The Impact of UV Radiation on Paramecium Populations from Alpine Lakes.

Paramecium populations from a clear and a glacier-fed turbid alpine lake were exposed to solar simulated ultraviolet (UVR) and photosynthetically active radiation (PAR) at 8 and 15 °C. The ciliates were tested for DNA damage (comet assay), behavioral changes, and mortality after UVR + PAR exposure. High DNA damage levels (~58% tail DNA) and abnormal swimming behavior were observed, although no significant changes in cell numbers were found irrespective of the lake origin (clear, turbid), and temperatures. We conclude that environmental stressors such as UVR and their effects may influence the adaptation of ciliates living in alpine lakes.

808-nm laser therapy with a flat-top handpiece photobiomodulates mitochondria activities of Paramecium primaurelia (Protozoa).

Photobiomodulation is proposed as a non-linear process, and only low-level laser therapy (LLLT) is assumed to stimulate exposed cells, whereas high powered laser and fluences can cause negative effects, exhausting the cell's energy reserve as a consequence of excessive photon-based stimulation. In our work, we investigated and compared the effects of 808-nm diode laser (CW) with a new flat-top handpiece. To this purpose, we tested the photobiomodulation effects of 1 and 3 J/cm(2) fluence, both generated by 100 mW or 1 W of laser power and of 64 J/cm(2) of fluence generated by 100 mW, 1 W, 1.5 W or 2 W, as expressed through oxygen consumption and ATP synthesis of Paramecium. Data collected indicates the incremental consumption of oxygen through irradiation with 3 J/cm(2)-100 mW or 64 J/cm(2)-1 W correlates with an increase in Paramecium ATP synthesis. The Paramecium respiration was inhibited by fluences 64 J/cm(2)-100 mW or 64 J/cm(2)-2 W and was followed by a decrease in the endogenous ATP concentration. The 1 J/cm(2)-100 mW or 1 W and 3 J/cm(2)-1 W did not affect mitochondrial activity. The results show that the fluence of 64 J/cm(2)-1 W more than the 3 J/cm(2)-100 mW causes greater efficiency in Paramecium mitochondria respiratory chain activity. Our results suggest that thanks to flat-top handpiece we used, high fluences by high-powered laser have to be reconsidered as an effective and non-invasive therapy. Possible associated benefits of deeper tissue penetration would increase treatment effectiveness and reduced irradiation time.

The protozoan, Paramecium primaurelia, as a non-sentient model to test laser light irradiation: The effects of an 808nm infrared laser diode on cellular respiration.

Photobiomodulation (PBM) has been used in clinical practice for more than 40 years. Unfortunately, conflicting literature has led to the labelling of PBM as a complementary or alternative medicine approach. However, past and ongoing clinical and research studies by reputable investigators have re-established the merits of PBM as a genuine medical therapy, and the technique has, in the last decade, seen an exponential increase in the numbers of clinical instruments available, and their applications. This resurgence has led to a clear need for appropriate experimental models to test the burgeoning laser technology being developed for medical applications. In this context, an ethical model that employs the protozoan, Paramecium primaurelia, is proposed. We studied the possibility of using the measure of oxygen consumption to test PBM by irradiation with an infrared or near-infrared laser. The results show that an 808nm infrared laser diode (1W; 64J/cm²) affects cellular respiration in P. primaurelia, inducing, in the irradiated cells, a significantly (p < 0.05) increased oxygen consumption of about 40%. Our findings indicate that Paramecium can be an excellent tool in biological assays involving infrared and near-infrared PBM, as it combines the advantages of in vivo results with the practicality of in vitro testing. This test represents a fast, inexpensive and straightforward assay, which offers an alternative to both traditional in vivo testing and more expensive mammalian cellular cultures.

Photoreactivation in vivo of pyrimidine dimers in paramecium DNA.

Cells of Paramecium aurelia labeled with tritiated thymidine were irradiated with ultraviolet light and then were either exposed to photoreactivating light or kept in the dark as controls. In the controls, the level of thymine-containing pyrimidine dimers did not change, but in cells exposed to photoreactivating light such dimers were destroyed. This is the first demonstration in a eukaryote of in vivo photoreactivation of thymine-containing pyrimidine dimers.

DNA repair and longevity assurance in Paramecium tetraurelia.

