Stereospecific effects of opiate antagonists on superficial and deep nociception and on motor activity suggest involvement of endorphins on different opioid receptors.

It is known that various opiate antagonists enhance stereospecifically reactions to superficial nociceptive stimuli (e.g. in the hot plate test) suggesting the involvement of endogenous ligands in these reactions. In mice and rats the writhing responses to deep nociceptive stimuli (intraperitoneal test) were also enhanced stereospecifically by (-) naloxone, Mr 2266 and GPA 2163 but some other antagonists (naltrexone, levallorphan, diprenorphine) were inactive probably as a consequence of interfering agonist (antinociceptive) properties. An another antagonist, (-) Win 44441 suggested to bind principally with κ receptors did not enhance either superficial or deep nociception indicating that the former antagonists are probably interfering with endorphins at the level of μ receptors. The motor reaction of mice to a novel environment was stereospecifically depressed by opioid antagonists including (-) Win 44441 suggesting an involvement of endorphins at the level of κ receptors ; Mr 2266 and GPA 2163 were ineffective in this test and hyperalgesic in the two antinociceptive tests ; they might be relatively pure μ antagonists.     

Respiration,ATP level,and sugar accumulation in potato tubers during storage at 4°

A kinetic study was made of the relationship between respiration rate, sugar content and ATP levels, in fresh and aged potato tubers stored at 4°. The ATP content in tubers rose rapidly immediately after the chilling stress, while respiration rate decreased below the initial rate and sugar accumulation was not detected. After 4 days of storage, the ATP level declined and the sugars started to accumulate. The typical increase in respiration rate that usually follows chilling stress, appeared only in fresh tubers (at about the 6th day of storage). In dinitrophenol-treated tubers, the ATP level remained below the initial level and sugar accumulation was blocked completely. The evidence presented suggests that ATP elevation is not generated by the respiration burst.     

Incubation in Mice Provides a Signal for the Differentiation of Trypanosoma cruzi Epimastigotes to Trypomastigotes1

The differentiation of Trypanosoma cruzi epimastigotes into trypomastigotes was studied in diffusion chambers sub-cutaneously implanted in mice. Using epimastigotes of the Tulahuén strain, transformation was first evident at 16 h after implantation and reached its maximum (92% trypomastigotes) by 24 h. Shortly before their differentiation into trypomastigotes, epimastigotes were found to develop resistance to lysis by the alternative pathway of complement. Furthermore, implantation of stationary-phase (as opposed to log-phase) parasites resulted in the accumulation of large numbers of complement-resistant epimastigotes in the chambers. These observations suggest that epimastigotes pass through a complement-resistant transitional stage before differentiating into trypomastigotes and that transformation may require cell division. In a further series of experiments, epimastigotes recovered 7 h after implantation in mice were found to differentiate into trypomastigotes when cultured in vitro for an additional 17 h at 37°C. This observation indicates that the events which trigger the morphologic transformation of epimastigotes into trypomastigotes can be dissociated operationally from the differentiation process itself.     

Axotomy accelerates slow component b of axonal transport.

Because the integrity of an axon depends on the supply of proteins synthesized in the cell body, we examined the effect of axotomy on the transport of structural proteins in rat motor axons, and the effect of altered transport on the rate of outgrowth after a subsequent testing axotomy. To examine the axonal transport of structural proteins, we labeled newly synthesized proteins with 35S-methionine 7 days after a "conditioning" lesion of the sciatic nerve, and removed the nerve 7-21 days later for SDS-PAGE. Tubulin, actin, calmodulin, and the 68-kD light neurofilament protein (NF-L) were identified by fluorography and removed for liquid scintillation counting. The fastest moving structural proteins were carried by slow component b (SCb) of axonal transport, which advanced 20% faster in conditioned axons: 4.2 versus 3.5 mm/day (p less than 0.01). NF-L was not accelerated, indicating that the motor for subcomponent a (SCa) of slow axonal transport was unaffected by axotomy. To measure outgrowth distances, the testing lesions was made 7 days after the conditioning lesion, and growth cones were located by the fast transport method 3 or 9 days later. The regression analysis of outgrowth distance on time showed that sprouts elongated 25% faster in conditioned axons: 4.0 versus 3.2 mm/day (p less than 0.001). These accelerated sprouts were formed too far from the spinal cord to contain SCb proteins that were synthesized after axotomy. Because the rate of outgrowth correlated closely with the rate of SCb in outgrowing sprouts (McQuarrie and Jacob, J. Comp. Neurol. 305:139-147, 1991), we conclude that SCb is accelerated throughout the length of the axon by 7 days after axotomy.     

