Modulating effect of cystamine and unsaturated fatty acids on gamma-glutamyl transpeptidase: Relation to cellular oxidative status

Summary Cell surface gamma-glutamyl transpeptidese activity in cultured neoplastic astrocytes was significantly increased upon treatment of the cells with the hepatoprotective disulfide, cystamine. The cystamine effect was sensitive to cycloheximide and could be significantly depressed by exogenous glutathione. Surface gamma-glutamyl transpeptidase activity was also modulated by the presence in the culture medium of the unsaturated fatty acids, linoleic acid and arachidonic acid. Metabolism of the fatty acids via the cyclooxygenase pathway was not a prerequisite for their modulation of the glycoprotein ectoenzyme. Lipoxygenase, however, was found to potentiate the unsaturated fatty acid effect in neoplastic astrocytes. Lipoxygenase is reported to catalyze the conversion of unsaturated fatty acids to their corresponding peroxides. The data indicate an oxidative influence on the control of gamma-glutamyl transpeptidase activity.     

Axonal Transport Characteristics of Gangliosides in Sensory Axons of Rat Sciatic Nerve

The distribution of axonally transported gangliosides and glycoproteins along the sciatic nerve was examined from 3 h to 4 weeks following injection of[3H]glucosamine into the fifth lumbar dorsal root ganglion of adult rats. Incorporation of labeled precursor into these glycoconjugates reached a maximal level in the ganglion within 6 h. Outflow patterns of radioactivity for glycoproteins showed a well-defined crest with a transport rate of approximately 330 mm/day. In contrast, the crest of transported gangliosides was continuously attenuated, implying a significant deposition along the axon, and an alternative method of calculating velocity was required. Analysis of accumulation of labeled material at double ligatures demonstrated both anterograde and retrograde transport of glycoproteins and gangliosides and allowed for the calculation of an anterograde transport rate of about 270 mm/day for each. Additional evidence of ganglioside transport is provided in that the TLC pattern of transported radioactive gangliosides accumulating at a ligature is significantly different from the pattern seen in the dorsal root ganglion or following intraneural administration of the labeled precursor. These data indicate that gangliosides are transported at the same rapid rate as glycoproteins but are subject to a more extensive exchange with stationary material than are glycoproteins.     

Enzymatic Protection Against Peroxidative Damage in Isolated Brain Capillaries

The content of polyunsaturated fatty acids, the activities of superoxide dismutase (SOD), glutathione peroxidase, glutathione reductase, and catalase, and the concentration of reduced glutathione were measured in cerebral microvessels isolated from rat brain. Polyunsaturated fatty acids, mainly arachidonic, linoleic, and docosahexaenoic acids, accounted for 32% of total fatty acids in cerebral microvessels. Whereas total SOD activity in the microvessels was slightly lower than that found in cerebrum and cerebellum, glutathione peroxidase and glutathione reductase activities were twice as high and catalase activity was four times higher. Glutathione peroxidase in microvessels is active on both hydrogen peroxide and cumen hydroperoxide, and it is strongly inhibited by mercaptosuccinate. After several hours of preparation, the concentration of reduced glutathione in isolated microvessels was 0.7 mumol/mg of protein, which corresponds to a concentration of approximately 3.5 mM. Our results indicate that the blood-brain barrier contains large amounts of peroxide-detoxifying enzymes, which may act, in vivo, to protect its highly polyunsaturated membranes against oxidative alterations.     

Time Course of Adaptations in Dopamine Biosynthesis, Metabolism, and Release Following Nigrostriatal Lesions: Implications for Behavioral Recovery from Brain Injury

Alterations in neostriatal dopamine metabolism, release, and biosynthesis were determined 3, 5, or 18 days following partial, unilateral destruction of the rat nigrostriatal dopamine projection. Concentrations of dopamine and each of its metabolites, 3,4-dihydroxyphenylacetic acid (DOPAC), homovanillic acid (HVA), and 3-methoxytyramine (3-MT) were markedly decreased in the lesioned striata at 3, 5, or 18 days postoperation. The decline in striatal high-affinity [3H]dopamine uptake closely matched the depletion of dopamine at 3 and 18 days postoperation. However, neither DOPAC, HVA, nor 3-MT concentrations were decreased to as great an extent as dopamine at any time following lesions that depleted the dopamine innervation of the striatum by greater than 80%. In these more severely lesioned animals, dopamine metabolism, estimated from the ratio of DOPAC or HVA to dopamine, was increased two- to four-fold in the injured hemisphere compared with the intact hemisphere. Dopamine release, estimated by the ratio of 3-MT to dopamine, was more increased, by five- to sixfold. Importantly, the HVA/dopamine, DOPAC/dopamine, and 3-MT/dopamine ratios did not differ between 3 and 18 days postlesioning. The rate of in vivo dopamine biosynthesis, as estimated by striatal DOPA accumulation following 3,4-dihydroxyphenylalanine (DOPA) decarboxylase inhibition with NSD 1015, was increased by 2.6- to 2.7-fold in the surviving dopamine terminals but again equally at 3 and 18 days postoperation. Thus, maximal increases in dopamine metabolism, release, and biosynthesis occur rapidly within neostriatal terminals that survive a lesion. This mobilization of dopaminergic function could contribute to the recovery from the behavioral deficits of partial denervation by increasing the availability of dopamine to neostriatal dopamine receptors. However, these presynaptic compensations are not sufficient to account for the protracted (at least 3-week) time course of sensorimotor recovery that has been observed following partial nigrostriatal lesion.     

