Emission of velocity-correlated clusters in fullerene-solid single collision and diagnostics of the impact energized subsurface nanovolume.

We have measured kinetic energy distributions (KEDs) of large clusters emitted from five different solid targets following a single impact of C60 - ion at 14 keV kinetic energy. It was found that all the large clusters emitted from a given target move with nearly the same velocity and that their KEDs can be described by a thermal distribution riding on a common center-of-mass velocity (shifted Maxwellian) of some precursor. This behavior is in sharp contrast to that observed when the incoming projectile ion is monoatomic. Different trends were observed when comparing the behavior of the KED families of group 5 early transition metal elements (Ta and Nb) with those of group 11 late transition metals (Cu, Ag, and Au). We propose a model for the initial phase of formation of the precursor and show that the measured KEDs can serve as both pressure and temperature probes for the impact excited, highly energized subsurface nanovolume, driving the ejection of the clusters. It is also shown that under the proposed impact scenario, thermally equilibrated conditions (of the atomic subsystem) can be established at the subsurface nanovolume on the early subpicosecond time scale relevant for the emission process. This conclusion is demonstrated both experimentally by the KEDs of the emitted large clusters (very high temperatures and center-of-mass velocity) and by molecular dynamics simulation of the temporal evolution of the thermal characteristics of the impact energized subsurface nanovolume.

Tin-carbon clusters and the onset of microscopic level immiscibility: Experimental and computational study.

We report the experimental observation and computational analysis of the binary tin-carbon gas phase species. These novel ionic compounds are generated by impact of C60(-) anions on a clean tin target at some kiloelectronvolts kinetic energies. Positive Sn(m)C(n)(+) (m = 1-12, 1 ≤ n ≤ 8) ions were detected mass spectrometrically following ejection from the surface. Impact induced shattering of the C60(-) ion followed by sub-surface penetration of the resulting atomic carbon flux forces efficient mixing between target and projectile atoms even though the two elements (Sn/C) are completely immiscible in the bulk. This approach of C60(-) ion beam induced synthesis can be considered as an effective way for producing novel metal-carbon species of the so-called non-carbide forming elements, thus exploring the possible onset of molecular level miscibility in these systems. Sn2C2(+) was found to be the most abundant carbide cluster ion. Its instantaneous formation kinetics and its measured kinetic energy distribution while exiting the surface demonstrate a single impact formation/emission event (on the sub-ps time scale). Optimal geometries were calculated for both neutral and positively charged species using Born-Oppenheimer molecular dynamics for identifying global minima, followed by density functional theory (DFT) structure optimization and energy calculations at the coupled cluster singles, doubles and perturbative triples [CCSD(T)] level. The calculated structures reflect two distinct binding tendencies. The carbon rich species exhibit polyynic/cummulenic nature (tin end capped carbon chains) while the more stoichiometrically balanced species have larger contributions of metal-metal bonding, sometimes resulting in distinct tin and carbon moieties attached to each other (segregated structures). The Sn2C(n) (n = 3-8) and Sn2C(n)(+) (n = 2-8) are polyynic/cummulenic while all neutral Sn(m)C(n) structures (m = 3-4) could be described as small tin clusters (dimer, trimer, and tetramer, correspondingly) attached to a nearly linear carbon chain. For example, the 1:1 (Sn:C) Sn3C3 and Sn4C4 clusters are composed of all-tin triangle and rhombus, correspondingly, with a short carbon chain (C3, C4) attached on top. The cationic Sn3C(n)(+) (n = 1-5) and Sn4C(n)(+) (n = 1-4) species exhibit various intermediate geometries. Structure calculations at the CCSD(T) level are essential since the segregation effect is not as easily evident based on the most stable structures calculated by DFT alone. Dependences of bond energies (per atom) reflect the evolution of the segregation effect. The mass spectral abundances could be reasonably rationalized in terms of calculated stabilities of the cluster ions with respect to various dissociation channels.

The effects of excitatory amino acids and their transporters on function and structure of the distal retina in albino rabbits.

