Low-energy visible light (LEVL) has been shown to stimulate cell functions. This is called "photobiostimulation" and has been used successfully over the last three decades for treating a range of conditions, including soft tissue injuries, severe wounds, chronic pain, and more. Nevertheless, the mechanism of photobiostimulative processes is still being debated. It is obvious that, in order to interact with the living cell, light has to be absorbed by intracellular chromophores. In a search for chromophores responsible for photobiostimulation, endogenous porphyrins, mitochondrial and membranal cytochromes, and flavoproteins were found to be suitable candidates. The above-mentioned chromophores are photosensitizers that generate reactive oxygen species (ROS) following irradiation. As the cellular redox state has a key role in maintaining the viability of the cell, changes in ROS may play a significant role in cell activation. In the present review, we summarize evidence demonstrating that various ROS and antioxidants are produced following LEVL illumination. We found that very little evidence for NO formation in illuminated non-vascular smooth muscle cells exists in the literature. We suggest that the change in the cellular redox state which plays a pivotal role in maintaining cellular activities leads to photobiostimulative processes.

Low energy visible light (LEVL) irradiation has beenshown to exert some beneficial effects on various cell cultures. For example, it increases the fertilizing capability of sperm cells, promotes cell proliferation, induces sprouting of neurons, and more. To learn about the mechanism of photobiostimulation, we studied the relationship between increased intracellular calcium ( Life on earth is entirely dependent upon the interaction of sunlight with cells especially in plant photosynthesis (1). Sunlight also has medical benefits, which have been exploited for over thousands of years in ancient Egypt, India, and China in treating skin diseases, psoriasis, vitiligo, and even cancer (2). Recent observations show that even low energy visible light (LEVL) 1 can serve as a medical tool. For example, LEVL increases the rate of wound healing (3), enhances the fertilizing capability of sperm cells (4), and increases the rate of healing bone defects (5). In vitro studies have found that LEVL increases proliferation of cells as fibroblasts (6), keratinocytes (7), and lymphocytes (8) and induces the respiratory burst in neutrophils (9). The mechanism of photobiostimulation by LEVL is still unclear. It has been suggested that reactive oxygen species (ROS), which can be produced by photosensitization of endogenous cell chromophores such as cytochromes (10) [Ca 2ϩ ] i followed by myosin phosphorylation and cell contractions (26). Growth factors and hormones were shown to stimulate ROS production, which were dependent on [Ca 2ϩ ] i rise (27). The relationship between ROS and [Ca 2ϩ ] i has been suggested to involve the redox-sensitive transcription factor N␤, which was found to change [Ca 2ϩ ] i homeostasis in response to changes in the redox state of thiol groups (28). The kinetics that characterize the [Ca 2ϩ ] i elevation have been shown to be an important parameter determining the kind of signal that will be evoked. Livingston et al. (29) showed that high concentrations of oxidants (Ͼ50 M) caused a sustained increase in [Ca 2ϩ ] i , whereas a transient increase in [Ca 2ϩ ] i was observed following administration of a low concentration of oxidants. More than a 4-fold increase in the [Ca 2ϩ ] i level was obtained in photodynamic treatment of mouse myeloma cells that had been enriched with exogenous photosensitizers before illumination, whereas only a slight increase in [Ca 2ϩ ] i was observed in irradiated cells without exogenous photosensitizers (30).Only a few works have dealt with calcium/ROS changes following LEVL irradiation. In this study, we have investigated * The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.§ The article describes research performed by Ronit Lavi as partial fulfillment of the requirements for her Ph.D. degree at Bar-Ilan University. To whom correspondence should be addressed. Tel.: 972-3-5317797; Fax: 972-3-...

A small, water soluble photosensitizer active only at wavelengths shorter than 500 nm is consistent with a flavin, which reproduces cellular signals at physiological concentrations. Thus, flavins are responsible for the photosensitization of the observed oxyradicals in cells.

Site-directed mutagenesis provides a straightforward means of creating specific targets for chemical modifications of proteins. This capability enhanced the applications of spectroscopic methods adapted for addressing specific structural questions such as the characterization of partially folded and transient intermediate structures of globular proteins. Some applications such as the steady state or time-resolved fluorescence resonance energy transfer (FRET) detection of the kinetics of protein folding require relatively large quantities (approximately 10-100 mg) of site-specific doubly labeled protein samples. Engineered cysteine residues are common targets for labeling of proteins. The challenge here is to develop methods for selective modification of one of two reactive sulfhydryl groups in a protein molecule. A general systematic procedure for selective labeling of each of two cysteine residues in a protein molecule was developed, using Escherichia coli adenylate kinase (AKe) as a model protein. Potential sites for insertion of cysteine residues were selected by examination of the crystal structure of the protein. A series of single-cysteine mutants was prepared, and the rates of the reaction of each engineered cysteine residue with a reference reagent [5,5'-dithiobis(2-nitrobenzoic acid) (DTNB)] were determined. Two-cysteine mutants were prepared by selection of pairs of sites for which the ratio of this reaction rate constant was high (>80). The conditions for the selective labeling reaction were optimized. In a first cycle of labeling, the more reactive cysteine residue was labeled with a fluorescent probe (donor). The second probe was attached to the less reactive site under unfolding conditions in the second cycle of labeling. The doubly and singly labeled mutants retained full enzymatic activity and the capacity for a reversible folding-unfolding transition. High yields (70-90%) of the preparation of the pure, site-specific doubly labeled AK mutant were obtained. The procedure described herein is a general outline of procedures, which can meet the double challenge of both site specificity and large-scale preparation of doubly labeled proteins.

It was concluded that the stimulation of alpha 2 adrenoceptors with UK14,304 produced a dilation in the rat middle cerebral artery that (1) was dependent on intact endothelium, (2) involved nitric oxide, and (3) acted via a pertussis toxin-sensitive G protein.