At given doses and clonal ages, ultraviolet irradiation-induced DNA damage reduced clonal life-span, but when followed by photoreactivation, extension of clonal life-span was observed. If photoreactivation preceded the ultraviolet treatment, no significant beneficial effect was detected. Because studies of others have shown that photoreactivation repair monomerizes the ultraviolet-induced cyclobutane dimers in DNA, but does not affect the other photoproducts, these results indicate that DNA damage can influence the duration of clonal life-span unless that damage is repaired. Repeated treatment with ultraviolet and photoreactivation resulted in significant mean and maximal clonal life-span extension when compared with untreated controls, and it is assumed that the rejuvenation effect was due to the correction or prevention of some age damage.

Cycloheximide induces synchronous swelling of perialgal vacuoles enclosing symbiotic Chlorella vulgaris and digestion of the algae in the ciliate Paramecium bursaria.

Cycloheximide is known to inhibit preferentially protein synthesis of symbiotic Chlorella of the ciliate Paramecium bursaria, but to hardly host protein synthesis. Treatment of algae-bearing Paramecium cells with cycloheximide induces synchronous swelling of all perialgal vacuoles that are localized immediately beneath the host's cell membrane. In this study, the space between the symbiotic algal cell wall and the perialgal vacuole membrane widened to about 25 times its normal width 24 h after treatment with cycloheximide. Then, the vacuoles detached from beneath the host's cell membrane, were condensed and stained with Gomori's solution, and the algae in the vacuoles were digested. Although this phenomenon is induced only under a fluorescent light condition, and not under a constant dark condition, this phenomenon was not induced in paramecia treated with cycloheximide in the light in the presence of the photosynthesis inhibitor 3-(3,4-dichlorophenyl)-1,1-dimethylurea. These results indicate that algal proteins synthesized in the presence of algal photosynthesis serve some important function to prevent expansion of the perialgal vacuole and to maintain the ability of the perialgal vacuole membrane to protect itself from host lysosomal fusion.

Mendelian and non-mendelian mutations affecting surface antigen expression in Paramecium tetraurelia.

A screening procedure was devised for the isolation of X-ray-induced mutations affecting the expression of the A immobilization antigen (i-antigen) in Paramecium tetraurelia. Two of the mutations isolated by this procedure proved to be in modifier genes. The two genes are unlinked to each other and unlinked to the structural A i-antigen gene. These are the first modifier genes identified in a Paramecium sp. that affect surface antigen expression. Another mutation was found to be a deletion of sequences just downstream from the A i-antigen gene. In cells carrying this mutation, the A i-antigen gene lies in close proximity to the end of a macronuclear chromosome. The expression of the A i-antigen is not affected in these cells, demonstrating that downstream sequences are not important for the regulation and expression of the A i-antigen gene. A stable cell line was also recovered which shows non-Mendelian inheritance of a macronuclear deletion of the A i-antigen gene. This mutant does not contain the gene in its macronucleus, but contains a complete copy of the gene in its micronucleus. In the cytoplasm of wild-type animals, the micronuclear gene is included in the developing macronucleus; in the cytoplasm of the mutant, the incorporation of the A i-antigen gene into the macronucleus is inhibited. This is the first evidence that a mechanism is available in ciliates to control the expression of a gene by regulating its incorporation into developing macronuclei.

Short-pulse neodymium:yttrium-aluminium garnet (Nd:YAG 1064nm) laser irradiation photobiomodulates mitochondria activity and cellular multiplication of Paramecium primaurelia (Protozoa).

Few studies exist to explore the potential photobiomodulation (PBM) effect of neodymium:yttrium-aluminium garnet (Nd:YAG) laser irradiation using a flat-top handpiece delivery system. In this study, we explored the photobiomodulation effect of that laser, on Paramecium primaurelia. The parameters for the different study groups were: 0.50W, 10Hz, 100msp, 30J/cm2; 0.75W, 10Hz, 100msp, 45J/cm2; 1.00W, 10Hz, 100msp, 60J/cm2; 1.25W, 10Hz, 100msp, 75J/cm2 and 1.50W, 10Hz, 100msp, 90J/cm2. Our results suggest that only the parameter 0.5W, 10Hz, 100msp, 30J/cm2 positively photobiomodulates the Paramecium cells inducing an increment in oxygen consumption, endogenous ATP synthesis and fission rate rhythm. Applying the laser energy with parameters of 1.25W, 10Hz, 100msp, 75J/cm2 and 1.50W, 10Hz, 100msp, 90J/cm2, induce adverse effect on the Paramecium cells, which protect themselves through the increase in Heat Shock Protein-70 (HSP70). The data presented in our work support our assumption that, when using appropriate parameters of irradiation, the 1064nm Nd:YAG laser with flat-top handpiece could be a valuable aid for effective clinical application of PBM.