PURPOSE: To study the physiological and pathological roles of excitatory amino acid transporters in the distal retina of albino rabbits. METHODS: Albino rabbits were injected intravitreally in one eye with different doses of L- or D-isomers of glutamate or aspartate, with mixtures of L-glutamate and antagonists to glutamate receptors or with inhibitors of glutamate transporters. The other eye was injected with saline, and served as a control. The electroretinogram (ERG) was recorded 4 h and 2 weeks after injection. At the end of the ERG follow-up period, retinas were prepared for light microscopy. RESULTS: The ERG b-wave was reduced and the a-wave augmented by both isomers of EAAs when tested 4 h after injection. Long-term (2-week) follow-up indicated severe damage to the retina by both isomers of EAAs. Antagonists to glutamate-gated ionic channels failed to protect the rabbit distal retina from permanent damage. Competitive inhibitors of GLAST-1 transporter were highly effective in blocking synaptic transmission in the OPL and in inducing permanent ERG deficit. Selective inhibition of the GLT-1 transporter caused short-term augmentation of the ERG and no permanent ERG deficit. CONCLUSION: GLAST-1, the glutamate transporter of Müller cells, plays a major role in synaptic transmission within the OPL of the rabbit retina. Over-activation of GLAST-1 seems to induce permanent damage to the distal rabbit retina via yet unidentified mechanism.

Early retinal damage in experimental diabetes: electroretinographical and morphological observations.

A growing body of evidence indicates that impairment of retinal function precedes the earliest signs of vascular complications. The aim of this study was to follow the development of retinopathy both functionally and morphologically in a rat model of diabetes mellitus. Diabetes was induced in rats by intravenous injection of streptozotocin (STZ). Age-matched rats raised under similar conditions served as control. The electroretinogram (ERG) was recorded in order to assess retinal function. The expression of glial fibrillary acidic protein (GFAP) in Müller cells was used as a cellular marker for retinal damage. The ERG responses of the diabetic rats were reduced in amplitude compared to the responses recorded from the control rats as early as 2 weeks after onset of diabetes. The b-wave was more affected than the a-wave. GFAP expression in the diabetic retina did not differ from that in the control retina during the first 5 weeks of diabetes. GFAP was demonstrated only in astrocytes in the vitreo-retinal border. After 6-7 weeks of diabetes, GFAP expression in the retinas of the diabetic rats was also detected in the endfeet of the Müller cells. With the progression of diabetes, GFAP expression spreads throughout the entire length of the Müller cells. In the retinas from control rats, GFAP expression was limited to astrocytes and was not detected in Müller cells even at 40 weeks of follow-up. The observations indicate that the functional integrity of retinal cells is compromised already at short time intervals after onset of experimental diabetes in rats.

Retinal toxicity of gentamicin after subconjunctival injection performed adjacent to thinned sclera.

OBJECTIVE: To investigate the potential toxicity to the retina of gentamicin injected near surgically thinned scleral areas in a rabbit model. DESIGN: Experimental study. METHODS: Scleral scraping to half thickness was performed in the superotemporal scleral area in both eyes of adult rabbits (n = 10). Gentamicin sulfate was injected subconjunctivally to the right eye and saline to the left eye, which always served as a control eye. Four weeks after the procedure, electroretinography (ERG) was performed to assess retinal function. Then, the eyes were enucleated and prepared for histologic evaluation of structural damage. In four eyes of two additional rabbits, vitreous gentamicin concentrations were measured using a fluorescence polarization assay. MAIN OUTCOME MEASURES: Dark- and light-adapted ERG responses and histopathologic damage. RESULTS: Dark- and light-adapted ERG responses in all rabbits were similar in the experimental and control eyes. Gentamicin levels were more than 10 microg/ml after subconjunctival injection of gentamicin with scraping and 0.29 microg/ml after subconjunctival injection of gentamicin with no scraping. Histopathologic examination revealed significant local damage to the photoreceptors adjacent to the area of scraping and subconjunctival injection. A significantly lesser degree of damage was seen if gentamicin was injected in pigmented rabbits or in albino rabbits, but only 4 weeks after scleral scraping. CONCLUSIONS: Increased penetration of gentamicin through thinned sclera may lead to toxic levels of the drug in a localized area adjacent to the site of injection. These toxic effects are also influenced by the degree of pigmentation and acute inflammation.

