Search

Toxicological and environmental issues are associated with the extensive use of agricultural pesticides, although the knowledge of their toxic effects as commercial formulations is still far from being complete. This work investigated the impact of three herbicides as commercial formulations on the oxidative status of a wild type Saccharomyces cerevisiae strain. With yeast being a well-established model of eukaryotic cells, especially as far as regards the stress response, these results may be indicative of potential damages on higher eukaryotes. It was found that herbicide-mediated toxicity towards yeast cells could be the result of an increased production of hydroperoxides (like in the case of the herbicides Pointer and Silglif) or advanced oxidation protein products and lipid peroxidation (especially in the case of the herbicide Proper Energy). Through a redox-proteomic approach it was found also that, besides a common signature, each herbicide showed a specific pattern for protein thiols oxidation.

Quercetin uptake in Jurkat cells is extremely rapid and associated with a remarkable accumulation of the flavonoid, dependent on its binding to intracellular components. Cell-associated quercetin is biologically active, quantitatively consumed to promote survival in the presence of reactive species, such as peroxynitrite (ONOO(-)), or reduction of extracellular oxidants via activation of plasma membrane oxidoreductases. In alternative, quercetin is very slowly released upon post-incubation in drug-free medium, an event significantly accelerated by extracellular albumin. Quercetin uptake is also observed in isolated mitochondria, resulting in an enormous accumulation of the flavonoid, consumed under conditions associated with prevention of lipid peroxidation induced by ONOO(-). Interestingly, remarkable quercetin accumulation is also detected in the mitochondria isolated from quercetin-pre-loaded cells, and exposure to either ONOO(-) or extracellular oxidants caused the parallel loss of both the mitochondrial and cytosolic fractions of the flavonoid. In conclusion, Jurkat cells accumulate large amounts of quercetin and even larger amounts of the flavonoid further accumulate in their mitochondria. Intramitochondrial quercetin appears to be functional for prevention of mitochondrial damage as well as for redistribution to the cytosol, when the fraction of the flavonoid therein retained is progressively consumed either by cell-permeant oxidants or by activation of plasma membrane oxidoreductases., (Copyright 2010 Elsevier Inc. All rights reserved.)

We investigated the color vision pattern in Cebus apella monkeys by means of electroretinogram measurements (ERG) and genetic analysis. Based on ERG we could discriminate among three types of dichromatic males. Among females, this classification is more complex and requires additional genetic analysis. We found five among 10 possible different phenotypes, two trichromats and three dichromats. We also found that Cebus present a new allele with spectral peak near 552nm, with the amino acid combination SFT at positions 180, 277 and 285 of the opsin gene, in addition to the previously described SYT, AFT and AFA alleles.

Monocytes/macrophages respond to peroxynitrite with the triggering of events leading to prevention of an otherwise prompt lethal response. This survival signaling regulated by molecules of the arachidonate cascade however presents a hypothetical critical limitation. In human promonocytic cell lines, peroxynitrite indeed promotes ryanodine receptor-derived Ca(2+)-dependent mitochondrial formation of H2O2, entirely responsible for the ensuing DNA strand scission. The occurrence of the same events in monocytes/macrophages at the inflammatory sites would therefore enhance the extent of DNA strand scission in viable cells, thereby increasing the rate of mutation and neoplastic transformation. The present study illustrates the details of a novel strategy based on a differentiation-associated loss of expression of ryanodine receptors. These cells simply do not accumulate mitochondrial Ca(2+) in response to peroxynitrite and therefore fail to generate superoxide/H2O2, thereby preserving the integrity of their DNA. We propose that an important component of the overall strategy adopted by monocytes/macrophages to survive to peroxynitrite, with no increased risk of neoplastic transformation, involves down-regulation of ryanodine receptor expression.

Rat astrocytes accumulate extensive DNA single-strand breakage in response to agents promoting activation of NADPH oxidase. Proinflammatory stimuli, as bacterial lipopolysaccharide associated with interferon-gamma, caused a rapid/robust burst of superoxide radicals, sensitive to NADPH oxidase inhibition, followed by dismutation to H2O2, the species resulting in DNA damage via a Fenton-type reaction. There was no contribution of superoxide radical/H2O2 of mitochondrial origin and there was no evidence for the formation/involvement of peroxynitrite. On the other hand, astrocytes were virtually invulnerable to the DNA-damaging effects of exogenous peroxynitrite, an agent causing DNA strand scission in other cell types, via the Ca2+-dependent mitochondrial formation of superoxide radical/H2O2. Resistance was not dependent on scavenging of peroxynitrite but, rather, on insufficient mitochondrial Ca2+ accumulation. Hence, different manipulations resulting in an increase of the mitochondrial Ca2+ pool were invariably associated with the formation of DNA-damaging levels of H2O2. In conclusion, it appears that the strategy adopted by astrocytes to avoid inflammation-dependent genotoxic events, in particular those mediated by peroxynitrite, is to prevent mitochondrial Ca2+ accumulation, critical for the formation of secondary species largely responsible for DNA damage induced by peroxynitrite.

We investigated the distribution pattern of SMI-32-immunopositive cells in the lateral geniculate nucleus (LGN) and in the primary (V1) and middle temporal (MT) cortical visual areas of the adult New World monkey Cebus apella. In the LGN, the reaction for SMI-32 labeled cells in both the magnocellular (M) and parvocellular (P) layers. However, the cellular label was heavier in M layers, which also showed a more intense labeling in the neuropil. In V1, the reaction showed a lamination pattern, with the heaviest labeling occurring in layer 4B and upper layer 6 (layers that project to area MT). Area MT shows a dense band of labeled neuropil and large pyramidal neurons in layer 3, large darkly labeled but less densely packed neurons in layer 5, and a population of small, lightly labeled cells in layer 6. These results resemble those found in other New and Old World monkeys, which suggest that the preferential labeling of projection neurons associated with fast-conducting pathways to the extrastriate dorsal stream is a common characteristic of simian primates. In the superficial layers of V1 in Cebus monkeys, however, SMI-32-labeled neurons are found in both cytochrome oxidase blobs and interblob regions. In this aspect, our results in Cebus are similar to those found in the Old World monkey Macaca and different from those described for squirrel monkey, a smaller New World Monkey. In Cebus, as well as in Macaca, there is no correlation between SMI-32 distribution and the blob pattern.

