Zvi Malik

Inactivation of gram-negative bacteria by photosensitized porphyrins.

Abstract— Photosensitization of Escherichia coli and Pseudomonas aeruginosa cells by deuteroporphyrin (DP) is shown to be possible in the presence of the polycationic agent polymyxin nonapeptide (PMNP). Previous studies established complete resistance of Gram‐negative bacteria to the photodynamic effects of porphyrins. The present results show that combined treatment of E. coli or P. aeruginosa cultures with DP and PMNP inhibit cell growth and viability. No antibacterial activity of PMNP alone could be demonstrated and cell viability remained unchanged. Spectroscopically, PMNP was found to bind DP, a mechanism which probably assists its penetration into the cells membranes. Insertion of DP into the cells was monitored by the characteristic fluorescence band of bound DP at 622 nm. Binding times were 5–40 min and the extent of binding increased with decreasing the pH from 8.5 to 6.5. DP binding constants, as well as the concentrations of PMNP which were required for maximal effect on the various Gram‐negative bacteria, were determined fluorometrically. By the treatment of DP, PMNP and light the growth of E. coli and P. aeruginosa cultures was stopped and the viability of the culture was dramatically reduced. Within 60 min of treatment the survival fraction of E. coli culture was 9 × 10–6 that of P. aeruginosa was 5.2 × 10–4. Electron microscopy depicted ultrastructural alterations in the Gram‐negative cells treated by DP and PMNP. The completion of cell division was inhibited and the chromosomal domain was altered markedly.

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.

FOURIER TRANSFORM MULTIPIXEL SPECTROSCOPY FOR QUANTITATIVE CYTOLOGY

A Fourier transform multipixel spectroscopy system was set up and applied to fluorescence microscopy of single living cells. Continuous fluorescence spectra for all pixels of the cell image were recorded simultaneously by the system. Multiple frames of data were first acquired and stored as a set of interferograms for each pixel of the image; they were then Fourier transformed and used as a spatially organized set of fluorescence spectra. Practical spectral resolution of 5 nm was achieved, typically, for 104 pixels in a single cell. The net result was I(xy,λ), the fluorescence intensity (I) for each pixel of the image (xy), as function of wavelength (λ). The present study demonstrates that multipixel spectroscopy can reveal dynamic processes of the food‐digestive cycle in the unicellular Paramecium vulgaris fed with algae. Spectral variability of fluorescence intensity at different cytoplasmic sites pinpointed the location of cellular deposits of chlorophyll (630 nm) and of pheophytin (695 nm), a digestive product of the chlorophyll. Localization of compartmental spectral changes was achieved using a ‘similarity mapping’ algorithm, followed by enhanced image construction. Similarity mapping based on the fluorescence spectrum of native chlorophyll revealed a highlighted image of the cell cytopharynx structure where algae were ingested. Phagolysosomes, migrating vacuoles and the cytoproct, each containing different ratios of pheophytin, were similarly imaged.

Topical application of 5-aminolevulinic acid, DMSO and EDTA: protoporphyrin IX accumulation in skin and tumours of mice.

Topical 5-aminolevulinic acid (ALA) application in three different creams was carried out on mice bearing subcutaneously transplanted C26 colon carcinoma. The creams contained (a) 20% ALA alone, (b) ALA with 2% dimethylsulphoxide (DMSO) and (c) ALA, DMSO and 2% edetic acid disodium salt (EDTA). Protoporphyrin IX (PP) production in the tumour and in the skin overlying the tumour was studied by two methods: laser-induced fluorescence (LIF) and chemical extraction. The kinetics of PP production in the skin and in the tumour, as studied by the LIF method, was similar for all three cream preparations. The PP fluorescence intensity in the tissues reached its maximum 4-6 h after application of the creams. Quantitative analysis showed that the PP concentration after treatment was more pronounced in the skin than in the tumour. The efficiency of porphyrin production in the skin by the creams used was in the following order: ALA-DMSO-EDTA > ALA-DMSO > ALA. In the tumour the enhancing effect of DMSO and EDTA on PP accumulation induced by ALA was observed mainly in the upper 2 mm section. However, the concentration of PP in the tumour was found to be approximately the same for ALA-DMSO and ALA-DMSO-EDTA cream combinations. The possible mechanisms of the effect of DMSO and EDTA are discussed.

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.

Green fluorescent protein photobleaching: a model for protein damage by endogenous and exogenous singlet oxygen.

Abstract Characterization of protein damage during photosensitization of chlorin e6-treated cells was performed using the green fluorescent protein (GFP). The GFP-chromophore damage caused by singlet oxygen was studied in COS 7 kidney cells and E. coli bacteria following light irradiation. Electron spin resonance (ESR) revealed the generation of endogenous singlet oxygen (1O2) by photoactivated GFP, an effect similar to that produced by the exogenous photosensitizer chlorin e6. A light dose-dependent photobleaching effect of GFP was pronounced at low pH or upon photosensitization with chlorin e6. However, the 1O2 quenchers β-carotene and sodium azide minimized GFP photobleaching. Gel electrophoresis of photosensitized GFP followed by fluorescence multi-pixel spectral imaging revealed the binding of chlorin e6 to GFP, affecting the photobleaching efficacy. Fluorescence multi-pixel spectral imaging of GFP-transfected COS 7 cells demonstrated the presence of GFP in the cytoplasm and nucleus, while chlorin e6 was found to be concentrated in the perinuclear vesicles. Exposure of the cells to light induced GFP photobleaching in the close vicinity of chlorin e6 vesicles. We conclude that photoactivated GFP generates endogenous 1O2, inducing chromophore damage,, which can be enhanced by the cooperation of exogenous chlorine6.