NADPH diaphorase activity in the rabbit retina is modulated by glutamatergic pathways.

NADPH diaphorase activity in the rabbit retina is modulated by the state of visual adaptation. In this study, we tested possible glutamatergic control of this phenomenon. Rabbits were injected intravitreally with agonists and antagonists of glutamate. After adaptation (3 hours) to either room light or darkness, the rabbits were killed and the retinae were prepared for NADPH diaphorase histochemistry. Kainic acid significantly reduced the number of NADPH diaphorase amacrine cells but augmented NADPH diaphorase activity in horizontal cells in both light- and dark-adapted animals. 6,7-Dinitroquinoxaline-2,3(1H,4H)-dione exerted no effect on amacrine cells but eliminated NADPH diaphorase activity in horizontal cells. 2-Amino-4-phosphono butyric acid did not affect NADPH diaphorase activity in horizontal cells but reduced the degree of staining in the neuronal processes of amacrine cells. MK-801 and N-methyl-D-aspartic acid (NMDA) had no effect on NADPH diaphorase activity in horizontal cells. However, MK-801 reduced staining in the neuronal processes of amacrine cells but not in their cell bodies. NMDA effects were expressed in a significant reduction in the number and size of amacrine cells that were NADPH diaphorase positive. These results indicate that activation of NADPH diaphorase in horizontal cells by darkness is mediated by the activation of alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA)/kainic acid (KA)-type glutamate receptors. The ON pathway in the retina is probably involved in modulation of NADPH diaphorase in the neuronal processes of amacrine cells. Amacrine cells that are NADPH diaphorase positive contain NMDA-type and AMPA/KA-type receptors and are highly susceptible to NMDA and kainic acid toxicity.

Development of laser-induced retinal damage in the rabbit.

BACKGROUND: Laser lesions may induce retinal damage that is larger than expected from the size of the coagulated area. This study was designed to follow the development of laser-induced reduction in retinal function and to correlate it with structural changes. METHODS: Pigmented rabbits were treated in one eye with 225 argon laser lesions. The ERG responses were recorded at different times after treatment. The effect of the laser treatment upon the functional integrity of the retina was assessed from the ERG responses. Structural damage was examined by light microscopy. RESULTS: Shortly (1-2 h) after laser treatment, the ERG responses were reduced by about 50%. ERG deficit continued to develop and reached a maximal level about 24 h after treatment. Thereafter, slow recovery was observed but permanent deficit, relative to the initial laser effect, was seen even 30 days after treatment. Histological observations indicated extensive serous retinal detachment between laser lesions that developed within 24 h after treatment. At 30 days post-treatment, lesioned areas were completely destroyed and heavily pigmented. The retina between lesions was attached to the pigment epithelium but exhibited different degrees of structural damage. CONCLUSIONS: The immediate laser damage is confined to the coagulated areas while secondary functional damage develops within 24 h and probably reflects serous retinal detachment between lesions. The serous retinal detachment completely resolves with time but may induce permanent structural abnormalities in non-coagulated retinal areas that is reflected in a functional deficit larger than the initial laser effect.

NADPH diaphorase activity in mammalian retinas is modulated by the state of visual adaptation.

NADPH diaphorase histochemistry is commonly used to identify cells containing nitric oxide synthase (NOS), the enzyme catalyzing the production of nitric oxide from L-arginine. NADPH diaphorase activity and NOS immunostaining was demonstrated in different cells of the vertebrate retina; photoreceptors, horizontal cells, amacrine cells, ganglion cells, and Müller cells. However, the physiological role of nitric oxide (NO) in the retina has yet to be elucidated. In this study, we tested the assumption that NADPH diaphorase activity in the retinas of rabbits and rats depended on the state of visual adaptation. In the rabbit, light adaptation enhanced NADPH diaphorase activity in amacrine cells and practically eliminated it in horizontal cells. Dark adaptation induced the opposite effects; the NADPH diaphorase activity was reduced in amacrine cells and enhanced in horizontal cells. Retinas from eyes that were injected intravitreally with L-glutamate exhibited a pattern of NADPH diaphorase activity that was similar to that seen in dark-adapted retinas. In rats, the NADPH diaphorase activity of amacrine and horizontal cells exhibited adaptation dependency similar to that of the rabbit retina. But, the most pronounced effect of dark adaptation in the rat's retina was an enhancement of NADPH diaphorase activity in Müller cells, especially of the endfoot region. Assuming that NADPH diaphorase activity is a marker for NOS, these findings suggest that NO production in the mammalian retina is modulated by the level of ambient illumination and support the notion that NO plays a physiological role in the retina.