The Effect of Free Radicals Induced by Paraquat and Copper on the In Vitro Development of Plasmodium falciparum

The role of transition metals in paraquat toxicity was studied in cultures of Plasmodium falciparum. We showed that addition of copper led to an enhancement of the plasmodium killing, whereas addition of chelating agents. such as desferrioxamine and diethylenetriamine pentaacetic acid markedly reduced the toxic effects. Parsitized G6PD deficient erythrocytes were more sensitive than parasitized normal eryth-rocytes to copper and to the combination of copper and paraquat.     

Cytotoxic activity of tumor necrosis factor is mediated by early damage of mitochondrial functions. Evidence for the involvement of mitochondrial radical generation.

Structural mitochondrial damage accompanies the cytotoxic effects of several drugs including tumor necrosis factor (TNF). Using various inhibitors of mitochondrial electron transport we have investigated the mechanism of TNF-mediated cytotoxicity in L929 and WEHI 164 clone 13 mouse fibrosarcoma cells. Inhibitors with different sites of action modulated TNF cytotoxicity, however, with contrasting effects on final cell viability. Inhibition of mitochondrial electron transport at complex III (cytochrome c reductase) by antimycin A resulted in a marked potentiation of TNF-mediated injury. In contrast, when the electron flow to ubiquinone was blocked, either at complex I (NADH-ubiquinone oxidoreductase) with amytal or at complex II (succinate-ubiquinone reductase) with thenoyltrifluoroacetone, cells were markedly protected against TNF cytotoxicity. Neither uncouplers nor inhibitors of oxidative phosphorylation nor complex IV (cytochrome c oxidase) inhibitors significantly interfered with TNF-mediated effects, ruling out the involvement of energy-coupled phenomena. In addition, the toxic effects of TNF were counteracted by the addition of antioxidants and iron chelators. Furthermore, we analyzed the direct effect of TNF on mitochondrial morphology and functions. Treatment of L929 cells with TNF led to an early degeneration of the mitochondrial ultrastructure without any pronounced damage of other cellular organelles. Analysis of the mitochondrial electron flow revealed that TNF treatment led to a rapid inhibition of the mitochondria to oxidize succinate and NADH-linked substrates. The inhibition of electron transport was dose-dependent and became readily detectable 60 min after the start of TNF treatment, thus preceding the onset of cell death by at least 3-6 h. In contrast, only minor effects were observed on complex IV activity. The different effects observed with the mitochondrial respiratory chain inhibitors provide suggestive evidence that mitochondrial production of oxygen radicals mainly generated at the ubisemiquinone site is a causal mechanism of TNF cytotoxicity. This conclusion is further supported by the protective effect of antioxidants as well as the selective pattern of damage of mitochondrial chain components and characteristic alterations of the mitochondrial ultrastructure.     

Role of thyroid hormone in intermediary metabolism of propranolol or alloxan-treated Calotes versicolor.