An 808-nm Diode Laser with a Flat-Top Handpiece Positively Photobiomodulates Mitochondria Activities.

OBJECTIVE: Photobiomodulation is proposed as a non-linear process. Only the action of light at a low intensity and fluence is assumed to have stimulation on cells; whereas a higher light intensity and fluence generates negative effects, exhausting the cell's energy reserve as a consequence of a too strong stimulation. In our work, we detected the photobiomodulatory effect of an 808-nm higher-fluence diode laser [64 J/cm2-1 W, continuous wave (CW)] irradiated by a flat-top handpiece on mitochondria activities, such as oxygen consumption, activity of mitochondria complexes I, II, III, and IV, and cytochrome c as well as ATP synthesis. MATERIALS AND METHODS: The experiments are performed by standard procedure on mitochondria purified from bovine liver. RESULTS: Our higher-fluence diode laser positively photobiomodulates the mitochondria oxygen consumption, the activity of the complexes III and IV, and the ATP production, with a P/O = 2.6. The other activities are not influenced. CONCLUSION: Our data show for the first time that even the higher fluences (64 J/cm2-1 W), similar to the low fluences, can photobiostimulate the mitochondria respiratory chain without uncoupling them and can induce an increment in the ATP production. These results suggest that the negative effects of higher fluences observed to date are not unequivocally due to higher fluence per se but might be a consequence of the irradiation carried by handpieces with a Gaussian profile.

Age-correlated changes in expression of micronuclear damage and repair in Paramecium tetraurelia.

In Paramecium, age is defined as the number of mitotic divisions which have elapsed since the previous cross-fertilization (conjugation) or self-fertilization (autogamy). As the mitotic interval between fertilizations increases, the percentage of nonviable progeny clones increases. In the current study, resolution of conflicting previous reports on the pattern of increase of death and reduced viability in progeny from aging parent cells is found. Some exautogamous clones exhibit a high mortality at young clonal ages, others show no mortality throughout their life span, but most (73%) show an abrupt increase in the percent death and reduced viability in progeny from cells 50-80 fissions old. Ultraviolet-irradiation-induced micronuclear mutations, repairable by photoreactivation, increased with increased clonal age when monitored by percent death and reduced viability of exautogamous progeny of irradiated cells. Loss of dark repair is considered a contributor to the increased expression of micronuclear mutations with increased clonal age.

Photosensitive signal transduction induces membrane hyperpolarization in Paramecium bursaria.

The protozoan ciliate Paramecium bursaria exhibits membrane hyperpolarization in response to photostimulation, accompanied with an increased swimming speed. The external addition of cyclic nucleotide phosphodiesterase (PDE) inhibitors, either theophylline (1,3-dimethylxanthine) or 3-isobutyl-1-methylxanthin (IBMX), increased in both amplitudes of the membrane hyperpolarization and the increase in swimming speed. Moreover, the addition of membrane permeable cyclic nucleotide analogs, either 8-bromo-adenosine 3',5'-cyclic monophosphate (Br-cAMP) or 8-Br-guanosine 3',5'-cyclic monophosphate (Br-cGMP), increased these amplitudes. On the other hand, the addition of l-cis-diltiazem, known to block the conductance of cyclic nucleotide-gated channels, partially decreased both amplitudes of the membrane hyperpolarization and the increase in swimming speed. An enzyme immunoassay of cellular cyclic nucleotide contents showed that photostimulation induced a rapid increase in adenosine 3',5'-cyclic monophosphate (cAMP), but little increase in guanosine 3',5'-cyclic monophosphate (cGMP), raising the possibility that a rapid increase in cAMP mediates the light-induced hyperpolarization in P. bursaria.

Circadian rhythm of photoaccumulation in Paramecium bursaria.