Ocular pigmentation protects the rabbit retina from gentamicin-induced toxicity.

PURPOSE: This study was designed to investigate the possibility that gentamicin-induced retinal toxicity is dependent on ocular pigmentation by comparing the effects of the drug on the functional and morphologic integrity of the retina in albino and pigmented rabbits. METHODS: In each rabbit, a solution of gentamicin sulfate was injected into the vitreous of one eye, and saline was injected into the other eye. Retinal function was assessed by electroretinogram (ERG) at different time intervals after injection. Retinal structure was examined at the light microscopic level. RESULTS: In albino and pigmented rabbits, functional retinal damage developed to a maximal level within the first week after gentamicin injection. Thereafter, gradual recovery could be seen in eyes that suffered less than 80% maximal reduction in the ERG b-wave. For each dose >0.1 mg studied, retinal damage was more severe in the albino rabbits than in the pigmented ones. The degree of damage was not affected by the level of ambient illumination, nor was it reduced by the administration of N-acetylcystein, a free radical scavenger, together with gentamicin. CONCLUSIONS: Ocular pigmentation partially protects the rabbit retina from the toxic action of gentamicin. This protection probably reflects binding of the drug by the melanin, which thereby reduces the concentration of the free gentamicin. When the initial gentamicin-induced retinal damage is expressed in < 80% reduction in the ERG, substantial recovery may occur in both strains of rabbits.

The effects of lidocaine and bupivacaine on the rabbit retina.

The toxic action of two commercial anesthetics, lidocaine and bupivacaine, on the functional and morphologic integrity of the retina was investigated in albino and pigmented rabbits. The experimental drug was injected into the vitreous of one eye, while saline solution was injected into the fellow eye. Retinal function was assessed from the electroretinogram and the visual evoked potential. Retinal structure was examined at the light microscopic level. Ten milligrams of lidocaine did not affect the electroretinogram and the visual evoked potential responses, though structural damage could be detected close to the site of injection. A lower dose of 5 mg did not produce any detectable physiologic or morphologic damage. The only dose of bupivacaine used, 0.5 mg, was not toxic to the albino and pigmented rabbit retinas, as assessed by the electroretinogram, visual evoked potential, and light microscopy. The results of this study demonstrate that lidocaine and bupivacaine are nontoxic to the rabbit retina at concentrations that are effective for retrobulbar anesthesia.

Drug-induced retinal toxicity in albino rabbits: the effects of imipenem and aztreonam.

PURPOSE: To test the toxic action of two antibiotics, imipenem and aztreonam, on the functional and morphologic integrity of the albino rabbit retina. METHODS: Two commercial drugs were used--Tienam, which contains imipenem, and Azactam, which contains aztreonam. Different doses of these drugs were injected intravitreally. Retinal function was assessed from the electroretinogram (ERG) and the visual evoked potential (VEP). Retinal structure was examined at the light microscopic level. RESULTS: Imipenem did not affect the ERG and the VEP responses or the morphology of the retina up to a total injected dose of 0.98 mg (2 mg Tienam). Aztreonam was not toxic to the albino rabbit retina up to a total injected dose of 2.8 mg (5 mg of Azactam). Severe functional and morphologic retinal damage was seen when 10 mg of Azactam was injected. A similar degree of damage was seen when a dose of 5 mg L-arginine, an ingredient of Azactam, was injected into the vitreous. CONCLUSIONS: Imipenem and aztreonam are nontoxic to the albino rabbit retina at concentrations that are 500-fold higher than their effective dose against bacterial infection. Azactam is highly toxic at high levels (more than 10 mg injected into the vitreous). Most of the toxicity could be explained by the L-arginine content of the drug.

