Glycolysis in permeabilized L-929 cells.

Mouse L-929 cells permeabilized with dextran sulphate (DSP cells) carry out glycolysis when supplemented with glucose, ATP and NAD+ in a suitable incubation buffer. Glycolytic rates were linear and generally independent of cell density over the range examined (1 x 10(6)-10 x 10(6) cells/ml). Electron microscopy revealed characteristic changes in DSP cell ultrastructure, notably for nuclei and mitochondria. Some cells lacked plasma membranes, while others appeared intact. In the latter case, estimates of the lesion size in plasma membranes were obtained from volume of distribution studies using 14C-labelled proteins, and infiltration of fluorescein isothiocyanate dextran. The results indicated the presence of lesions large enough to allow globular proteins of about 400 kDa to cross the cell surface. In spite of that, only about 10% of total cell protein exited from DSP cells during a 30 min incubation period. We propose that none of the glycolytic enzymes in DSP cells can exist completely in solution in the 'cytosol', suggesting extensive enzyme organization. The results are interpreted within the broader picture of metabolic organization in animal cells and the nature of the 'cytosol'.     

Structural correlates of imbibitional injury in Typha pollen

The ultrastructure of Typha latifolia pollen was examined as a function of pollen moisture content and incubation temperature, in order to identify possible lesions induced by imbibitional chilling. A syndrome of structural traits was found which characterizes damaged grains. Compared to viable grains, the protoplast of damaged pollen has a higher proportion of its volume occupied by vesicles, and less volume occupied by cytoplasm. Damaged grains also tend to have dilated cisternae of endoplasmic reticulum, larger starch grains and lipid bodies, poorly preserved mitochondria and membranes, and, sometimes, numerous electron-dense globules associated with membranes. The percentage of grains exhibiting this damage syndrome correlates closely with the number of ungerminated grains in most samples, regardless of moisture content or incubation temperature. Injury due to rapid imbibition from the dry state or to imbibitional chilling appear to be similar structurally, regardless of whether the stresses are imposed singly or together. The injury is not confined to one cell component (e.g., mitochondria), but may involve a generalized disruption of membranes. These results suggest that similar stress responses are elicited by imbibition from the dry state and by imbibitional chilling.     

Characterization of a rapid cellular-fractionation technique for hepatocytes. Application in the measurement of mitochondrial membrane potential in situ

A rapid cellular-fractionation technique [ Hoek , Nicholls & Williamson (1980) J. Biol. Chem. 255, 1458-1464] was further characterized by using hepatocytes. Of the mitochondrial marker-enzyme activity, 80% was routinely separated from 71-98% of the total cell activities of marker enzymes for plasma membranes, Golgi-membranes, endoplasmic reticulum, lysosomes and cytosol. The mitochondria were contaminated with 53% of cell nuclei. [3H]Triphenylmethylphosphonium ion (TPMP+) was added to hepatocytes in an attempt to measure cellular transmembrane electrical potentials. After rapid cell fractionation the electrical potential between mitochondria in situ and the incubation medium was found to be 202 mV. This value was slightly increased when hepatocytes were treated with oligomycin, but substantially decreased by oligomycin plus an uncoupler of oxidative phosphorylation. Although estimates of TPMP+ binding were obtained, substantial difficulties prevented the accurate measurement of the electrical potential across the plasma membrane. It is concluded that TPMP+ may be employed to demonstrate the integrity of mitochondria during the fractionation procedures. However, the cation is inadequate for the determination of the separate components of the electrical potential between the mitochondrial matrix and the incubation medium.     

The effect of extracellular Ca and Mg on mitochondrial ultrastructure in isolated intact neurons

The ultrastructural transformations of mitochondria in isolated crayfish neurons were studied after incubation of the cells in saline media containing different Ca2+ and Mg2+ concentrations. Incubation in a 5-fold higher Ca concentration resulted in the swelling of mitochondria that was prevented by the addition of the calcium channel blocker, verapamil. Exposure of the cells to Mg2+-depleted medium induced swelling of all the mitochondria, followed by substantial shrinkage of most of them. The absence of Ca as well as the presence of verapamil in Mg2+-free medium led to the inhibition of mitochondrial swelling and to a strong contraction of the mitochondria after 1 h incubation. The omission of Ca2+ from the saline medium or the addition of Ca2+-ionophore A23187 in the presence of Ca2+ resulted in strong mitochondrial shrinkage. These structural alterations of mitochondria are interpreted as an osmotic response of the inner mitochondrial membranes to changes in their potassium transport, induced by a disturbance in the cellular and mitochondrial Ca2+-Mg2+ homeostasis.     