Populations of Paramecium bursaria cells display a circadian rhythm of photoaccumulation. Both Chlorella-containing cells and Chlorella-free cells exhibit this rhythm. Several other species of Paramecium do not express rhythmic photoaccumulation when tested under the same conditions. In P. bursaria, photoaccumulation rhythms persist in continuous conditions (constant temperature and either continuous light or continuous darkness). The period of this rhythm is "temperature-compensated," with a Q10 of 1.10-1.12. The rhythm can be reset by pulses of light or darkness in a phase-dependent manner. Therefore, an endogenous circadian oscillator controls photoaccumulation behavior in P. bursaria.

Paramecium: a promising non-animal bioassay to study the effect of 808 nm infrared diode laser photobiomodulation.

OBJECTIVE: Photobiostimulation and photobiomodulation (PBM) are terms applied to the manipulation of cellular behavior using low intensity light sources, which works on the principle of inducing a biological response through energy transfer. The aim of this investigation was to identify a laboratory assay to test the effect of an infrared diode laser light (808 nm) on cell fission rate. MATERIALS AND METHODS: Sixty cells of Paramecium primaurelia were divided in two groups of 30. The first group (test group) was irradiated, at a temperature of 24°C, for 50 sec by a 808 nm diode laser with a flat top handpiece [1 cm of spot diameter, 1 W in continuous wave (CW), 50 sec irradiation time, 64 J/cm(2) of fluence]. The second group (control group) received no laser irradiation. All cells were transferred onto a depression slide, fed, and incubated in a moist chamber at a temperature of 24°C. The cells were exposed and monitored for 10 consecutive fission rates. Changes in temperature and pH were also evaluated. RESULTS: The exposed cells had a fission rate rhythm faster than the control cells, showing a binary fission significantly (p<0.05) shorter than unexposed cells. No significant effects of laser irradiation on pH and temperature of Paramecium's lettuce infusion medium were observed. CONCLUSIONS: The 808 nm infrared diode laser light, at the irradiation parameters used in our work, results in a precocious fission rate in P. primaurelia cells, probably through an increase in metabolic activity, secondary to an energy transfer.

Effect of 808 nm Diode Laser on Swimming Behavior, Food Vacuole Formation and Endogenous ATP Production of Paramecium primaurelia (Protozoa).

Photobiomodulation (PBM) has been used in clinical practice for more than 40 years. To clarify the mechanisms of action of PBM at cellular and organism levels, we investigated its effect on Paramecium primaurelia (Protozoa) irradiated by an 808 nm infrared diode laser with a flat-top handpiece (1 W in CW). Our results led to the conclusion that: (1) the 808 nm laser stimulates the P. primaurelia without a thermal effect, (2) the laser effect is demonstrated by an increase in swimming speed and in food vacuole formation, (3) the laser treatment affects endogenous adenosine triphosphate (ATP) production in a positive way, (4) the effects of irradiation dose suggest an optimum exposure time of 50 s (64 J cm(-2) of fluence) to stimulate the Paramecium cells; irradiation of 25 s shows no effect or only mild effects and irradiation up to 100 s does not increase the effect observed with 50 s of treatment, (5) the increment of endogenous ATP concentration highlights the positive photobiomodulating effect of the 808 nm laser and the optimal irradiation conditions by the flat-top handpiece.

Mechanism of action of moderate-intensity static magnetic fields on biological systems.

There is substantial evidence indicating that moderate-intensity static magnetic fields (SMF) are capable of influencing a number of biological systems, particularly those whose function is closely linked to the properties of membrane channels. Most of the reported moderate SMF effects may be explained on the basis of alterations in membrane calcium ion flux. The mechanism suggested to explain these effects is based on the diamagnetic anisitropic properties of membrane phospholipids. It is proposed that reorientation of these molecules during moderate SMF exposure will result in the deformation of imbedded ion channels, thereby altering their activation kinetics. Channel inactivation would not be expected to be influenced by these fields because this mechanism is not located within the intramembraneous portion of the channel. Patch-clamp studies of calcium channels have provided support for this hypothesis, as well as demonstrating a temperature dependency that is understandable on the basis of the membrane thermotropic phase transition. Additional studies have demonstrated that sodium channels are similarly affected by SMFs, although to a lesser degree. These findings support the view that moderate SMF effects on biological membranes represent a general phenomenon, with some channels being more susceptible than others to membrane deformation.

