Some UV-bystander effects are mediated through induction of antioxidant defense in mammalian cells.

Bystander effect is the communication of signals from irradiated to unexposed neighboring cells which is often mediated through factors released from irradiated cells. We have attempted to investigate whether UV-bystander phenomenon can modulate the sensitivity of A375 cells and its mechanism. For this purpose, the conditioned medium from UVC-irradiated cells, which contained these released factors, was used to treat non-exposed cells. These cells were then subsequently treated with UVC or another genotoxicant H2O2. Cell viability was determined by Trypan blue-exclusion assay, DNA damage by flow cytometry analysis, ROS production by flow cytometry and microscopic analysis. Lipid peroxidation and antioxidant defense were assayed biochemically. Our findings revealed that exposure of non-irradiated cells to these factors induced increased in SOD and catalase activities which reverted to normal levels by 8 h. During this period, the released factors-treated cells were resistant to killing by UVC or H2O2 and induced DNA damage and lipid peroxidation were also lowered. This protection from cell killing was not present 8 h after exposure to these released factors. Our results suggested UV-bystander effect increased viability of cells through induction of antioxidant defense. This indicated UV-bystander phenomenon triggers protective response in cells.

Conservation and divergence of Grb7 family of Ras-binding domains

Ras proteins are signal-transducing GTPases that cycle between inactive GDP-bound and active GTP-bound forms. Ras is a prolific signaling molecule interacting with a spectrum of effector molecules and acting through more than one signaling pathway. The Ras-effector proteins contain a Ras-associating (RA) domain through which these associate with Ras in a GTP-dependent manner. The RA domain is highly conserved among the members of the growth factor receptor-bound (Grb) 7 family of proteins which includes Grb7, Grb10 and Grb14. Our laboratory has reported an unusual observation that RA domain of Grb14 binds to the C-terminal nucleotide binding site of cyclic nucleotide gated channel (CTRCNGA1) and inhibits the channel activity. Molecular modeling of the CTR-CNGA1 displays 50%-70% tertiary structural similarity towards Ras proteins. We named this region as Ras-like domain (RLD). The interaction between RA-Grb14 and RLD-CNGA1 is mediated through a simple protein-protein interaction temporally and spatially regulated by light and cGMP. It is interesting to note that Grb14 binds to GTPase-mutant Rab5, a Ras-related small GTPase whereas Grb10 binds only to GTP-bound form of active Rab5 but not to GTPase-defective mutant Rab5. These results suggest that Grb14 might have been evolved later in the evolution that binds to both Ras and nucleotide binding proteins such as CNGA1. Our studies also suggest that eukaryotic CNG channels could be evolved through a gene fusion between prokaryotic ion channels and cyclic nucleotide binding proteins, both of which might have undergone several sequence variations for functional adaptation during evolution.

Identification of functionally important acidic residues in transducin by group-specific labeling

Transducin (T), a GTP-binding protein involved in phototransduction of rod photoreceptor cells, is a heterotrimer arranged as two units, the alpha-subunit (T alpha) and the beta gamma-complex (T beta gamma). The role of the carboxyl groups in T was evaluated by labeling with N,N'-dicyclohexylcarbodiimide (DCCD) and 1-ethyl 3-(3-dimethylaminopropyl) carbodiimide (EDC). Only a minor effect on the binding of beta, gamma-imido guanosine 5'-triphosphate (GMPpNp) to T was observed in the presence of the hydrophobic carbodiimide, DCCD. Similarly, the GMPpNp binding activity of the reconstituted holoenzyme was not significantly affected when T alpha was combined with DCCD-treated T beta gamma. However, the binding of guanine nucleotides to the reconstituted T was approximately 50 per cent inhibited when DCCD-labeled T alpha was incubated with T beta gamma. In contrast, treatment of T with the hydrophilic carbodiimide, EDC, completely impaired its GMPpNp-binding ability. EDC-modified T was incapable of interacting with illuminated rhodopsin, as determined by sedimentation experiments. However, rhodopsin only partially protected against the inactivation of T. Additionally, analyses of trypsin digestion patterns showed that fluoroaluminate was not capable of activating the EDC-labeled T sample. The function of the reconstituted holoenzyme was also disrupted when EDC-modified T alpha was combined with T beta gamma, and when EDC-treated T beta gamma was incubated with T alpha.