Nucleoside phosphatase (ATPase) activity in myocardial cells of rat embryos. An electron-microscopic study.

Timed pregnancies were obtained in Sprague-Dawley rats, and cardiac tissues from embryos of days 10, 11, 12, 13, 14 and from newborn rats were used for the cytochemical localization of ATPase activity utilizing a lead phosphate precipitation procedure. Following incubation with ATP as the substrate, granular deposits of reaction product are discernible on the cell membranes of the embryonic myocardium. There is a noticeable decrease in the intensity of reaction product as visualized in the electron micrographs from the 10th day of gestation to the 14th day. No granular reaction product is recognizable in myofibrils, mitochondria or other organelles in the cytoplasm. It appears that there is a selective deposition of the reaction product on the cell membranes or structures derived from it. The intense ATPase activity seen on 10th and 11th days seems to be correlated with the initial appearance of myofilaments and fibrils in the myocardial cells.     

Synthesis of NAADP and cADPR in mitochondria.

Here we investigated whether cADPR and NAADP are synthesized in mitochondria. We found that ADPR-cyclase activity is present in mitochondria. In addition, we describe for the first time synthesis of NAADP in this intracellular organelle. ADPR-cyclase activities (V(MAX)) and NAADP synthesis in mitochondria were about 4-fold lower than that in plasma membranes. Otherwise, ADPR-cyclases in mitochondria and in plasma membranes have similar catalytic properties in terms of apparent K(m) for the substrate NGD and K(i) values for inhibition by dithiotreitol, beta-NAD, and nicotinamide. ADPR-cyclase in plasma membranes and to a lesser degree mitochondrial enzyme, was inhibited by Zn(2+) and Cu(2+); ADPR-cyclase from mitochondria was more stable upon thermal inactivation. CD38 antigen, determined by Western blot, was well-expressed in plasma membranes but was far less so (17-fold less) in mitochondria. The major difference between ADPR-cyclase activity in mitochondria and plasma membranes is that mitochondrial cyclase activity was increased by incubation with nonionic detergents. Conversely, the incubation with phosphatidylinositol-specific phosphodiesterase C (PI-PLC) released ADPR-cyclase activity from plasma membranes, but not from mitochondria. We conclude that ADPR-cyclase in mitochondria and in plasma membranes are both multifunctional enzymes with similar catalytic properties; however, the two ADPR-cyclases differ in the mode of anchoring to the membrane: by glycosylphosphoinositol anchor in plasma membranes and by hydrophobic interactions in mitochondria. In addition, synthesis of NAADP can also be found in intracellular organelles via mitochondria. We propose that independent mitochondrial cADPR and NAADP systems may have an intracrine signaling function that is not dependent on direct input by extracellular hormonal stimuli, but rather responds to changes of intermediary cellular metabolism.     

Micro-managing the executioner: pathogen targeting of mitochondria

Eukaryotic cell viability is largely regulated at the level of mitochondria, with cell death executed by endogenous proteins that act to increase the permeability of the inner and/or outer membranes of these organelles. The gastric pathogen, Helicobacter pylori, can mimic this mechanism by producing the pro-apoptotic toxin, VacA, which was recently demonstrated to (i) localize to mitochondria within epithelial cells, (ii) rapidly transport into mitochondria in vitro, and (iii) induce changes consistent with permeabilization of mitochondrial membranes by a mechanism dependent on cellular entry and toxin membrane channel activity. The targeting of mitochondrial membranes is emerging as a strategy used by pathogenic microbes to control cell viability while circumventing upstream pathways and checkpoints of cell death.     