Comparative ultraviolet action spectra (254-320 nm) of five "wild-type" eukaryotic microorganisms and Escherichia coli.

The action spectra of five eukaryotic organisms and the prokaryote, Escherichia coli, were examined over the wavelength range, 254-320 nm. Both the repair competent and three repair defective strains (E. coli, Caenorhabditis elegans, Saccharomyces) were examined. Tetrahymena pyriformis action spectra were performed with and without the excision repair inhibitor caffeine present. Others have observed that lethality, mutation, and the production of pyrimidine dimers show much the same wavelength dependence as DNA absorption. The results presented here demonstrate several action spectra which deviate from the DNA absorption spectra. Ultraviolet sensitization ratios (repair competent/repair defective) were also examined and were shown to change over the wavelength range. These findings suggest that DNA may not be the only important chromophore leading to cell death in the uv wavelength range studied. Since uv-B is of major importance in solar uv damage, these findings may also yield important implications for solar uv studies.

Dihydroxy-carotenoid liposomes inhibit phototoxicity in Paramecium caudatum.

The phototoxic effects of hematoporphyrin derivative, using Paramecium caudatum as a model system, are significantly reduced in the presence of carotenoid-containing liposomes. Multilammelar large or small unilammelar vesicles, containing specific carotenoids, were effective in protecting the organism, whether administered exogenously in the bathing solution, or via incubation of paramecia in starved culture media containing carotenoid liposomes. The effectiveness of the carotenoids as inhibitors of phototoxic effects was found to depend on the mode of administration, with small unilammelar being more effective than multilammelar large vesicles for all carotenoids tested. Small unilammelar vesicles containing the dihydroxy-carotenoids zeaxanthin or astaxanthin afforded the greatest protection in both exogenous and endogenous studies. The results of this study suggest that carotenoid efficacy may be determined, in part, by the environment of the carotenoid molecules.

The photodynamic action of methylene blue on the ion channels of Paramecium causes cell damage.

The photodynamic effects of methylene blue (MB) on wild-type and mutant strains of Paramecium Were studied. From measurements of survival and cell motility under the continuous application of light in the presence of MB, the mutant strains remained alive for about three times longer than the wild-type strain. Although the resting potential of the mutant cells was similar to that of wild-type cells, the continuous photodynamic action shifted the membrane potentials of the mutant and wild-type cells to a depolarized level and a hyperpolarized level, respectively, from that before light application. Under voltage clamping, the mutant cells reduced not only the outward current elicited by the photodynamic action but also the outward tail current elicited by the preceding pulse of hyperpolarization. We conclude that the mutant strain is defective in the activation of Ca(2+)-dependent K+ channels. This defect might cause a reduction in the Ca2+ influx because of the suppression of the membrane hyperpolarization, which results in the elongation of survival time under the photodynamic action.

Influence on cell proliferation of background radiation or exposure to very low, chronic gamma radiation.

Investigations carried out on the protozoan Paramecium tetraurelia and the cyanobacteria Synechococcus lividus, which were shielded against background radiation or exposed to very low doses of gamma radiation, demonstrated that radiation can stimulate the proliferation of these two single-cell organisms. Radiation hormesis depends on internal factors (age of starting cells) and external factors (lighting conditions). The stimulatory effect occurred only in a limited range of doses and disappeared for dose rates higher than 50 mGy/y.

Proliferation kinetics of paramecium tetraurelia in balloon-borne experiments.

Experiments were carried out to demonstrate the effect of cosmic radiation, at a balloon-flight ceiling of about 36,500 m (120,000 ft) on single-cell organism proliferation. Paramecium tetraurelia were placed in air-tight containers and maintained at 25 degrees +/- 0.1 degrees C. Cellular growth was determined by cell count, either after recovery or during the flight, by means of an automatic fixation device. Dosimetry was performed by a tissue equivalent proportional counter and was of about 0.5 mrad/h. Flight ceiling duration ranged from 48 min - 22 h. A secondary stimulating effect of growth rate, preceded by a temporary decrease, was observed after recovery. Because of the high bacterial concentration in the trans-Mediterranean flight culture medium, the temporary drop of the growth rate, due to the radiolysis products, disappears. We consider that the stimulating effect can be the result of enzymatic intracellular scavenging of radiolysis products generated in the cell.