Dexterous hands, used to manipulate food, tools, and other objects, are one of the hallmarks of primate evolution. However, the neural substrate of fine manual control necessary for these behaviors remains unclear. Here, we describe the functional organization of parietal cortical areas 2 and 5 in the cebus monkey. Whereas other New World monkeys can be quite dexterous, and possess a poorly developed area 5, cebus monkeys are the only New World primate known to use a precision grip, and thus have an extended repertoire of manual behaviors. Unlike other New World Monkeys, but much like the macaque monkey, cebus monkeys possess a proprioceptive cortical area 2 and a well developed area 5, which is associated with motor planning and the generation of internal body coordinates necessary for visually guided reaching, grasping, and manipulation. The similarity of these fields in cebus monkeys and distantly related macaque monkeys with similar manual abilities indicates that the range of cortical organizations that can emerge in primates is constrained, and those that emerge are the result of highly conserved developmental mechanisms that shape the boundaries and topographic organizations of cortical areas.

Several proteins have their normal patterns of distributions altered by monocular visual deprivation. We studied the distribution of the calcium-binding proteins calbindin-28kD (Cb) and parvalbumin (Pv) in V1 in normal adult Cebus apella monkeys and in monkeys with monocular retinal lesions. In normal monkeys, the interblobs regions in layers 2/3 and the layer 4B are intensely labeled for Cb, while Pv reaction showed a complementary labeling pattern with a stronger staining in layers 4A, 4C and in the blob regions in layers 2/3. In monkeys with monocular retinal lesion, the laminar distribution of these proteins was differentially affected, although both reactions resulted in stronger labeling in non-deprived ocular dominance columns. While Cb reaction resulted in stronger labeling in layers 1 through 5, Pv labeling was heavier in layers 2/3, 4A and 4C. There was a clear reduction in the intensity of neuropil staining for both Pv and Cb in deprived ocular dominance columns with little or no reduction in number of labeled cells. This reduction could thus be attributed to activity-dependent changes at synapses level.

In this study we investigated some biological properties of flavonoids recovered in the aqueous (AqE) and ether (EtE) extracts from four Italian multifloral honeys. In particular, a cell-free assay was employed to detect direct reduction of ferricyanide, whereas an assay using intact human erythrocytes was used to measure the ability to donate electrons to a trans-plasma membrane oxidoreductase. It was found that the AqE displays greater "in vitro" ferricyanide-reducing activity than the EtE but, unlike the latter, is virtually ineffective in the cell-based assay. Uptake studies employing high-performance liquid chromatography/mass spectrometry (HPLC/MS) showed that the different results were explained by the inability of AqE components to cross the erythrocyte plasma membrane and by the excellent uptake of EtE flavonoids, which, once within the cell, donate electrons to the membrane oxidoreductase to efficiently reduce extracellular oxidants. The latter property appears to depend on the content of ether-soluble flavonoids in the starting honeys.

Previous studies performed in our laboratory indicated that non-toxic concentrations of peroxynitrite nevertheless commit U937 cells to a rapid necrosis that is however prevented by a survival signaling driven by cytosolic phospholipase A(2)-released arachidonic acid. Toxicity was mediated by concentrations of peroxynitrite resulting in H(2)O(2)-dependent inhibition of arachidonic acid release. The present study shows that U937 cells differentiated to monocytes by prolonged exposure to dimethyl sulfoxide are resistant to peroxynitrite because able to respond with enhanced release of arachidonic acid. An additional important observation was that these cells require more arachidonate than the undifferentiated cells to support the survival signaling. The enhanced arachidonic acid release was not associated with changes in cytosolic phospholipase A(2) expression but was rather dependent on the increased responsiveness of the enzyme to calcium-dependent stimulation as well as on reduced mitochondrial formation of H(2)O(2). The latter event was found to be critical, since differentiated and undifferentiated cells were equally sensitive to peroxynitrite when the accumulation of H(2)O(2) was enhanced via depletion of catalase, or addition of a complex III inhibitor. Thus, the strategy selected by the differentiation process to allow monocytes to cope with peroxynitrite appears to involve some specific mechanism preventing the mitochondrial formation of H(2)O(2).

The plasma membrane oxidoreductase (PMOR) activity, which mainly utilises ascorbate as intracellular electron donor, represents a major mechanism for cell-dependent reduction of extracellular oxidants and might be an important process used by the erythrocytes to keep a reduced plasma environment. We previously reported that in human erythrocytes, myricetin and quercetin act as intracellular substrates of a PMOR showing a novel mechanism whereby these flavonoids could exert beneficial effects under oxidative stress conditions. Here, we evaluated the ability of different flavonoids (quercetin, myricetin, morin, kaempferol, fisetin, catechin, luteolin, apigenin, acacetin, rutin, taxifolin, naringenin, genistein) and of two in vivo O-methylated metabolites of quercetin (isorhamnetin and tamarixetin) to be substrates of PMOR, by comparing their antioxidant capacity (i.e. direct interaction with the oxidant ferricyanide or with the free radical 1,1-diphenyl-2-picryl-hydrazil) with their ability to penetrate the erythrocytes and donate electrons to the PMOR. The results obtained indicate that, although most of the flavonoids display significant antioxidant activities, only those (quercetin, myricetin, fisetin) that combine the cathecol structure of the B ring (responsible for the reducing activity) with the 2,3 double bond and 4-oxo function of the C ring (responsible for the uptake by erythrocytes) can act as intracellular substrates for PMOR. It is of note that the metabolites of quercetin enter erythrocytes and donate electrons to the PMOR as the parent compound. The present data show a relationship between the flavonoid structures and their ability to provide electrons to the PMOR, suggesting an additional mechanism whereby dietary flavonoids may exert beneficial effects in man.