Binding of anti-fibronectin to early amphibian ectoderm does not result in inhibition of neural induction under in vitro conditions

Summary Antibodies directed to fibronectin (anti-FN) were injected into the blastocoel of late blastulae of Xenopus laevis. Two animal caps (ectoderm) were isolated, when control embryos reached the early gastrula stage, and were combined with untreated upper blastopore lip in the sandwich method. In two control series fibronectin or Holtfreter solution was injected into the blastocoel. The results of the experiments suggest that neural induction cannot be prevented by binding anti-FN to fibronectin, which covers the blastocoelic side of the ectoderm. The data support the view that extracellular matrix proteins are not themselves responsible for neural induction. However, in comparison with the control series a slight shift of the differentiation pattern in the spinocaudal direction could be observed in the anti-FN series. The possible role of extracellular proteins in the formation of a close juxtaposition of mesodermal and ectodermal target cells as a prerequisite for shortdistance transmission of neural inducers is discussed.     

Application of a new method for detecting the phenotype of target binding cells

Summary A new method was developed for detecting the phenotype of target binding cells (TBC) in a single-cell assay system. This methodology was evaluated during a clinical trial of recombinant interferon alfa-2a (rIFN alfa-2a) for the treatment of 10 metastatic renal cell carcinoma patients. Total TBC with K562 targets, HNK-1+ TBC, and HLA-DR+ TBC were quantitated during rIFN alfa-2a therapy. A significantly increased proportion of lymphocytes bound to target cells on day 9 of therapy bore the HNK-1 marker. This proportion subsequently declined to pretreatment levels. Total TBC paralleled the rise and fall in HNK-1+ TBC. HLA-DR+ TBC binding to targets remained constant and low throughout therapy. These findings suggest that rIFN alfa-2a early in therapy (day 9) caused the recruitment of additional HNK-1+ cells into binders. However, with continued therapy, this proportion reverts to pretreatment levels. The results of this clinical trial served to illustrate the ability of the modified single-cell assay system to detect TBC phenotype.Supported in part by Hoffman-La Roche, NIH grant CA 12582, and Jonsson Comprehensive Cancer Center grant CA 15866Dr. Figlin is a recipient of an American Cancer Society Junior Faculty Fellowship-JFCF 762-A     

Study of human chromosome V

Summary The induction of fragile sites on human chromosomes has been demonstrated under various conditions that cause thymidylate stress, including exposure to uridine. In this study, we examined common fragile site expression by initially exposing peripheral lymphocytes to uridine, followed by repair of the fragile sites with media containing various concentrations of thymidine. Lymphocytes were cultured in medium 199 with 2 mM uridine. At 0.5, 1, 2, 3, 8, 10, 12, and 18 h before harvest, the uridine medium was removed and replaced by medium containing thymidine at various concentrations. Our results demonstrate that the effect of uridine on chromosome fragility can be reversed by low concentrations of thymidine (2 M up to 200 M) and the rescuing effect of thymidine can be achieved if the cells were treated prior to 2–3 h before harvest. No repair was found if thymidine was added to culture within 2 h prior to harvesting, suggesting that packing of chromosomes is also an important factor in the expression and repair of fragile sites.     

Ecological analyses of nesting success in evening grosbeaks

Summary We studied the nesting success of Evening Grosbeaks (Coccothraustes vespertinus) inhabiting two areas of the Front Range of the Rocky Mountains of Colorado from 1983–1987. Sixty-four nests were followed during building, incubating, brooding, and fledging; 54.7% were successful (young fledged). The largest number of nests failed during incubation. Nests started later were more successful than nests begun earlier in the season. Failure was most likely due to severe weather, abandonment during building, or predation. Specific habitat characteristics of grosbeak nesting sites and where nests were placed in trees were consistently associated with nesting success. Successful nests, when compared with nests that failed, were: (1) built in more open areas characterized by dispersed vegetation and a higher minimum canopy, (2) oriented in more southerly directions, (3) built closer to the main trunk of the nest tree, and (4) built in larger trees. Current ideas about whether or not birds actually select nest-sites are briefly discussed. We conclude that some grosbeaks optimally select nest sites where the likelihood of producing fledglings is higher than in other areas.