Pupil size following dark adaptation in patients with retinitis pigmentosa

According to the equivalent light hypothesis, molecular defects in the photoreceptor lead to a continuous activation of the photoreceptor cascade in a manner equivalent to real light. The consequences in diseases such as retinitis pigmentosa (RP) are as disruptive to the cells as real light. Two forms of the equivalent light hypothesis can be distinguished: strong - mutations in rhodopsin or other cascade proteins in some forms of RP continuously excite the visual phototransduction cascade; weak - disruption of outer segments in all patients with RP eliminates circulating dark current and blocks neurotransmitter release in a manner similar to real light. Both forms of the equivalent light hypothesis predict that pupils of patients with RP will be constricted like those of normal subjects in the light. The purpose of this study was to test the equivalent light hypothesis by determining whether steady-state pupil diameter following full dark adaptation is abnormally small in any of a sample of patients with RP. Thirty-five patients with RP and 15 normal subjects were tested. Direct steady-state pupillometric measures were obtained from one eye in a full-field dome after 45 min of dark adaptation by videotaping the pupil with an infrared camera. Mean pupil diameter in the dark was comparable (t = -0.15, P = 0.88) between patients with RP (6.85 Ý 0.58 mm) and normal subjects (6.82 Ý 0.76 mm). The results of the present study are clearly counter to the prediction of the second (weaker) form of the equivalent light hypothesis

Resultados das derivaçöes infra-inguinais até segmento isolado de artéria / Results of infra-inguinal by-passes to isolated artery segment

Os autores apresentam sua experiência com 23 casos operados e nos quais foi feita revascularização do membro inferior por meio de derivaçöes em que a anastomose distal foi implantada num segmento isolado de artéria. A perviedade cumulativa no grupo em que foram usadas próteses (7 casos), a taxa foi de 21 'por cento'. Os resultados obtidos quanto à preservação do membro foram elevados, sendo de 81 'por cento' e 87 'por cento' respectivamente para as lesöes estáveis e instáveis.

Safety of intravitreally injected ciprofloxacin in phakic rabbit eyes

This study was designed to determine the maximal safe drug concentration of intravitreal ciprofloxacin in phakic rabbit eyes. Twenty-two eyes of New Zealand pigmented rabbits received midvitreal ciprofloxacin of 100, 200, 400, 600 or 800 microgram in BSS Plus, or BSS Plus only. Retinal toxicity was dose-dependent as determined with electroretinography, light microscopy, and transmission electron microscopy. At a dose of greater than 400 microgram, disorganization of the outer segments was a main pathological finding in transmission electron microscopy. We evaluated retinal function by measuring the electroretinograms for a graded series of flash intensities and by fitting electroretinogram b-wave amplitudes to the Naka-Rushton equation. At a dose of greater than 600 microgram, Rmax was significantly decreased and log K was significantly increased. N-value tended to decrease. A decrease of b-wave amplitudes caused by retinal toxicity could be detected very sensitively with lower luminance stimuli. Determination of retinal toxicity with lower luminance electroretinography revealed a significant decrease of b-wave amplitudes at a dose of greater than 400 microgram. We concluded that a safe dose of intravitreal ciprofloxacin in phakic rabbit eyes was 200 microgram in phakic eyes.

Improved purification of transducin subunits from bovine retinal rod outer segments

Transducin serves as a mediator between the receptor protein, rhodopsin, and the effector protein, cGMP phosphodiesterase, in the visual process. Transducin is a protein composed of three polypeptides: T alpha, T beta, and T gamma, and acts as two functional units, the alpha-subunit and the beta gamma-complex. In the present study, I describe an efficient and fast method of purifying T alpha and T beta gamma using chromatography on a blue agarose column connected in tandem with an omega-amino octylagarose column. The recombination of T alpha and T beta gamma reconstitutes the functional heterotrimeric holoprotein, as demonstrated by the recovery of three native properties of transducin: 1) its capacity to exchange guanine nucleotide, 2) its GTP hydrolytic activity, and 3) the ADP-ribosylation of T alpha catalysed by pertussis toxin

Photobleaching and cyclic GMP dependences of rhodopsin phosphorylation in rod outer segment.