FLUORESCENCE SPECTRAL CHANGES OF HEMATOPORPHYRIN DERIVATIVE UPON BINDING TO LIPID VESICLES, Staphylococcus aureus AND Escherichia coli CELLS

Abstract— The binding of hematoporphyrin derivated (Hpd) to lipid vesicles and bacterial membranes was determined by fluorescence spectroscopy. The fluorescence measurements of Hpd in aqueous solutions showed two bands at 613 and 677 nm. In lipid environments of lecithin vesicles the fluorescence spectrum was shifted to 631 and 692 nm, respectively. Hpd was rapidly bound to the cell membrane of Staphylococcus aureus while much less binding occurred in the presence of Escherichia coli. At the same time, spheroplasts of both bacteria were shown to bind Hpd to a similar extent. These results are well correlated with the photoinactivation of the gram positive bacteria with Hpd while the gram negative cells were shown to be resistant. The pH dependence of both Hpd binding to S. aureus as well as the photodynamic inhibitory effect of the same bacteria are similar. It is concluded that the segregation of Hpd to the cell membrane is a prerequisite for its photodynamic effect.

Influence of Environmental Temperature on the Immune Response in Fish

At the end of the last century and the beginning of this century, the problems of immunity in lower vertebrates and the influence of environmental temperature attracted attention for the first time (Ernst, 1890; Widal and Sicard, 1897; Metchnikoff, 1901). However, relatively little work has been done on this subject until recently. The early investigators were chiefly interested in the immuno-pathological problems. They immunized various species of lower vertebrates essentially with bacterial vaccines; agglutinating, neutralizing and protective antibodies were detected in their blood. The influence of environmental temperature on the immune response was investigated, since this subject represented great economical and theoretical importance. Epizootic diseases were observed to occur in relation to the cold season of the year, when the decrease or spontaneous increase of water temperature occurred (Schaperclaus, 1965; Besse et al., 1965; Klontz et al., 1965 Wood, 1966). The immunological deficiency of fish, caused by their natural or experimental stay in cold water, is now evident for both humoral and cellular immunity. In this review we will focus on two points: We shall attempt (1) to explain the mechanism by which the environmental temperature influences the immune resistance of fish to pathogens, (2) to determine the chronological location of this temperature-sensitive stage in the process of antibody formation, and to make some approaches to the general antibody formation mechanism.

Treatment of viral infections with 5-aminolevulinic acid and light

When 5‐aminolevulinic acid (ALA) is exogenously supplied, protoporphyrin IX (PpIX) is accumulated in various cells and makes them light sensitive. The possibility of using such an approach for the treatment of viral infections was studied in this work.

ALA induced photodynamic effects on Gram positive and negative bacteria

In the present study we examined the production of high amounts of porphyrins upon induction by delta-aminolevulinic acid (ALA) in 9 bacterial strains. This was performed by solely inducing the porphyrin biosynthesis pathway. Four of the strains were Gram positive bacteria and five were Gram negative strains. All strains, except Streptococcus faecalis, produced porphyrins when incubated in PBS with 0.38 mM ALA for 4 h. Excess porphyrin production was excreted to the medium. Gram positive bacteria exhibited fluorescent emission peaks at 622 nm for the endogenous and 617 nm for the excreted porphyrins. Gram negative bacteria exhibited a 630 nm emission peak for the endogenous and a 615 nm emission peak for the excreted extracellular porphyrins. Upon illumination of the ALA induced Staphylococcal strains with 407-420 nm blue light, a decrease of five orders of magnitude was demonstrated with a light dose of 50 J cm(-2). Total eradication of the Staphylococcal strains could be achieved with a 100 J cm(-2) dose, which resulted in a decrease in viability of seven orders of magnitude. The viability of all the induced Gram negative strains and B. cereus decreased by one or two orders of magnitude upon illumination with 50 and 100 J cm(-2), respectively. This difference in the photoinactivation rate was found to be due to the distribution and amounts of the various porphyrins in the bacterial strains. The predominant porphyrin in the Staphylococcal strains was coproporphyrin (68.3-74.6%). In the Gram negative strains there was no predominant porphyrin and the porphyrins found were mostly 5-carboxyporphyrin, uroporphyrin, 7- carboxyporphyrin, coproporphyrin and protoporphyrin. In the B. cereus(Gram positive) strain the predominant porphyrin was uroporphyrin (75.8%). Although the total production of porphyrins in the Gram negative bacteria was higher than in the Staphylococcal strains, the amount of coproporphyrin produced by the latter was twice to three times higher than in the Gram negative strains. The extracellular excreted porphyrins did not contribute to the photoinactivation in any of the tested strains. Significant decreases in the Na(+) and K(+) content were detected in induced S. aureus after illumination while only small changes were observed in E. coli B. The green fluorescent protein within the cytoplasm of induced E. coli strains was only partially disrupted (by 60% only). These results indicate a partial yield of the effects generated by (1)O(2) radicals resulting from the photoinactivation of Gram negative bacteria and a successful generation of the same effects in the Staphylococcal strains.