Exposure of U937 cells to an otherwise nontoxic concentration of peroxynitrite promotes a rapid necrotic response in the presence of pharmacological inhibitors of phospholipase A2. A 12-fold higher concentration of the oxidant, in the absence of additional treatments, caused remarkably greater DNA single-strand breakage, delayed formation of H2O2, and depletion of reduced glutathione but an identical level of toxicity. Cell death was prevented in both circumstances by nanomolar levels of arachidonic acid or by cyclosporin A via mechanisms unrelated to elimination of the above effects and was causally linked to prevention of mitochondrial permeability transition. Treatment with a high dose of peroxynitrite for 30 min caused an approximately 40% decline in ATP, both in the absence and presence of arachidonate, whereas only a small, arachidonic acid-sensitive reduction of the ATP pool was detected in cells treated with the low dose of peroxynitrite and the phospholipase A2 inhibitor. ATP-predepleted cells, however, were hypersensitive to peroxynitrite, and under these conditions, toxicity was not prevented by arachidonate. The above findings were reproduced in another promonocytic cell line, THP-1 cells. We concluded that the rapid necrotic response triggered by peroxynitrite in monocytes is mediated by a regulated process, not by ATP depletion, associated with reduced arachidonate availability. Supplementation of exogenous arachidonic acid always rescued cells via an ATP-dependent survival pathway.

The visual system is constantly challenged to organize the retinal pattern of stimulation into coherent percepts. This task is achieved by the cortical visual system, which is composed by topographically organized analytic areas and by synthetic areas of the temporal lobe that have more holistic processing. Additional visual areas of the parietal lobe are related to motion perception and visuomotor control. V1 and V2 represent the entire visual field. MT represents only the binocular field, and V4 only the central 30 degrees-40 degrees. The parietal areas represent more of the periphery. For any eccentricity, the receptive field grows at each step of processing, more at anterior areas in the temporal lobe. Minimal point image size increases towards the temporal lobe, but remains fairly constant toward the parietal lobe. Patterns of projection show asymmetries. Central V2 and V4 project mainly to the temporal lobe, while peripherals V2 (more than 30 degrees) and V4 (more than 10 degrees) also project to the parietal lobe. Visual information that arrives at V1 projects to V2, MT and PO, which then project to other areas. Local lateral propagation and recursive loops corroborate to perceptual completion and filling in. Priority connections to temporal, parietal and parieto-temporal cortices help construct crude early representations of objects, trajectories and movements.

The transcription factors c-Fos and Zif268 have been used as markers of neuronal activity, and they also have been implicated in neuronal plasticity. In this study, we investigated the expression of c-Fos and Zif268 proteins in the lateral geniculate nucleus (LGN) and in the cortical primary visual area (V1) of normal adult Cebus apella monkeys and in animals with monocular lesions. In the LGN, the reaction for c-Fos showed immunopositive cells in both magnocellular (M) and parvocellular (P) layers; however, the label was heavier in P layers. In animals that suffered monocular lesions, the immunocytochemistry for c-Fos showed more labeling in layers related to the normal eye compared with those of the lesioned eye. No specific label was observed after the reaction for Zif268 in the LGN. In V1, the reaction for both Zif268 and c-Fos showed a pattern of lamination in which heavier labeling was found in layers 2/3, 4A, 4C, and 6. After monocular lesions, we observed a clear pattern of ocular dominance columns in V1 for both c-Fos and Zif268, in which the columns related to the normal eye are more heavily labeled than those related to the lesioned eye. This pattern is more evident in layer 4C after c-Fos reaction, whereas, after Zif268, it is more clearly observed in layers 2/3. These results suggest that, in addition to be regulated by functional activity, these transcription factors are involved in different processes during cortical reorganization., (2004 Wiley-Liss, Inc.)

We recently found that long-term exposure to nitric oxide (NO) triggers mitochondrial biogenesis in mammalian cells and tissues by activation of guanylate cyclase and generation of cGMP. Here, we report that the NO/cGMP-dependent mitochondrial biogenesis is associated with enhanced coupled respiration and content of ATP in U937, L6, and PC12 cells. The observed increase in ATP content depended entirely on oxidative phosphorylation, because ATP formation by glycolysis was unchanged. Brain, kidney, liver, heart, and gastrocnemius muscle from endothelial NO synthase null mutant mice displayed markedly reduced mitochondrial content associated with significantly lower oxygen consumption and ATP content. In these tissues, ultrastructural analyses revealed significantly smaller mitochondria. Furthermore, a significant reduction in the number of mitochondria was observed in the subsarcolemmal region of the gastrocnemius muscle. We conclude that NO/cGMP stimulates mitochondrial biogenesis, both in vitro and in vivo, and that this stimulation is associated with increased mitochondrial function, resulting in enhanced formation of ATP.

A short-term pre-exposure to dehydroascorbic acid (DHA) promotes U937 cell death upon exposure to otherwise non-toxic levels of peroxynitrite (ONOO-). Toxicity is mediated by a saturable mechanism and cell death takes place as a consequence of mitochondrial permeability transition. The following lines of evidence are consistent with the notion that the enhancing effects of DHA were related to mitochondrial events resulting in inhibition of complex III upon exposure to otherwise inactive concentrations of ONOO-. First, DHA, as well as bona fide complex III inhibitors, similarly enhanced toxicity and subsequent formation of H2O2 induced by ONOO- via a rotenone- or catalase-sensitive mechanism. Secondly, bona fide complex III inhibitors were ineffective in DHA-pre-loaded cells. In addition, respiration-deficient cells were resistant to toxicity elicited by ONOO- and their supplementation with increasing concentrations of DHA, although resulting in the accumulation of vitamin C levels identical with those observed in respiration-proficient cells, failed to affect ONOO- toxicity. Finally, oxygen-consumption experiments demonstrated that pre-exposure to DHA promotes the ONOO--dependent inhibition of complex III. In conclusion, the above results collectively demonstrate that increasing the intracellular accumulation of vitamin C promotes mitochondrial events leading to ONOO--dependent formation of H2O2 and resulting in a rapid necrotic response.

Quercetin is rapidly and avidly taken up by human red blood cells (RBC) via a passive diffusion mechanism, driven by flavonoid binding to haemoglobin and resulting in an almost quantitative accumulation of the flavonoid. Heamoglobin-free resealed ghosts accumulated quercetin exclusively in the membrane fraction. Cell-associated quercetin was biological active and could be quantitatively utilised to support the reduction of extracellular oxidants mediated by a transplasma-membrane oxido-reductase. Additional experimental evidence revealed that quercetin uptake declined in the presence of albumin and that, under these conditions, the amount of cell-associated quercetin is enhanced by increasing the RBC number. Quercetin release from flavonoid-preloaded RBC was observed only in the presence of albumin (or in human plasma) and this response was progressively inhibited upon incubation in solutions containing albumin previously exposed to increasing concentrations of quercetin and cleared of the unbound fraction of the flavonoid. Furthermore, exposure to quercetin pre-saturated albumin promoted accumulation of the flavonoid in fresh RBC and this response was a direct function of the extent of albumin saturation. These results, indicating a flow of quercetin from albumin to haemoglobin, and vice versa, are therefore consistent with the possibility that human RBC play a pivotal role in the distribution and bioavailability of circulating flavonoids.

