By extensively examining the experimental conditions for time-resolved spectrophotometry of non-transparent light scattering systems, we demonstrated the feasibility of quantitative analysis of both the fluorescence lifetime and intensity of reduced pyridine nucleotides in living tissues, suspensions of isolated liver mitochondria, and hepatocytes, as well as hemoglobin-free perfused rat liver being used systematically for measurements. The fluorescence decay was analyzed by the maximum likelihood method with a 4-component decay model. The lifetime of NADH observed in mitochondria (mean: 2.8 +/- 0.2 ns) was much longer than that of the free form in an aqueous solution (mean: 0.43 +/- 0.01 ns), and it was characterized as a protein-bound form. The lifetime was not affected by either aerobic or anaerobic conditions nor by the energy state, though the intensity changed markedly. The decay curves of isolated hepatocytes under normal aerobic conditions were the same as those of isolated mitochondria, though cytosolic NADH and NADPH were superimposed. Under the conditions of "unphysiological" acidosis, the mean lifetime became about 1.5 times longer than that under normal conditions. With perfused liver, the relative contributions of cytosolic NADH and NADPH were determined by infusing lactate and tert-butylhydroperoxide. Cytosolic NADH did not contribute to the overall fluorescence of pyridine nucleotides. In contrast, about 70% of the total fluorescence intensity was due to cytosolic NADPH, but its decay parameters were essentially the same as those of mitochondrial NADH. No free form of either NADH or NADPH was detected in the cytosolic and mitochondrial spaces. We concluded that the changes in fluorescence intensity observed under the various conditions can be simply explained by a change in the amount of reduced pyridine nucleotides in tissues, rather than by changes in the microscopic environment. The wide applicability of time-resolved fluorescence photometry to in vivo studies is well documented.

In this report, we describe a laser-latex combination system that enables membrane-impermeable molecules to penetrate cell membranes. Laser light (Q-switched Nd:YAG laser, 532.5 nm) was used to irradiate a mixture of commercial latex particles (blue dyed, 1 micro m in diameter) and mouse fibrosarcoma (Meth-A) cells. After irradiation, membrane permeability was evaluated by flow cytometric assaying using propidium iodide (PI) and fluorescein diacetate (FDA). The proportion of permeabilized-resealed cells was affected by changes in the light intensity (approximately 780 mW/cm(2)), the irradiation time (approximately 240 s), and/or the particle concentration (approximately 10(9) particles/ml). The permeability persisted up to 20 min after light irradiation. Near the sites of individual particles, the permeability of the cell membrane is modified, probably due to localized temperature changes. These results suggest that this laser-induced permeabilization strategy constitutes a new means of delivering exogenous materials into living cells.

Low-energy laser irradiation has positive effects on bone fracture healing, osteoblast proliferation, bone nodule formation, and alkaline phosphatase activity. However, the mechanism by which low-energy laser irradiation affects bone is not clearly known. It was recently found that light at a low radiation dosage is absorbed by intracellular chromophores. High-intensity pulsed laser irradiation can produce acoustic waves in the target surface by rapidly heating the tissue. We considered that the acoustic waves induced by high-intensity pulsed laser irradiation, in addition to the photochemical effects that are induced, accelerate bone formation. To clarify whether high-intensity pulsed laser irradiation accelerates bone formation, we investigated bone formation in the irradiated femur of rat, using histomorphometric analysis. Rat femurs were irradiated with a Q-switched Nd: YAG laser, which has a wavelength of 1064 nm, under two conditions: once a day, with the average fluence rate set at 100 mW/cm(2) (LA1), and twice a day, i.e., every 12 h, with the average fluence rate set at 50 mW/cm(2) (LA2). The mean bone volume and mineral apposition rate in the LA1 group were significantly higher than those in the nonirradiated group (control). These values were highest for the LA2 group, and were about 1.52 and 1.25-fold those of the control, respectively. These data demonstrated that the number of pulses, rather than the intensity of the laser irradiation, affects bone formation. Thus, this study indicated that high-intensity pulsed laser irradiation accelerates bone formation in the metaphysis. This bone formation induced by high-intensity pulsed laser irradiation might be due to laser-induced pressure waves.

The solution X-ray scattering technique has been applied to examine the conformations of the proteolytic fragment F34 (78Asp-148Lys) of calmodulin in the absence of both Ca2+ and mastoparan, in the presence of Ca2+ only, and in the presence of both Ca2+ and mastoparan. The radius of gyration and the molecular weight for the F34 fragment increased by 1.1 +/- 0.3 A and 19%, respectively, upon binding of both 2 mol of Ca2+/mol to the F34 fragment and mastoparan to form the tertiary complex. A smaller change was found for the Ca(2+)-saturated F34 fragment in the absence of mastoparan (0.3 +/- 0.3 A) without any change of the molecular weight. The analysis based on the small-angle scattering data showed that the F34 fragment in the presence of Ca2+ alone preserved the tertiary structure of the globular domain in the crystal to a great extent. Further analyses based on a two-domain model showed that the center-to-center distance between F34 and mastoparan is about 12.7 A, if the structure of the F34 fragment in the presence of mastoparan resembles that in the absence of mastoparan and if mastoparan in the complex retains an alpha-helical conformation. The modeling studies using their crystal structure coordinates have been made on the basis of the solution X-ray scattering data. The combined results support a model proposed by Persechini and Kretsinger [Persechini, A., & Kretsinger, R. H. (1988) J. Cardiovasc. Pharmacol. 12 (Suppl. 5), S1-S12], although the center-to-center distance between mastoparan and the F34 fragment is shorter by about 5 A than that in their model.(ABSTRACT TRUNCATED AT 250 WORDS)

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