from TEM observations). In addition, the intercellular hyphae in cv. PI, but not in AY, sometimes produced haustoria (and necrosis) in epidermal cells, which gave rise to the early appearance (72 h) of water-soaked lesions. It is proposed that the containment of host cells and haustoria by callose- and lignin-like materials may interrupt the flow of nutrients from and into the invaded host cells.

Dopamine-melanin is actively phagocytized by PC12 cells and cerebellar granular cells: possible implications for the etiology of Parkinson's disease

Neuromelanin in the substantia nigra may be associated with the pathogenesis of nigral cell death in Parkinsons disease. We used synthetic dopamine-melanin (DA-M) as a model compound for neuromelanin and examined its toxic effects on mice cerebellar granule cells and a rat pheochromocytoma cell line (PC12). The DA-M and dopamine-treated cells showed an accumulation of black deposits when examined by light microscopy. Electron microscopy revealed different stages of DA-M phagocytosis, starting with DA-M binding, engulfment of the particles and the formation of phagosomes located in the cytoplasm. Using absorption assays, we found that NaN3 and low temperature inhibit the internalization of DA-M, pointing to an energy-dependent phagocytosis mechanism. These results suggest that neuromelanin can be phagocytised by neuronal cells which may thus be subjected to its toxic effects. These findings may contribute to our understanding of the formation and disposition of neuromelanin and its possible role in the etiology of Parkinsons disease.

Mesosomal structures and antimicrobial activity induced by hemin oxidation or porphyrin photodynamic sensitization inStaphylococci

The porphyrin-dependent inactivation ofStaphyloccocus aureus and the induction of mesosomal structures are described. The antimicrobial activity of different photoactivated porphyrins was compared with the dark cytotoxic effect of hemin. Deuteroporphyrin, protoporphyrin, hematoporphyrin, and hematoporphyrin derivative (Hpd) markedly reduced cell growth upon irradiation with light. Photofrin II, the polymerized fraction of Hpd, and other synthetic porphyrins had no effect on staphylococcal growth. Hemin immediately inhibited cell viability in the dark and induced the development of an irregular cell wall, analyzed by scanning electron microscopy (SEM). Inside the cytoplasm a multivesicular mesosome was formed near the septum, as detected by transmission electron microscopy (TEM). The mesosomal volume and its frequency in the cells was increased in a time-dependent manner. The mesosome appearance was not related to fixation by glutaraldehyde or to post-fixation by osmium tetroxide. Glycosyl moieties stained by ruthenium red revealed the formation of membrane-like structures in the mesosome. It is concluded that oxygen-dependent reactions potentiated by porphyrins may induce disturbances in the synthesis of staphylococcal membrane and cell wall, revealed as mesosomes.

Multipixel spectral imaging of green fluorescent protein (GFP) in COS-7 cells: folding kinetics and chromophore formation

Spectrally resolved imaging of Green fluorescent protein (GFP) expressed in living COS-7 kidney cells distinguished the subcellular localization and demarcated the processes of protein folding and chromophore formation. COS-7 kidney cells were transfected by a plasmid pEGFP-N1 plasmid followed by incubation for 15 hours for gen expression. At different intervals the cells were examined by fluorescence microscopy and analyzed by a spectral imaging system. After 7 hours, GFP was detected in the cytoplasm, concentrated in a localized form. Spectra of the initial GFP evinced several components that belong both tot he typical fluorescent signal as well as to unspecific fingerprints. At 10 and 15 hours, GFP was seen spread in the cytoplasm as well as in the nucleus, and the specific spectra of the GFP were dominant at the later time. The typical GFP spectral fingerprint is the result of protein folding and chromophore formation following internal oxidation reactions. This folding and chromophore formation process, up to final conformation, was detected by spectral imaging as localized in the nucleus rather than in the cytosol. Thus, the method of fluorescence microscopy combined with multiplex spectral imaging demonstrates distinct biochemical pathways leading to GFP conformation.