Gattass and collaborators (Gattass R, Rosa MGP, Souza APB, Piñon MCG, Neuenschwander S [1990a] Braz J Med Biol Res 23:375-393) proposed that the dorsal stream of visual processing, as defined by Ungerleider and Mishkin (Ungerleider LG, Mishkin M [1982] In: Ingle DJ, Goodale MA, Mansfield RJW, editors. Analysis of visual behavior. Cambridge: Massachusetts Institute of Technology. p 549-586), can be subdivided into dorsolateral and dorsomedial streams, and suggested that they may be involved in different aspects of the processing of motion and spatial perception, respectively. The goal of the present study was to provide additional evidence for this hypothesis by using cytochrome oxidase immunohistochemistry combined with retrograde tracing techniques. In Old World monkeys, the locations of visual area 4 (V4; ventral stream) and middle temporal area (MT; dorsal stream) projecting neurons in V2 supports the hypothesis that the cytochrome oxidase (CytOx)-rich thin stripes and the CytOx-poor interstripes are associated with the ventral stream, and that the CytOx-rich thick stripes belong to the dorsal stream. In this study we describe, in the New World monkey Cebus, the distribution of retrogradely labeled cells in V2 relative to the CytOx compartments after fluorescent tracers were placed in areas V4, MT, and the parietooccipital area (PO). We found PO-projecting neurons in CytOx-rich thick stripes and CytOx-poor interstripes in V2, whereas MT-projecting neurons appeared almost exclusively in thick stripes. In contrast, V4-projecting neurons were located mostly in CytOx-poor interstripes and CytOx-rich thin stripes. In addition, V4- and MT-projecting neurons were located mainly in supragranular layers, whereas PO-projecting neurons were located in supragranular and infragranular layers. These results support the hypothesis for the existence of three distinct streams of visual processing: ventral (including V4), dorsolateral (including MT), and dorsomedial (including PO)., (Copyright 2003 Wiley-Liss, Inc.)

In rodents, enriched environments drive the expression of the immediate early gene NGFI-A, a regulator of the plasticity marker Synapsin I. Both proteins have been implicated as mediators of plasticity in the rat mammalian retina. In the present work immunocytochemistry directed against these proteins was used to explore their basal activity in the retina of a more visual species, the New World monkey Cebus apella. In contrast to rat, monkey retina displayed high basal expression of both NGFI-A and Synapsin I. The greatest number of NGFI-A -expressing cells was observed within the inner nuclear layer, although NGFI-A positive nuclei were also found in the ganglion cell layer. High levels of Synapsin I were found in the inner plexiform layer and outer plexiform layer. Our findings are consistent with the postulate that the retinas of highly visual animals may experience ongoing reorganization as part of normal visual processing, and that NGFI-A and Synapsin I may be well positioned to regulate some of these changes.

We investigated the effect of a scene on the activity of cells in the anterior inferotemporal (AIT) cortex while the monkey performed a saccadic eye movement (SEM) task with and without the context of a scene (gray frame). Most neurons did not code for the presence of a scene when it appeared alone (monkey free viewing) or when the monkey was fixating. Nevertheless, when a peripheral target was turned on and the monkey had to make a SEM to it, some cells were capable of differentially coding the presence of the scene before and after the saccade.

Exposure of rabbit red blood cells to dehydroascorbic acid (DHA) caused a significant decline in glutathione content which was largely prevented by quercetin, whereas it was insensitive to various antioxidants, iron chelators or scavengers of reactive oxygen species. This response was not mediated by chemical reduction of either extracellular DHA or intracellular glutathione disulfide. In addition, the flavonoid did not affect the uptake of DHA or its reduction to ascorbic acid. Rather, quercetin appeared to specifically stimulate downstream events promoting GSH formation.

A well-established protocol to increase the intracellular content of ascorbic acid was used to investigate the effects of the vitamin on DNA single-strand breakage and toxicity mediated by authentic peroxynitrite (ONOO(-)) in U937 cells. This protocol involved exposure for 60 min to 100 microM dehydroascorbic acid, which was taken up by the cells and converted into ascorbic acid via a GSH-independent mechanism. At the time of exposure to ONOO(-), which was performed in fresh saline immediately after loading with dehydroascorbic acid, the vitamin present in the cells was all in its reduced form. It was found that, in cells that are otherwise ascorbate-deficient, an increase in their ascorbic acid content does not prevent, but rather enhances, the DNA-damaging and lethal responses mediated by exogenous ONOO(-). These results therefore suggest that acute supplementation of ascorbic acid can be detrimental for individuals with pathologies associated with a decrease in ascorbic acid and in which ONOO(-) is known to promote deleterious effects.

Based on cytoarchitectonic criteria, the primate pulvinar nucleus has been subdivided into medial (PM), lateral (PL), and inferior (PI) regions. However, these subdivisions show no correlation with those established by electrophysiological, immunocytochemical, or neuroanatomical tracer studies. In this work, we studied the connections of the pulvinar nucleus of Cebus monkey with visual areas V1, V2, V4, MT, and PO by means of retrograde fluorescent tracers injected into these areas. Based on the projection zones to cortical visual areas, the visual portion of the pulvinar of Cebus monkey was subdivided into three subregions: P1, P2, and P3, similar to those described in the macaque (Ungerleider et al., 1984). In Cebus, P1 includes the centrolateral portion of traditionally defined PI and adjacent portion of PL. P2 is located in the dorsal portion of PL and P3 includes the medial portion of PI and extends dorsally into adjacent PL and PM. In addition, we studied the histology of the pulvinar using multiple criteria, such as cytoarchitecture and myeloarchitecture; histochemistry for cytochrome oxidase, NADPH-diaphorase, and acetylcholinesterase; and immunocytochemistry for two calcium-binding proteins, calbindin and parvalbumin, and for a neurofilament recognized by the SMI-32 antibody. Some of these stains, mainly calbindin, showed additional subdivisions of the Cebus pulvinar, beyond the traditional PI, PL, and PM. Based on this immunohistochemical staining, the border of PI is moved dorsally above the brachium of the superior colliculus and PI can be subdivided in five regions (PI(P), PI(M), PI(C), PI(L), and PI(LS)). Regions P1, P2, and P3 defined based on efferent connections with cortical visual areas are not architectonically/neurochemically homogeneous. Rather they appear to consist of further chemoarchitectonic subdivisions. These distinct histochemical regions might be related to different functional modules of visual processing within one connectional area.