The effects of myristyl gamma-picolinium chloride on the rabbit retina: morphologic observations.

PURPOSE: This study was designed to localize the site of action of myristyl gamma-picolinium chloride (MGP) in the rabbit retina and to evaluate the extent of the structural damage induced by the drug. METHODS: The structural damage was assessed at the light microscopic level in eyes treated with various concentrations of MGP at different time intervals after intravitreal injection of the drug. Glial fibrillary acidic protein (GFAP) immunoreactivity was tested in the same eyes and served as an index of retinal damage. RESULTS: The rabbit retinas, examined about 1 mo after MGP injection, exhibited loss of photoreceptors and thinning of the retina in the regions close to the site of injection; remote retinal areas appeared morphologically intact or only slightly affected. Immunocytochemical analysis demonstrated the presence of GFAP in Müller (glial) cells throughout the entire retina. When the effects of MGP were examined at short time intervals (24 and 72 hr) after injection, severe morphologic damage in areas adjacent to the site of drug injection developed in parallel with the electroretinographic findings. However, GFAP could not be demonstrated. CONCLUSIONS: MGP, the preservative used in Depo-Medrol (Upjohn, Kalamazoo, MI), is highly toxic to the rabbit retina.

The effects of Depo-Medrol preservative on the rabbit visual system.

Periocular injections of corticosteroids play an important role in the management of various ophthalmologic diseases. The Depo-Medrol vehicle, injected into the vitreous, was shown to be toxic to the lens and to the retina when applied at double strength. The authors examined the effects of Depo-Medrol and one of the components of its vehicle, myristyl-gamma-picolinium chloride (MGP), on the functional integrity of the rabbit visual system. Visual function was assessed objectively from the electroretinogram (ERG) and the visual evoked potential (VEP). The experimental drugs were injected into the vitreous humor of one eye while saline was injected into the fellow eye for control. Depo-Medrol did not produce any measurable effects on the ERG or the VEP. When MGP solutions were injected in concentrations at least twice as large as that in the Depo-Medrol, significant reductions in the light- and dark-adapted ERG responses were seen. The effects of the drug on the ERG responses was seen as early as 3 days postinjection and developed to its maximal level within 1-2 weeks. No ERG recovery was seen over a period of more than 2 months. The VEP, elicited by applying light stimuli to the experimental eye, was characterized by low amplitude and delayed implicit time compared with the response obtained from the control eye.

Uridine diphosphoglucose dehydrogenase activity in normal and osteoarthritic human chondrocytes.

The activity of uridine diphosphoglucose dehydrogenase (UDPGD), a key enzyme in the synthesis of proteoglycans was measured by a quantitative cytochemical method in normal and in osteoarthritic (OA) human cartilage. Normal adult chondrocytes showed low UDPGD activity, which was about half the activity of glucose-6-phosphate dehydrogenase of the same specimens. No significant increase in UDPGD activity was noted in OA chondrocytes. The lack of significantly enhanced UDPGD activity in OA indirectly agrees with studies showing normal 35S uptake in this disease and might explain in part the inability of chondrocytes to cope with continuous proteoglycan loss.

Photosystem II in Guard Cells of Vicia faba: Immunological Detection.

Antibodies were raised against individual polypeptides of the oxygen-evolving photosystem II (PSII) complex from mesophyll chloroplasts of Vicia faba (Long Pod). These antibodies were used to probe immunologically for the presence of the main structural components of the PSII complex in guard cell chloroplasts, using both immunofluorescence microscopy and Western blotting. Immunofluorescence of epidermal peels with antibodies raised against the extrinsic 33 kilodalton polypeptide, as well as the 47 and the 44 kilodalton subunits and the light-harvesting chlorophyll a/b protein, resulted in intense fluorescence indicating the presence of these polypeptide components in guard cell chloroplasts. Results obtained with Western blot analysis showed that the relative amounts of the 33 kilodalton and light-harvesting complex protein polypeptides are between 60 and 80% of that found in mesophyll cells (on chlorophyll basis). These results provide evidence for the existence of structural components associated with PSII activity in guard cell similar to those of mesophyll chloroplasts.