Mechanism of cadmium-induced cytotoxicity in rat hepatocytes

Exposure of rat hepatocytes to cadmium below 50 μM for a short period (10 min) resulted in cellular acidification. Conversely, exposure to Cd more than 50 μM for a long period (60 min) caused cellular alkalinization accompanied by membrane damage as reflected by decrease in cellular K content and loss of intracellular lactic dehydrogenase. In hepatocytes exposed to 5 μM Cd, a concentration sufficient to induce acidification without cytotoxicity, the metal was preferentially associated with the crude nuclei and cell debris fractions, suggesting an interaction between Cd and cell membranes to cause acidification. Omission of bicarbonate from the incubation medium induced cellular acidification. The presence of Cd in this medium did not potentiate the medium-induced acidification. Mg-ATP (25 μM) induced cellular acidification in relation to an increase in the concentration of cytosolic free Ca. The coexistence of Mg-ATP and Cd at the concentrations which had no effect on cellular pH in the presence of either agants induced cellular acidification. These observations suggest that Cd induced cellular acidification by modulating the process connected with the rise in cytosolic free Ca via interaction with plasma membranes. This acidification had no strong immediate cytotoxic actions but led to subsequent cellular alkalinization accompanied with severe cytotoxicity and membrane breakage.     

Permeabilization of rat hepatocytes with Staphylococcus aureus alpha- toxin

Pathogenic staphylococci secrete a number of exotoxins, including alpha-toxin. alpha-Toxin induces lysis of erythrocytes and liposomes when its 3S protein monomers associate with the lipid bilayer and form a hexomeric transmembrane channel 3 nm in diameter. We have used alpha-toxin to render rat hepatocytes 93-100% permeable to trypan blue with a lactate dehydrogenase leakage less than or equal to 22%. Treatment conditions included incubation for 5-10 min at 37 degrees C and pH 7.0 with an alpha-toxin concentration of 4-35 human hemolytic U/ml and a cell concentration of 13-21 mg dry wt/ml. Scanning electron microscopy revealed signs of swelling in the treated hepatocytes, but there were no large lesions or gross damage to the cell surface. Transmission electron microscopy indicated that the nucleus, mitochondria, and cytoplasm were similar in control and treated cells and both had large regions of well-defined lamellar rough endoplasmic reticulum. Comparisons of the mannose-6-phosphatase and glucose-6-phosphatase activities demonstrated that 5-10 U/ml alpha-toxin rendered cells freely permeable to glucose-6-phosphate, while substantially preserving the selective permeability of the membranes of the endoplasmic reticulum and the functionality of the glucose-6-phosphatase system. Thus, alpha-toxin appears to have significant potential as a means to induce selective permeability to small ions. It should make possible the study of a variety of cellular functions in situ.     

P-Glycoprotein is not present in mitochondrial membranes

Recent reports have indicated the presence of P-glycoprotein in crude mitochondrial membrane fractions, leading to the assumption that P-glycoprotein is present in mitochondrial membranes, and may be involved in transport across these membranes. To determine the validity of this claim, two cell lines overexpressing endogenous P-glycoprotein were investigated. Using various centrifugation steps, mitochondria were purified from these cells and analyzed by Western blot reaction with the anti-P-glycoprotein antibody C219 and organelle-specific antibodies. While P-glycoprotein is present in crude mitochondrial fractions, these fractions are contaminated with plasma membranes. Further purification of the mitochondria to remove plasma membranes revealed that P-glycoprotein is not expressed in mitochondria of the KB-V1 (vinblastine-resistant KB-3-1 cells) or MCF-7(ADR) (adriamycin-resistant MCF-7 cells) cell lines. To further substantiate these findings, we used confocal microscopy and the anti-P-glycoprotein antibody 17F9. This demonstrated that in intact cells, P-glycoprotein is not present in mitochondria and is primarily localized to the plasma membrane. These findings are consistent with the role of P-glycoprotein in conferring multidrug resistance by decreasing cellular drug accumulation. Therefore, contrary to previous speculation, P-glycoprotein does not confer cellular protection by residing in mitochondrial membranes.     

Interrelationships among fluorometric analyses of spermatozoal function, classical semen quality parameters and the fertility of frozen-thawed bovine spermatozoa