Treatment of U937 cells with a sublethal concentration of tert-butylhydroperoxide generates DNA single strand breakage in U937 cells and this response is increased by caffeine, ATP, pyruvate or antimycin A. As we previously reported (Guidarelli, Clementi, Brambilla and Cantoni, (1997) Biochem. J. 328, 801-806), the enhancing effects of antimycin A are mediated by inhibition of complex III and the ensuing formation of superoxides and hydrogen peroxide in a reaction in which ubisemiquinone serves as an electron donor. Active electron transport was required in pyruvate-supplemented cells since the increased genotoxic response occurred as a consequence of enforced mitochondrial Ca2+ accumulation, a process driven by the increased electrochemical gradient. The enhancing effects of caffeine or ATP were also the consequence of mitochondrial Ca2+ accumulation but these responses were independent on electron transport. The increased formation of DNA lesions resulting from exposure to tert-butylhydroperoxide associated with the Ca2+-mobilizing agents or the respiratory substrate was mediated by arachidonic acid generated by Ca2+-dependent activation of phospholipase A2. Melittin, a potent phospholipase A2 activator, and reagent arachidonic acid mimicked the effects of caffeine, ATP or pyruvate on the tert-butylhydroperoxide-induced DNA single strand breakage.

A large body of experimental evidence suggests that DNA damage and cytotoxicity mediated by peroxynitrite are linked by a causal relationship and important events in various pathological conditions. In the present study, we investigated the mechanism whereby peroxynitrite causes DNA single strand breakage in intact cells and found that the respiratory chain plays a pivotal role in this response. In particular, peroxynitrite mediates inhibition of complex III and, under these conditions, electrons are directly transferred from ubisemiquinone to molecular oxygen. Hydrogen peroxide produced by the dismutation of superoxides is the species mediating the peroxynitrite-dependent DNA cleavage.

The oxidized form of vitamin C (dehydroascorbic acid, DHA) completely and irreversibly inactivates recombinant human hexokinase type I, in a pseudo-first order fashion. The inactivation reaction occurs without saturation, indicating that DHA does not form a reversible complex with hexokinase. Further characterization of this response revealed that the inactivation does not require oxygen and that dithiothreitol, while able to prevent the DHA-mediated loss of enzyme activity, failed to restore the activity of the DHA-inhibited enzyme. Inactivation was not associated with cleavage of the peptide chain or cross-linking. The decay in enzymatic activity was however both dependent on deprotonation of a residue with an alkaline pKa and associated with covalent binding of DHA to the protein. In addition, inactivation of hexokinase decreased or increased, respectively, in the presence of the substrates glucose or MgATP. Finally, amino acid analysis of the DHA-modified hexokinase revealed a decrease of cysteine residues. Taken together, the above results are consistent with the possibility that covalent binding of the reagent with a thiol group of cysteine is a critical event for the DHA-mediated loss of hexokinase activity.

We propose a framework for understanding visual perception based on a topographically organized, functionally distributed network. In this proposal the extraction of shape boundaries starts at retinal ganglion cells with concentric receptive fields. This information, relayed through the lateral geniculate nucleus, creates a neural representation of negative and positive boundaries in a set of topographically connected and organized visual areas. After boundary extraction, several processes involving contrast, brightness, texture and motion extraction take place in subsequent visual areas in different cortical modules. Following these steps of processing, filling-in processes at different levels, within each area, and in separate channels, propagate locally to transform boundary representations onto surfaces representations. These partial representations of the image propagate back and forth in the network, yielding a neural representation of the original image. We propose that completion takes places in a wide cortical circuit that heavily relies on V1, where long-range information helps determine contour responses at specific topographically organized locations. Neural representations of illusory contours would emerge in circuits involving primarily area V2. The neural representation of filling-in of a peripheral stimulus in a dynamic surround (such as in texture filling-in) would depend on circuits involving primarily cells in areas V2 and V3, and would include competitive mechanisms required for figure to ground segregation. Finally, we suggest that multiple representations of the stimulus engage competitive mechanisms that select the "most likely hypothesis". Such choice behavior would rely on winner-take-all mechanisms capable of constructing a single neural representation of perceived objects.

Exposure of intact rabbit erythrocytes or erythrocyte lysates to ascorbic acid/FeCl3 in a glucose-free saline promoted a rapid decline in reduced glutathione and this response was paralleled by inactivation of hexokinase. Under the same conditions, the activity of the enzymes glyceraldehyde-3-phosphate dehydrogenase and glucose-6-phosphate dehydrogenase did not show appreciablevariations in intact cells, but was severely inhibited in the cell-free system. Similar results were obtained by replacing ascorbic acid/FeCl3 with dehydroascorbic acid. In addition, both treatments effectively inhibited the activity of purified hexokinase as well as those of glucose-6-phosphate dehydrogenase and glyceraldehyde-3-phosphate dehydrogenase. Further studies using the cell-free system indicated that the inhibition of enzyme activities elicited by either of the two treatments was effectively counteracted by the specific substrates of these enzymes. The fact that the hexokinase substrate glucose freely permeates the plasma membrane, unlike the substrates of glucose-6-phosphate dehydrogenase and glyceraldehyde-3-phosphatedehydrogenase, explains the selective inhibition of hexokinase observed in intact cells. The above results also indicate that dehydroascorbic acid is an inhibitor of these enzymes and strongly suggest that it is at least in part responsible for the effects mediated by the cocktail ascorbic acid/FeCl3., (Copyright 1998 Academic Press.)