Administration of L-thyroxine (T4) to thyroidectomized Calotes versicolor significantly increased the activity of glucose-6-phosphatase (G-6-Pase) (liver and kidney), the concentrations of blood glucose and total protein (liver and kidney), and decreased hepatic cholesterol when compared to thyroidectomized lizards. Propranolol injections in thyroidectomized lizards increased the cholesterol concentration and did not change the other parameters. The activity of G-6-Pase and blood glucose content was stimulated, whereas the total protein and cholesterol contents were decreased after alloxan treatment. Administration of T4 to thyroidectomized animals pretreated with propranolol or alloxan significantly elevated the activity of G-6-Pase, the concentrations of blood glucose, and total protein, and reduced hepatic cholesterol level when compared to drug-treated lizards. From the results, it is evident that thyroid hormone has an independent stimulatory influence on intermediary metabolism in C. versicolor irrespective of the involvement of adrenaline or insulin.     

Dependence of photosynthesis of sunflower and maize leaves on phosphate supply, ribulose-1,5-bisphosphate carboxylase/oxygenase activity, and ribulose-1,5-bisphosphate pool size

Sunflower (Helianthus annuus L. cv Asmer) and maize (Zea mays L. cv Eta) plants were grown under controlled environmental conditions with a nutrient solution containing 0, 0.5, or 10 millimolar inorganic phosphate. Phosphate-deficient leaves had lower photosynthetic rates at ambient and saturating CO₂ and much smaller carboxylation efficiencies than those of plants grown with ample phosphate. In addition, phosphate-deficient leaves contained smaller quantities of total soluble proteins and ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco) per unit area, although the relative proportions of these components remained unchanged. The specific activity of Rubisco (estimated in the crude extracts of leaves) was significantly reduced by phosphate deficiency in sunflower but not in maize. Thus, there was a strong dependence of carboxylation efficiency and CO₂-saturated photosynthetic rate on Rubisco activity only in sunflower. Phosphate deficiency decreased the 3-phosphoglycerate and ribulose-1,5-bisphosphate (RuBP) contents of the leaf in both species. The ratio of 3-phosphoglycerate to RuBP decreased in sunflower but increased in maize with phosphate deficiency. The calculated concentrations of RuBP and RuBP-binding sites in the chloroplast stroma decreased markedly with phosphate deficiency. The ratio of the stromal concentration of RuBP to that of RuBP-binding sites decreased in sunflower but was not affected in maize with phosphate deficiency. We suggest that a decrease in this ratio made the RuBP-binding sites more vulnerable to blockage or inactivation by tight-binding metabolites/inhibitors, causing a decrease in the initial specific activity of Rubisco in the crude extract from phosphate-deficient sunflower leaves. However, the decrease in Rubisco specific activity was much less than the decrease in the RuBP content in the leaf and its concentration in the stroma. A large ratio of RuBP to RuBP-binding sites may have maintained the Rubisco-specific activity in phosphate-deficient maize leaves. We conclude that the effect of phosphate deficiency is more on RuBP regeneration than on Rubisco activity in both sunflower and maize.     

Definition of microsatellite size variants forTnfa andHsp70 in autoimmune and nonautoimmune mouse strains

We have cloned and sequenced the upstream regulatory region of tumor necrosis factor (Tnfa) gene in 12 different mouse strains and identified an allelic polymorphism in the upstream regulatory region of the mouseTnfa gene. TheTNF allele found in the NZW strain is distinct from those of all otherH-2 haplotypes, supporting our previous suggestion that this allele may be associated with a regulatory or structural defect. In addition, simple tandem repeat sequences (microsatellites) within the promoter region of theTnfa gene and the 3 untranslated region of one of the members of the HSP70 family (Hsp68c clone) were utilized as genetic markers. Ten TNF size variants and twelve HSP70 variants were identified in over forty mouse strains. Using these markers in a set of congenic mice, we mapped this member of the HSP70 family to the central portion of theH-2 complex, centromeric to theTnfa gene. The NOD and NZW strains carry uniqueHSP70 alleles based on the variability in the length of this marker. These findings raise the possibility that this protein may play a role in the association of the major histocompatibility complex with these autoimmune diseases.