Cryopreserved spermatozoa from 8 bulls were used to examine the interrelationships among flow cytometric spermatozoal quality assessments and classical semen quality parameters and nonreturn rate estimates of fertility. The integrity of the sperm cell membrane and the functional capacity of the mitochondria were quantified by flow cytometry after concurrent staining with carboxydimethylfluorescein diacetate (CDMFDA), propidium iodide (PI), and rhodamine 123 (R123). For each sample a total of 10,000 stained spermatozoa were simultaneously quantified for the intensity of their green and red fluorescence. Three straws from each bull were each examined initially and following incubation at 37 degrees C for 3 hours to assess the rate of senescence. The proportion of spermatozoa retaining membrane integrity and having functional mitochondria, as determined by CDMFDA and R123 staining, were compared with classical semen quality assessments (sperm motility, acrosomal status, cellular and head morphology, presence of vacuoles/craters and cytoplasmic droplets) and with fertility (nonreturn to estrus rates). For individual ejaculates nonreturn rates, the range was from 61.8 to 78.8%, whereas the cumulative rates of several ejaculates for each bull ranged from 71.3 to 83.5%. The proportion of spermatozoa with functional membranes and mitochondria were positively correlated with the percentage of spermatozoa with normal morphology (r=0.82; P=0.01) and motility after 4 hours of incubation (r=0.78; P=0.02), but not with the estimates of fertility. The actual number of spermatozoa per straw staining with CDMFDA and R123 after 4 hours of incubation at 37 degrees C was correlated with the percentage of spermatozoa with normal morphology (r=0.73; P=0.04). Multiple regression equations indicated that combinations of semen quality measurements could be useful in estimating fertilizing potential.     

The structural organization of haem synthesis in rat liver mitochondria

1. Anaerobic conditions are normally necessary for incorporation of iron into haems and only ferrous iron is used. After addition of succinate to an incubation mixture containing intact or ultrasonically treated mitochondria, Fe(3+) is used, but only if no inhibitors prevent the transfer of electrons from the mitochondrial respiratory chain to oxygen. 2. A dual-wavelength spectrophotometric assay for ferrochelatase is described that has been used for the continuous assay of incorporation of metal ions into porphyrins. Constants are given for the determination of rates of formation of protohaem and cobalt protoporphyrin, mesohaem, cobalt mesoporphyrin and zinc mesoporphyrin. For cobalt mesoporphyrin formation the K(m) for Co(2+) is 11x10(-6)m and that for mesoporphyrin is 5x10(-6)m. 3. An improved method for the separation of inner and outer membranes of mitochondria is described. Mitochondria swollen in hypo-osmotic media were contracted in hyperosmotic potassium chloride solution containing ATP and the outer membranes detached by mild ultrasonic treatment. Sucrose inhibited the ATP-induced contraction and decreased the yield of outer membranes. 4. Ferrochelatase is associated with cytochrome oxidase, which is used as a marker for inner mitochondrial membranes. 5. By using as substrate porphyrin dissolved in phospholipid micelles, ferrochelatase activity of intact mitochondria was shown to be latent, and to be liberated by ultrasonic treatment. 6. No ferrochelatase was detectable in microsomes or soluble cell components.     

Localization in the cell of the protein forming ATP-dependent potassium-selective channels in a bilayer lipid membrane. An ultrastructural study

The localization in cell of the protein forming in BLM the ATP-dependent potassium-selective channels was studied. The electron-microscopic investigation of rat liver and heart tissue sections after their incubation with Abs against the studied protein and visualization of the protein with secondary Abs conjugated with colloid gold were carried out. The colloid gold particles were observed both in mitochondrial membranes and in membranes of endoplasmic and sarcoplasmic reticulum. In heart mitochondria these particles were significantly greater then in liver mitochondria. The detection of the channel-protein localization both in mitochondria and reticulum, as well as structural similarity between the mitochondrial channel and the precursor of calreticulin suggests that the channel protein belongs to the calreticulin family. The possible function of the studied protein as a channel subunit of the mitochondrial ATP-dependent potassium channel is discussed.     

Localization in a cell of a protein forming the ATP-dependent potassium-selective channels in the bilayer lipid membrane. An ultrastructural study

The localization in the cell of the protein forming the ATP-dependent potassium-selective channels in the bilayer lipid membrane has been studied. The electron microscope investigation of rat liver and heart tissue sections after their incubation with Abs against this protein and the visualization of the protein with secondary Abs conjugated with colloid gold were carried out. Colloid gold particles were observed both in mitochondrial membranes and in membranes of endoplasmic and sarcoplasmic reticulum. In heart mitochondria, these particles were significantly greater than in liver mitochondria. The localization of the channel protein both in mitochondria and reticulum, as well as the structural similarity between the mitochondrial channel and the precursor of calreticulin suggest that the channel protein belongs to the family of calreticulins. The possible function of the protein as a channel subunit of the mitochondrial ATP-dependent potassium channel is discussed.     

Electron microscopic study of subcapsular cell hyperplasia in the adrenal glands of IQI/Jic mice.