Recent studies performed in our laboratory demonstrated that rabbit red blood cell hexokinase was remarkably inhibited by the cocktail ascorbic acid/Fe(II) (Stocchi et al., 1994, Arch. Biochem. Biophys. 311, 160-167) and that the formation of dehydroascorbic acid was a key event in this process (Fiorani et al., 1996, Arch. Biochem. Biophys, 334, 357-361). The present study was undertaken to determine the final hexokinase-inactivating species using cell-free extract as a model. Our results demonstrate superimposable kinetics of hexokinase decay promoted by either ascorbic acid/Fe(II) or dehydroascorbic acid in erythrocyte lysates in which the reduced glutathione (GSH) levels were variously manipulated. In particular, neither removal nor addition of this tripeptide was able to significantly alter the rate or extent of hexokinase inhibition. Thus, GSH-reductive processes are dispensable events in the process of hexokinase inhibition promoted by ascorbic acid/Fe(II) in red blood cells. As a consequence, dehydroascorbic acid appears to be the species which directly inhibits hexokinase. This inference is further supported by the observation that addition of dehydroascorbic acid to the purified enzyme leads to a remarkable inhibition in its activity.

The aim of this study was to investigate the effects of 50 Hz magnetic fields (0.2-0.5 mT) on rabbit red blood cells (RBCs) that were exposed simultaneously to the action of an oxygen radical-generating system, Fe(II)/ascorbate. Previous data obtained in our laboratory showed at the exposure of rabbit erythrocytes or reticulocytes to Fe(II)/ascorbate hexokinase inactivation, whereas the other glycolytic enzymes do not show any decay. We also observed depletion of reduced glutathione (GSH) content with a concomitant intracellular and extracellular increase in oxidized glutathione (GSSG) and a decrease in energy charge. In this work we investigated whether 50 Hz magnetic fields could influence the intracellular impairments that occur when erythrocytes or reticulocytes are exposed to this oxidant system, namely, inactivation of hexokinase activity, GSH depletion, a change in energy charge, and hemoglobin oxidation. The results obtained indicate the a 0.5 mT magnetic field had no effect on intact RBCs, whereas it increased the damage with Fe(II)/ascorbate to a 0.5 mT magnetic field induced a significant further decay in hexokinase activity (about 20%) as well as a twofold increase in methemoglobin production compared with RBCs that were exposed to the oxidant system alone. Although further studies will be needed to determine the physiological implications of these data, the results reported in this study demonstrate that the effects of the magnetic fields investigated are able to potentiate the cellular damage induced in vitro by oxidizing agents.

In this study we investigated the species involved in the process of hexokinase inactivation induced by ascorbic acid/Fe(II) in rabbit erythrocytes. Our results suggest a model in which divalent iron is first oxidized to the trivalent state and then triggers the oxidation of ascorbic acid. The H202 formed during this process accelerates the formation of dehydroascorbic acid, which appears to be necessary and sufficient to induce hexokinase inactivation. This model was validated by showing that: (a) H202-decomposing enzymes, unlike scavengers of the hydroxyl radicals, reduced the extent of hexokinase inactivation; (b) when H202 was used instead of ascorbate/Fe(II), it was unable, even at very high concentrations, to inhibit hexokinase activity; (c) replacing Fe(II) with either Fe(III) or H202 resulted in comparable levels of ascorbic acid-induced hexokinase inactivation; (d) expression of maximal hexokinase inhibiting activity was also triggered via enzyme-catalyzed oxidation of ascorbic acid or direct addition of dehydroascorbic acid; (e) the level of dehydroascorbic acid, which was actively generated in the external medium upon addition of ascorbic acid/Fe(II), increased as a function of time. Taken together, these results demonstrate that the process of hexokinase inactivation induced by ascorbic acid/Fe(II) is mediated by dehydroascorbate and that iron and H202 have the sole function of accelerating its formation.

Treatment of cultured mammalian cells with three different carcinogens, namely methylmethane sulphonate (MMS), chromate and 254 U.V. radiation, produces DNA single strand breaks (SSB) in cultured mammalian cells. The rate of removal of these lesions is not affected by exposure to 50 Hz electric (0.2 - 20 kV/m), magnetic (0.0002-0.2 mT), or combined electric and magnetic fields. These results indicate that, under the experimental conditions utilized in this study, 50 Hz electric, magnetic and electromagnetic fields (over a wide range of intensities) do not affect the machinery involved in the repair of DNA SSBs generated by different carcinogens in three different cultured mammalian cell lines, making it unlikely that field exposure enhances the ability of these carcinogens to induce transformation via inhibition of DNA repair.

In mammalian tissues hexokinase (ATP:D-hexose 6-phosphotransferase, EC 2.7.1.1) exists as four isoenzymes encoded by distinct genes. These proteins are homologous and are organized in two homologous domains, with the exception of hexokinase type IV which has only one. This organization is believed to be the result of a duplication and tandem fusion event involving the gene encoding for the ancestral hexokinase. In this study, we cloned the carboxyl-domain of human hexokinase type III and expressed it in Escherichia coli as a glutathione S-transferase fusion protein, using the pGEX-2T expression vector. The recombinant protein showed catalytic activity. A comparative study of the kinetic properties of the expressed carboxyl-domain and the enzyme partially purified from human lymphocytes is also shown. The results now allow a better understanding of the role of the carboxyl-domain in determining the catalytic properties of the enzyme.

In mammalian brain tissue, most hexokinase is bound to the mitochondria and only a small amount of the enzyme is present in soluble form. In this study we report that, in rabbit brain, hexokinase is present in two distinct molecular forms, which we designated HKH and HKL, both of which are separable using hydrophobic interaction or anion-exchange chromatography. These two molecular forms can be detected when hexokinase is prepared at pH 7.4, whereas at pH 10.0 only the more hydrophobic form, HKH, is present. The two subtypes of hexokinase do not show significant differences in Km values for glucose and ATP, in Ki values for glucose-6-phosphate or in their molecular weights. HKH is able to rebind mitochondrial membranes, while HKL has lost this ability, suggesting that the hydrophobic peptide at the N-terminal has been removed. The susceptibility of the N-terminal peptide to proteolysis is completely inhibited by using antiproteolytic compounds, such as leupeptin or E-64. The results reported in this paper suggest that a cysteine protease, probably belonging to a the class of cathepsins, may be involved in the processing of bindable hexokinase to the non-bindable form in rabbit brain, and that the activity of this protease is pH-dependent.