Immunological evidence for the presence of ribulose bisphosphate carboxylase in guard cell chloroplasts.

Ribulose bisphosphate carboxylase (Rubisco) has been found in Vicia faba L. guard cell chloroplasts by two immunological methods, using antibodies raised against highly purified subunits of ribulose bisphosphate carboxylase. Indirect cytoimmunofluorescence revealed binding of antibodies against both the small and the large subunits of ribulose bisphosphate carboxylase. Binding was observed only after partial digestion of guard cell walls by 4% Cellulysin to facilitate antibody penetration. After electrophoresis of a homogenate of guard cell protoplasts, the presence of both subunits was also revealed by immunolabeling technique. Positive response required the inhibition of proteolysis which appeared to be active upon homogenization.

Antigenic, electrophoretic and binding alterations of human C-reactive protein modified selectively in the absence of calcium.

C-Reactive protein (CRP) is a trace component of normal human serum which has a mol. wt of 105,000 and is composed of five apparently identical subunits arranged in cyclic symmetry. The serum concentration of this protein is known to increase dramatically in response to acute inflammatory or necrotic processes. We report here that in the presence of high concentrations of urea significant antigenic, electrophoretic and binding site modifications of CRP occur selectively in the absence of calcium. CRP treated in this way (designated F-CRP) had a pI of 5.4 and alpha-electrophoretic mobility in contrast to the native molecule which had a pI of 6.4 and gamma-mobility. F-CRP retained a partial antigenic identity to native CRP, displayed decreased intrinsic tryptophan fluorescence, and expressed a new antigenic reactivity. A similar neoantigen was expressed by heating CRP selectively in the absence if calcium (63 degrees C, 5 min). Treatment with guanidinium-HCl or deliberate carbamylation did not produce F-CRP or the expression of the F-antigen. The formation of F-CRP in urea or by heat was prevented by the presence of 0.7 mM or more calcium. CRP treated in this way retained identical characteristics to native CRP. F-CRP chromatographed through Sephadex G-150 in the presence or absence of 6M urea as a protein of apparent mol. wt 75, 000 with no evidence for free CRP subunits. The formation of F-CRP from native CRP resulted in a loss of capacity for calcium-dependent binding to the C-polysaccharide despite the persistence of calcium-independent binding reactivity for polycations. These data suggest that in the presence of sufficient calcium CRP can resist urea- or heat-induced structural denaturation, maintaining antigenic, electrophoretic and binding identity to the native molecule. In the absence of calcium, exposure to urea led to increased electrophoretic mobility and exposure of a new antigenic reactivity, and to alterations in the phosphocholine- but not the polycation-binding sites of the native CRP molecule; this new antigenic reactivity may be of value in further studies on the CRP molecule.

Effect of reduction on the structural, biologic, and immunologic properties of the third component of human complement.

A decrease of the hemolytic function of human C3 results from reduction with sodium borohydride (NaBH4) or with dithiothreitol (DTT) followed by alkylation with iodoacetamide. In the presence of 3.5 x 10(-2) M NaBH4, the loss of hemolytic activity (68%) is paralleled by a loss of C3b binding to EAC14oxy2 cells (67%), immune adherence (71%), and hemagglutinability (74%), but by no loss of C5 convertase function. Sulfhydryl analysis reveals that two disulfide bonds, both presumed to be situated in the C3 alpha subunit, are broken: one at 2.2 x 10(-2) M and one at 3.5 x 10(-2) M NaBH4. A contrasting inactivation profile is observed in the presence of 1.0 x 10(-1) M DTT. Loss of hemolytic function (76%) is paralleled by a decrease of C5 convertase function (87%), whereas C3b binding, immune adherence, and hemagglutinability are either unaffected or not significantly affected. Sulfhydryl analysis indicates the cleavage of four disulfide bonds; three bonds within C3 alpha and beta are broken at 5.0 x 10(-4) M DTT, and one additional bond within C3 alpha at 1.0 x 10(-3) M DTT. Antigenicity as determined by immunoelectrophoresis is unaltered at any of the above concentrations. The implication of these findings is discussed.