The experimental data on the cGMP decrease under continuous illumination of rod outer segment have been theoretically analysed to study the bleaching and hence the cGMP dependence of the rhodopsin phosphorylation. From the agreement of the theoretical results with the experimental observations it has been found that the rate of phosphorylation depends on the rate of cGMP hydrolysis. If the rate of cGMP hydrolysis increases the rate of phosphorylation also increases. The results of the theoretical treatment predict that (i) the presence of cGMP in rod outer segment inhibits the rhodopsin phosphorylation and (ii) rhodopsin phosphorylation process is much faster than what has been reported in the literature.

Studies on the Cellular Transfer of Autoimmune Uveitis

After the immunization of pigmented guinea pigs with bovine rod outer segments in complete Freund's adjuvant, the regional lymph nodes were excised, teased and sedimented by centrifugation. These fresh lymphocytes were injected intravitreally (a single injection of 10 X 10 cells in 50 micro l) in 18 eyes. The involvement of the ciliary body and the choroid with polymorphonuclear leucoytes and later with round cells was noted in 16 eyes. Destructions of the visual cells of the retina were demonstrated in 8 eyes. When the fresh lymphocytes were injected subconjunctivally (50 X 10 cells in 30 micro l) four times. four of the 8 eyes showed slight infiltrations of the ciliary body and the choroid with round cells. No retinal lesion was found. Following repeated freezing and thawing of the lymphocytes. transfer experiments wereperformed with the nonviable cells and their extracts respectively. In 8 eyes injected intravitreally with nonviable cells. 2 eyes showed the infiltration of the uvea with round cells, whereas. of 6 eyes injected subconjunctivally, only one eye showed such changes. The extracts of lymphocytes were injected intravitreally in 8 eyes of which 5 eyes demon-strated moderate degree of infiltration of the uvea. Of 2 eyes injected subconjunctivally, 2 eyes. showed slight histological changes. As control experiments, lymphocytes taken from animals immunized. only with the adjuvant were treated as before and transferred to normal animals. Only one eye of 3 eyes received single intravitreal injection of fresh cells showed slight degree of infiltration of the uvea. Thus it can be concluded that the experimental autoimmune uveitis in guinea pigs could be transferred to normal animals by the transfer of non-viable lymphocytes and especially by their extracts.

Transfer of Experimental Autoimmune Uveoretinitis by Serum

Passive transfer experiments were performed by local administrations of sera from guinea pigs hyperimmunized by bovine rod outer segments with complete Freund's adjuvant in random-bred guinea pigs. Routes of administrations were(1) single intravitreal injection of serum (25 micrl l, in 18 eyes), (2) the intravitreal injection associated with 2 subconjunctival injections (150 micrl l, in 9 eyes), (3) three subconjunctival injections of serum (150 micrl l, in 6 eyes), (4) five subconjunctival injections of serum with 2 paracenteses (in 14 eyes) and (5) four injections into the anterior chamber (50 micrl l) with 2 subconjunctival injections (in 4 eyes). Clinically animals showed cells in the anterior vitreous in 50-83% of cases. Histologically infltrations of round cells, focal or diffuse, were noted in the choroid with scattered polymorphonuclear, eosino and plasma cells. In the retina disappearance of visual cells was found, sometimes complicated by the involvement of the bipolar cells in severe cases. These findings were quite similar to the lesions produced in actively immunized animals. Choroidal lesions were most frequently found. Serum taken from the enucleated donors, and concentrated serum were more effective in the production of this transfer disese. The control experiments in which normal serum or serum immunized by adjuvant alone, revealed very low incidence of ocular lesions which were generally mild in degree. It can be concluded that humoral immunity also plays very important roles in the pathogenesis of autoimmune uveoretinitis.