Exposure of cultured mammalian cells to 50 Hz electric (0.2-20 kV/m), magnetic (0.002-2 G), or combined electric and magnetic fields did not affect the rate of repair of DNA single strand breaks (SSB) induced by hydrogen peroxide. The same lack of effect was observed on the repair of both DNA SSBs and DNA double strand breaks (DSBs) in cells treated with the cocktail hydrogen peroxide/L-Histidine. These results indicate that exposure to electric and/or magnetic fields does not affect the machinery involved in the repair of DNA lesions in oxidatively injured cells thus suggesting that it is unlikely that field exposure might induce changes in the response of the cells to the tumor promoting or carcinogenic effects elicited by reactive oxygen species.

Hydrogen peroxide and fetal bovine serum stimulate DNA synthesis in growth-arrested smooth muscle cells with remarkably similar kinetics and cell density dependence. However, while stimulation with fetal bovine serum results in cell proliferation, that by H2O2 is followed by cell death. Depletion of conventional and novel protein kinase C isoforms, resulting from a long treatment with phorbol-12-myristate-13-acetate, further increases H2O2-induced DNA synthesis. On the other hand, the specific protein kinase C inhibitor calphostin C abolished the increased DNA synthesis promoted by fetal bovine serum or H2O2. H2O2 increases protein kinase C activity in smooth muscle cells. This effect is markedly reduced, but not abolished, by down-regulation of the alpha, delta and epsilon protein kinase C isoforms. Thus, the zeta isoform of protein kinase C, which is not down-regulated, may be responsible for the residual H2O2 stimulation of protein kinase C. In conclusion, the results obtained show that H2O2 stimulates protein kinase C activity and DNA synthesis in growth-arrested smooth muscle cells: these events are not followed by cell proliferation but rather by cell death. This H2O2 stimulated DNA synthesis appears to be negatively controlled by alpha, delta and epsilon isoforms and positively controlled by the zeta isoform of protein kinase C.

Exposure of rabbit reticulocytes to Fe(II)/ascorbate induced a pronounced decay in hexokinase activity. In reticulocytes, this enzyme is present in at least three different molecular forms, Ia, Ia* and Ib, sub-types of hexokinase type I, which show different intracellular distribution. Hexokinase Ia and Ib are soluble, whereas hexokinase Ia* is almost entirely bound to the mitochondria. Anion exchange chromatography of hexokinase from intact reticulocytes exposed to Fe(II)/ascorbate revealed a selective inactivation of forms Ia and Ib, whereas the form Ia* did not show any decay. Binding to the mitochondrial membrane seems to be responsible for the observed resistance of the form Ia* to the inactivation elicited by Fe(II)/ascorbate. Indeed, by using a cell-free system in which hexokinase Ia* was solubilized using Triton X-100, the decay in hexokinase activity induced by iron/ascorbate involved all three enzymatic forms.

High performance capillary electrophoresis (HPCE) was applied to the separation of protein and peptide mixtures with molecular masses ranging from 1300 to 96000 Da using a new bonded hydrophilic phase capillary, CElect-P150. This coated capillary reduces the interaction between proteins and silanol groups in capillary walls, allowing a complete recovery of the proteins and peptides of interest. HPCE was also used for the analysis of a complex mixture of tryptic fragments and to monitor the process of enzymatic digestion. Moreover, using a CElect-P150 capillary, highly reproducible analysis was possible without preconditioning the capillary with acid or basic solutions before each new analysis.

Chinese hamster ovary (CHO) cells show a cell density-dependent modification of ATP levels during the growth cycle. Cells were seeded at a density of 500,000 cells/75 cm2 flask in 10 ml of growth medium and at various time intervals, samples were taken and assayed for cell number, for adenine (ATP, ADP, AMP) and pyridine (NAD+, NADP+) nucleotide levels and for the activity of some glycolytic enzymes. Glucose consumption was also evaluated. Experimental results indicated that the rate of cell growth was exponential for up to the 4th day of culture after which the cell number remained pratically unchanged up to the 9th day. Under these experimental conditions we found that, whereas the intracellular levels of NAD+ and some glycolytic enzymes were not significantly affected, a drop in ATP content was apparent after 48 hr of culture. The decline in ATP levels progressively increased, reaching a maximum after 4 days of culture, and then remained unchanged. In order to evaluate whether this effect on ATP was determined by a reduced availability of nutritional factors or was really a function of cell density, we also performed experiments similar to those reported above, with the exception that the cells were grown in 40 ml of culture medium. Under these experimental conditions, the exponential growth was longer (in comparison with the cell growth in 10 ml of medium) and a plateau was reached after 6 days of culture.(ABSTRACT TRUNCATED AT 250 WORDS)

Cortical projections to the middle temporal (MT) visual area were studied by injecting the retrogradely transported fluorescent tracer Fast Blue into MT in adult New World monkeys (Cebus apella). Injection sites were selected based on electrophysiological recordings, and covered eccentricities from 2-70 deg, in both the upper and lower visual fields. The position and laminar distribution of labeled cell bodies were correlated with myeloarchitectonic boundaries and displayed in flat reconstructions of the neocortex. Topographically organized projections were found to arise mainly from the primary, second, third, and fourth visual areas (V1, V2, V3, and V4). Coarsely topographic patterns were observed in transitional V4 (V4t), in the parieto-occipital and parieto-occipital medial areas (PO and POm), and in the temporal ventral posterior area (TVP). In addition, widespread or nontopographic label was found in visual areas of the superior temporal sulcus (medial superior temporal, MST, and fundus of superior temporal, FST), annectent gyrus (dorsointermediate area, DI; and dorsomedial area, DM), intraparietal sulcus (lateral intraparietal, LIP; posterior intraparietal, PIP; and ventral intraparietal, VIP), and in the frontal eye field (FEF). Label in PO, POm, and PIP was found only after injections in the representation of the peripheral visual field (> 10 deg), and label in V4 and FST was more extensive after injections in the central representation. The projections from V1 and V2 originated predominantly from neurons in supragranular layers, whereas those from V3, V4t, DM, DI, POm, and FEF consisted of intermixed patches with either supragranular or infragranular predominance. All of the other projections were predominantly infragranular. Invasion of area MST by the injection site led to the labeling of further pathways, including substantial projections from the dorsal prelunate area (DP) and from an ensemble of areas located along the medial wall of the hemisphere. In addition, weaker projections were observed from the parieto-occipital dorsal area (POd), area 7a, area prostriata, the posterior bank of the arcuate sulcus, and areas in the anterior part of the lateral sulcus. Despite the different nomenclatures and areal boundaries recognized by different models of simian cortical organization, the pattern of projections to area MT is remarkably similar among primates. Our results provide evidence for the existence of many homologous areas in the extrastriate visual cortex of New and Old World monkeys.