Hyperplastic cells in subcapsular cell hyperplasia (SCH) lesion in adrenal glands of female IQI/Jic mice were examined by electron microscopy. These cells were small and polygonal, and had irregular nuclei, elongated mitochondria with lamellar cristae and dense lipid droplets. While these cells showed different features, some of them had desmosomes and basement membranes, and a few round mitochondria with tubular cristae as endocrine cells. These findings suggest that hyperplastic cells in SCH lesions might originate from endocrine blastemic cells.     

Lipidg Peroxidation in Liver and Ehrlich Ascites Cell Mitochondria

Ehrlich ascites cell mitochondria are highly resistant to lipid peroxidation as compared to liver mitochondria from host animals. Succinate protects mitochondria from peroxidative damage, proteins from crosslinks, enzymes from inactivation of the enzymes and membranes from permeability changes. The sensitivity of Ehrlich ascites cell mitochondrial membranes to lipid peroxidation is significantly increased in sub-mitochondrial particles. Lipid peroxidation in tumour mitochondrial membranes can not be diminished by succinate as effectively as in liver mitochondria. Ascites cell mitochondria seems to be protected very efficiently from peroxidative damage by a glutathione-dependent mechanism.     

Increased Resistance to the Spread of Tobacco Mosaic Virus in Pinto Bean Leaves Caused by Sugar and Light

Ultraviolet light (UV) irradiation increased expansion of TMV lesions in detached Pinto bean primary leaves incubated in darkness. However, if after UV-irradiation the leaves were incubated in the light, no increase in lesion expansion occurred. The light effect was considered not to be due to photorepair of UV damaged DNA, since non-photorepairing treatments such as incubation in red light, or delayed exposure to white light after UV irradiation also prevented increase in lesion expansion. The effect of visible light in preventing TMV-lesion enlargement was shown to be related to photosynthetic energy supply to the host cell defense mechanism since incubation of infected leaves in the presence of the photosynthesis inhibitor 3-(3,4-dichlorophenyl)-l,l-dimethyl urea (DCMU) in light caused large lesions whether leaves were irradiated by UV or not. Supplying 0.1 M sucrose in the dark also inhibited lesion enlargement in UV-irradiated or nonirradiated leaves. Dinitrophenol (DNP) negated the sucrose effect in the dark. However, in light incubation, DNP did not induce large lesions indicating that DNP did not interfere with energy supply in the light. It is concluded that the Pinto bean leaf cells can use energy derived both from mitochondria and chloroplasts for building the resistance mechanism to virus spread. In this case, cellular resistance to virus spread seems to be correlated with callose deposition on the walls of noninfected cells adjacent to the necrotic cells. Energy supply in various forms will assist host cells in building the resistance mechanism as well as retarding senescence. Detachment, prolonged dark incubation, or exogenous supply of DNP led to accelerated senescence which in turn led to secondary enlargement of lesions. The cause of such secondary enlargement may be explained by starvation of cells and disappearance of callose.     

Über hydrophobe Acridinfarbstoffe zur Fluorochromierung von Mitochondrien in lebenden Zellen

