Human plasma-mediated hypoxic activation of indolequinone-based naloxone pro-drugs

Hypoxia is known to occur in tissues in response to narcotic analgesic therapy using as a result of respiratory depression. The aim of this study was to synthesize a narcotic antagonist pro-drug that can be activated by tissue hypoxia to prevent the damage associated with respiratory depression. We synthesized three different pro-drugs of the narcotic antagonist naloxone utilizing indolequinone as the hypoxia-sensitive moiety. The indolequinone structure in the pro-drugs was designed to have an open reactive point at the N-1 position offering the possibility of further conjugation with macromolecules to modify the bio-availability of these pro-drugs in vivo. A pro-drug (labeled 1) where naloxone and the indolequinone moiety were linked through a carbonate bond was rapidly hydrolyzed in phosphate buffered saline. However, two additional pro-drugs (labeled 2 and 3) having carbamate linkers were stable in phosphate buffered saline for 24 h. The reductive release of naloxone from the pro-drugs was achieved in the presence of the bio-reductive enzyme DT-Diaphorase, with about 80% release occurring from the two pro-drugs in 24 h. More than 99% of naloxone was released from pro-drug 2 in 30% human plasma, however the release only occurred under hypoxic conditions. This system provides a potential means for feedback control to counter critical respiratory depression induced by narcotic analgesics.     

Evidence for an essential arginine residue in the substrate binding site of the mammalian succinate dehydrogenase

Phenylglyoxal and 2,3-butanedione rapidly inactivate membrane-bound or soluble bovine heart succinate dehydrogenase. The inhibition of the enzyme by these reagents is completely prevented by saturating concentration of malonate. The modification of the active site sulfhydryl group by p-chloromercuribenzoate decreases the rate of the enzyme inhibition by phenylglyoxal and abolishes the protective effect of malonate. Kinetic data suggest that the inactivation by phenylglyoxal results from the modification of an essential arginine residue(s) which interacts with dicarboxylate to form the primary enzyme-substrate complex.     

Subcellular Localization of the Small GTPase Rab5a in Resting and Stimulated Human Neutrophils

The evidence that small GTPases of the Rab family are regulators of vesicle traffic which can influence various cell functions prompted us to investigate the potential role of one of these proteins, Rab5a, in human neutrophils. In this paper we show that a large amount of Rab5a is present in the cytosol of peripheral blood mature neutrophils. The remaining protein was found to be membrane and azurophilic granule associated. Upon neutrophil challenge with PMA for 10 min the amount of membrane-associated Rab5a was upregulated while the cytosolic content of the protein concomitantly decreased. These findings support the hypothesis that Rab5a could be involved in the mechanism of neutrophil activation by modulating the rate of endocytosis and/or vesicle fusion.     

Sensory input to neurons of the motor projection of biceps in the cat cortex

Correlation of cortical unit activity in the motor area for the biceps muscle was studied in chronic experiments on cats. In a group of neurons whose activity correlated with movement 68.1% of units had no sensory input from the working limb whereas in a group of neurons not correlating with movement there were 97.6% such units. In 24.2% of group I neurons cutaneous receptive fields of activation type were discovered on the distal part of the dorsal surface of the working limb. Five neurons responding to sensory input from the joints of the working limb were studied in this group.M. V. Lomonosov Moscow State University. Translated from Neirofiziologiya, Vol. 9, No. 6, pp. 563–569, November–December, 1977.     

Statistical analysis of unit activity in the cat motor cortex

Activity of 366 neurons in the motor area of the biceps brachii muscle was studied in chronic experiments on cats during an instrumental placing response. Of these cells, 59 received afferentation from the skin, joints, or deep structures of the limb participating in movement. Of 160 neurons which changed their discharge during the placing response, 52 (32.5%) had a peripheral input. Three types of motor cortical neurons are described: I) changing their discharge frequency before movement began, II) after movement began, and III) before and during movement. Positive correlation (r=0.3926) was found between the presence of a peripheral input for the neurons studied and changes in discharge frequency during the placing response. Correlation between the presence of a concrete input in the neurons and the type to which they belonged was studied.M. V. Lomonosov Moscow State University. Translated from Neirofiziologiya, Vol. 11, No. 3, pp. 201–207, May–June, 1979.     

Separation of 1–23-kb complementary DNA strands by urea–agarose gel electrophoresis

Double-stranded (ds), as well as denatured, single-stranded (ss) DNA samples can be analyzed on urea–agarose gels. Here we report that after denaturation by heat in the presence of 8 M urea, the two strands of the same ds DNA fragment of ∼1–20-kb size migrate differently in 1 M urea containing agarose gels. The two strands are readily distinguished on Southern blots by ss-specific probes. The different migration of the two strands could be attributed to their different, base composition-dependent conformation impinging on the electrophoretic mobility of the ss molecules. This phenomenon can be exploited for the efficient preparation of strand-specific probes and for the separation of the complementary DNA strands for subsequent analysis, offering a new tool for various cell biological research areas.     

Electrical characteristics of neurons in the cat motor cortex

Electrical characteristics of motor cortical neurons were studied in acute experiments on immobilized cats. Values of the input resistances varied from units to tens of megohms (mean 11.11±3.93 MΩ). The threshold current is a hyperbolic function of input resistance of the corresponding neurons and negative correlation was found between the axonal conduction velocity and input resistance. The time constant (τ₀) of the membrane was 7.1±3.46 msec. A time constant τ₁, of 1.65±0.36 msec, could also be distinguished in some neurons. Electrotonic lengths of dendrites of the cortical neurons were calculated by the use of Rall's model: mean 3.66±0.94 (in units of length constant).     

Ultrastructural changes of rat cortical neuron synapses for isolated and combined microapplications of transmitters

The ultrastructure of the presynaptic and postsynaptic components of the axo-dendritic synapses of layer V in the sensorimotor region of the rat neocortex is compared for microlonophoretic applications of 1-glutamate and norepinephrine, as well as for combined and successive administrations of these substances. A reliable increase is noted in the width of the postsynaptic density for combined electrophoresis of glutamate and norepinephrine, and a decrease in the number of synaptic vesicles at the terminals for applications of norepinephrine and combined and isolated administration of both transmitters. The results are discussed as an index of the modulating effect of norepinephrine on other neurotransmitter systems.M. V. Lomonosov State University, Moscow. Translated from Neirofiziologiya, Vol. 17, No. 4, pp. 476–481, July–August, 1985.