The authors describe a case of ECG alterations, indicating myocardial ischaemia, in a patient affected by mild cranial trauma. Both cycloergometric test and angiography of coronary vessels were normal. The authors conclude that every patient affected by brain injury should be monitored by the Cardiological Department, because of the high frequency of myocardial damage and lethal arrhythmias in these patients.

Escherichia coli cells challenged with low or high concentrations of hydrogen peroxide are killed via two different mechanisms and respond with morphological changes which are also dependent on the extracellular concentration of the oxidant. Treatment with low concentrations (less than 2.5 mM) of H2O2 is followed by an extensive cell filamentation which is dependent on the level of H2O2 or the time of exposure. In particular, addition of 1.75 mM H2O2 results in a growth lag of approximately 90 min followed by partial increase in optical density, which was mainly due to the onset of the filamentous response. In fact, microscopic analysis of the samples obtained from cultures incubated with the oxidant for various time intervals has revealed that this change in morphology becomes apparent after 90 min of exposure to H2O2 and that the length of the filaments gradually increases following longer time intervals. Analysis of the ability of these cells to form colonies has indicated a loss in viability in the first 90 min of exposure followed by a gradual recovery in the number of cells capable of forming colonies. Measurement of lactate dehydrogenase in culture medium (as a marker for membrane damage) has revealed that a small amount of this enzyme was released from the cells at early times (less than 150 min) but not after longer incubation periods (300 min). Cells exposed to high concentrations of H2O2 (greater than 10 mM) do not filament and their loss of viability is associated with a marked reduction in cell volume. In fact, treatment with 17.5 mM H2O2 resulted in a time-dependent decrease of the optical density, clonogenicity, and cellular volume. In addition, these effects were paralleled by a significant release in the culture medium of lactate dehydrogenase thus suggesting that the reduced cell volume may be dependent on membrane damage followed by loss of intracellular material. This hypothesis is supported by preliminary results obtained in electron microscopy studies. In conclusion, this study further demonstrates that the response of E. coli to hydrogen peroxide is highly dependent on the concentration of H2O2 and further stresses the point that low or high concentrations of the oxidant result in the production of different species leading to cell death via two different mechanisms and/or capable of specifically affecting the cell shape.

The representation of the visual field in the dorsal portion of the superior temporal sulcus (ST) was studied by multiunit recordings in eight Cebus apella, anesthetized with N2O and immobilized with pancuronium bromide, in repeated recording sessions. On the basis of visuotopic organization, myeloarchitecture, and receptive field size, area MT was distinguished from its neighboring areas. MT is an oval area of about 70 mm2 located mainly in the posterior bank of the superior temporal sulcus. It contains a visuotopically organized representation of at least the binocular visual field. The representation of the vertical meridian forms the dorsolateral, lateral, and ventrolateral borders of MT and that of the horizontal meridian runs across the posterior bank of ST. The fovea is represented at the lateralmost portion of MT, while the retinal periphery is represented medially. The representation of the central visual field is magnified relative to that of the periphery in MT. The cortical magnification factor in MT decreases with increasing eccentricity following a negative power function. Receptive field size increases with increasing eccentricity. A method to evaluate the scatter of receptive field position in multiunit recordings based on the inverse of the magnification factor is described. In MT, multiunit receptive field scatter increases with increasing eccentricity. As shown by the Heidenhain-Woelcke method, MT is coextensive with two myeloarchitectonically distinct zones: one heavily myelinated, located in the posterior bank of ST, and another, less myelinated, located at the junction of the posterior bank with the anterior bank of ST. At least three additional visual zones surround MT: DZ, MST, and FST. The areas of the dorsal portion of the superior temporal sulcus in the diurnal New World monkey Cebus are comparable to those described for the diurnal Old World monkey, Macaca. This observation suggests that these areas are ancestral characters of the simian lineage and that the differences observed in the owl monkey may be secondary adaptations to a nocturnal ecological niche.

Hypertensive emergencies, and to a certain extent their treatment, contribute to morbidity and mortality in elderly patients. We studied 22 hospitalized patients, aged 70-90 years, all of whom had moderate essential hypertensive. During acute hypertension, mean systolic and diastolic blood pressure rose to 230 +/- 24 and 120 +/- 22 mmHg, respectively. Symptoms of reduced tissue perfusion/oxygenation and/or organ failure occurred, forcing us to begin antihypertensive therapy. We administered 50 mg of the angiotensin converting enzyme (ACE) inhibitor captopril sublingually, and within 15 min, systolic blood pressure decreased by an average 60 +/- 16 mmHg and diastolic blood pressure by an average 25 +/- 14 mmHg. There was no significant change in the heart rate. In addition, we treated 22 comparable patients with 10 mg nifedipine sublingually and observed, in four cases, a greater fall in blood pressure (up to 90 mmHg) together with tachycardia. These results show the beneficial effects of captopril in the treatment of hypertensive emergencies in elderly patients. The absence of dangerous side effects indicates that ACE inhibitors can be used as first-choice drugs for the treatment of acute hypertensive crises, even in old age.

A recently observed case triggers an examination of Propafenone (P.) treatment of atrial flutter (AF). A review of the literature reveals that the question has not so far been sufficiently investigated. In fact only one paper reporting the inadequacy of P. in the treatment of certain forms of AF and atrial fibrillation caused by vagal hypertonia has apparently been published. The case presented here is of a 59 year old man with left bronchial pneumonia in a static phase and an ECG indicating AF. Treatment with P. in slow-release bolus form (2 mg/kg) converted the AF into sinus rhythm in the space of a few minutes. In the absence of any demonstrable cardiac pathology the condition in thought to have been caused by the patient's precarious metabolic status (hypoxaemia, sepsis).

The feasibility of the titration of some cations in molten sodium thiocyanate-potassium thiocyanate mixture with electrolytically generated sulphide ions has been tested at 443 K. Quantitative data are given for the titration of cadmium, mercury and zinc ions.