Summary The hydrophobic fluorescence dye 10-n-nonyl-acridinium-orange-chloride, NAO, stains specifically the mitochondria of living HeLa-cells. A dye concentration of 1·10^–8 M is sufficient for vital staining and at 5·10^–7 M an incubation time less than 1 min is enough to generate the bright green fluorescence of the mitochondria. The retention of NAO by the mitochondria is longer than 7 days.The dye accumulation is not affected by the ionophores valinomycin, nigericin, gramicidin, the uncoupling agents DNP, CCCP or by ouabain. In contrast to Rh 123 the trans-membrane potential is not the driving force of the NAO accumulation. We assume that NAO is bound to the hydrophobic lipids and proteins in the mitochondrial membranes by hydrophobic interaction.With valinomycin, 500 ng/ml, 10 min, the mitochondria in HeLa-cells swell. Now it is possible to observe some details in the enlarged mitochondria by light microscopy. After vital staining with NAO, 5·10^–7 M, 10 min, the periphery of the swollen mitochondria shows an intense green fluorescence, the inner part is dark. Obviously the dye is bound to the membranes. By electron microscopy it can be shown that the valinomycin treated and NAO stained mitochondria have outer and inner membranes and cristae. They differ from untreated mitochondria mainly in the size.After incubation of the HeLa-cells with relatively high NAO concentrations, 5·10^–6 M, 10 min, the mitochondria show a weak orange fluorescence. It is generated by the dimers D of NAO. Therefore the dye concentration in the mitochondrial membranes is locally very high and causes dye dimerisation. The weak orange fluorescence is instable and disappeares within a few seconds. Instead we observe a green fluorescence with growing intensity that is generated by the monomers M of NAO. The intensity has its maximum value after a few seconds. Using low NAO concentrations for incubation, 1·10^–7 M, 10 min, we observe only the green fluorescence with increasing intensity. In this case the orange fluorescence is too weak for observation (concentration quenching). It can be shown by experiments and quantum mechanics that the orange fluorescence is assigned to an optical forbidden, the green fluorescence to an allowed electronic transition of D or M respectively. Our results indicate a dissoziation of D in 2 M by irradiation of the mitochondria under the fluorescence microscope.The intensity changes of the orange and the green fluorescence of bound D and M by irradiation has been measured in living cells with a microspectrophotometer. The experimental data agree quantitatively with a first-order reaction mechanism for the dissoziation of D in 2 M by irradiation. There is some evidence for energy transfer between dimers at higher NAO concentration.The oxygen consumption of HeLa-cell suspensions has been measured electrochemically at various NAO concentrations and incubation times with an oxygen electrode. Up to 5·10^–7 M NAO, 10 min, the respiratory activity is not affected. After that we observe an increasing inhibition of the oxygen consumption with growing NAO concentration and incubation time. At 5·10^–6 M, 30 min, the inhibition is 40% relative to the untreated cells.The ultrastructure of the mitochondria in incubated HeLa-cells has been investigated by electron microscopy and compared with untreated cells. Similar to the resiratory experiments there is no difference in ultrastructure up to 5·10^–7 M NAO, 10 min. Then the ultrastructure changes rapidly with increasing NAO concentration and incubation time. At the final stage, 5·10^–6 M, 1 h, the cristae totally or partially disappeared. The outer and inner membranes are still visible. Obviously the mitochondria without cristae are instable and collapse. They change into liposomes with stacks of four, eight and more membranes on the periphery. They enclose cytoplasm. The genesis of the liposomes is discussed in some detail.These experiments show that the dye NAO is accumulated at the inner mitochondrial membrane and the cristae. It blocks the enzymes of the oxydative phosphorylation in the inner membranes and affects the self-organization of the cristae. NAO is specifically bound to the membranes of the mitochondria. Neither by fluorescence microscopy nor by electron microscopy we observe binding of NAO to the membranes of the nuclei.     

低温下转昆虫抗冻蛋白基因烟草的亚显微结构变化

分析在-1℃温度中处理1、2和3d的转昆虫抗冻蛋白基因MpAFP149烟草和野生型烟草亚细胞显微结构变化的结果表明,转基因烟草和野生型烟草的超微结构有差异,尤其是细胞膜、叶绿体和线粒体的膜。转基因烟草细胞器的膜结构伤害程度明显低于野生型烟草,这提示低温条件下转基因烟草中的抗冻蛋白可能有保护细胞膜和细胞器膜结构的作用,由此降低了低温的伤害。     

AN ELECTRON MICROSCOPE STUDY OF VITALLY STAINED SINGLE CELLS IRRADIATED WITH A RUBY LASER MICROBEAM

An electron microscope study has been made of vitally stained single cells whose cytoplasm has been subjected to a localized ruby laser microbeam. Light and moderate laser absorption (the resultant of stain concentration and laser energy density) produced restricted selective damage of mitochondria in cells stained with Janus green B; heavy laser absorption resulted in mitochondrial damage, as well as in nonselective interaction with other cell structures. With four other basic vital stains, the polysomes, ergastoplasm, mitochondria and other organelles at the irradiated site were uniformly damaged. Unstained cells showed no morphological alterations. With light primary damage (that restricted to the irradiation site), no secondary effects of the incident radiation were observed. With moderate primary damage, however, secondary damage of the mitochondria in the unirradiated cell portions was produced, which was reversible within 4 hr after irradiation. Heavy primary lesions caused severe secondary alteration of all cell structures that was irreversible and cell death occurred within 2 hr. Surviving cells examined 24 hr after light and moderate irradiation could not be distinguished from unirradiated controls. The possible mechanisms involved in the production of laser-induced cellular alterations are discussed.