Magnesium lithospermate B extracted from Salvia miltiorrhiza elevates intracellular Ca(2+) level in SH-SY5Y cells.

AIM: To examine if magnesium lithospermate B (MLB), a potent inhibitor of Na(+)/K(+)-ATPase, leads to the elevation of intracellular Ca(2+) level as observed in cells treated with cardiac glycosides. METHODS: Viability of SH-SY5Y neuroblastoma cells treated with various concentrations of ouabain or MLB was measured. Intracellular Ca(2+) levels were visualized using Fluo4-AM (fluorescent dye) when cells were treated with ouabain or MLB in the presence or absence of KB-R7943 (Na(+)/Ca(2+) exchanger inhibitor) and 2-APB (IP(3) receptor antagonist). Molecular modeling was conducted for the docking of ouabain or MLB to Na(+)/K(+)-ATPase. Changes of cell body and dendrite morphology were monitored under a microscope. RESULTS: severe toxicity was observed in cells treated with ouabain of concentration higher than 1 micromol/L for 24 h while no apparent toxicity was observed in those treated with MLB. Intracellular Ca(2+) levels were substantially elevated by MLB (1 micromol/L) and ouabain (1 micromol/L) in similar patterns, and significantly reduced in the presence of KB-R7943 (10 micromol/L) or 2-APB (100 micromol/L). Equivalent interaction with the binding cavity of Na(+)/K(+)-ATPase was simulated for ouabain and MLB by forming five hydrogen bonds, respectively. Treatment of ouabain (1 micromol/L), but not MLB (1 mumol/L), induced dendritic shrink of SH-SY5Y cells. CONCLUSION: Comparable to ouabain, MLB leads to the elevation of intracellular Ca(2+) level presumably via the same mechanism by inhibiting Na(+)/K(+)-ATPase. The elevated Ca(2+) levels seem to be supplied by Ca(2+) influx through the reversed mode of the Na(+)/Ca(2+) exchanger and intracellular release from endoplasmic reticulum.

Nocifensive behaviors components evoked by brief laser pulses are mediated by C fibers.

Nocifensive behavior involves several response elements that have been used to assess neuropharmacological effects in different animal models of pain. Our previous analysis of laser-evoked nocifensive behaviors suggested that hierarchically organized responses in the nocifensive motor system are recruited in varying degrees by noxious stimuli of different intensities. Nocifensive behaviors can be differentially elicited and mediated by different classes of nociceptors. Thus, the aim of this study was to test the hypothesis that nocifensive behavioral elements elicited by brief laser pulse stimuli are mediated by C nociceptors. Laser-evoked cortical potentials and nocifensive behavior elements were recorded concurrently. As stimulus energy increased, rats exhibited a larger number of different responses and a greater frequency of each response element. Applying the neurotoxin, capsaicin, which selectively inhibits C fibers, to the sciatic nerves of rats, differentially blocked nocifensive behavioral components of flinch, withdrawal and licking but not non-nocifensive responses, namely movement and head turning. Based on these results we suggest that flinch, withdrawal and licking are mediated by C fibers, which are temporally associated with the nocifensive motor system as well as spinal and cortical evoked potentials. These results link hierarchically organized nocifensive responses and the afferent C fibers in the nocifensive motor system.

Effects of polyamines on the DNA-reactive properties of dimeric mithramycin complexed with cobalt(II): implications for anticancer therapy.

Few studies have examined the effects of polyamines on the action of DNA-binding anticancer drugs. Here, a Co(II)-mediated dimeric mithramycin (Mith) complex, (Mith)(2)-Co(II), was shown to be resistant to polyamine competition toward the divalent metal ion when compared to the Fe(II)-mediated drug complexes. Surface plasmon resonance experiments demonstrated that polyamines interfered with the binding capacity and association rates of (Mith)(2)-Co(II) binding to DNA duplexes, while the dissociation rates were not affected. Although (Mith)(2)-Co(II) exhibited the highest oxidative activity under physiological conditions (pH 7.3 and 37 degrees C), polyamines (spermine in particular) inhibited the DNA cleavage activity of the (Mith)(2)-Co(II) in a concentration-dependent manner. Depletion of intracellular polyamines by methylglyoxal bis(guanylhydrazone) (MGBG) enhanced the sensitivity of A549 lung cancer cells to (Mith)(2)-Co(II), most likely due to the decreased intracellular effect of polyamines on the action of (Mith)(2)-Co(II). Our study suggests a novel method for enhancing the anticancer activity of DNA-binding metalloantibiotics through polyamine depletion.

Chicken heat shock protein 90 is a component of the putative cellular receptor complex of infectious bursal disease virus.

Infectious bursal disease virus (IBDV) causes a highly contagious disease in young chicks and leads to significant economic losses in the poultry industry. The capsid protein VP2 of IBDV plays an important role in virus binding and cell recognition. VP2 forms a subviral particle (SVP) with immunogenicity similar to that of the IBDV capsid. In the present study, we first showed that SVP could inhibit IBDV infection to an IBDV-susceptible cell line, DF-1 cells, in a dose-dependent manner. Second, the localizations of the SVP on the surface of DF-1 cells were confirmed by fluorescence microscopy, and the specific binding of the SVP to DF-1 cells occurred in a dose-dependent manner. Furthermore, the attachment of SVP to DF-1 cells was inhibited by an SVP-induced neutralizing monoclonal antibody against IBDV but not by denatured-VP2-induced polyclonal antibodies. Third, the cellular factors in DF-1 cells involved in the attachment of SVP were purified by affinity chromatography using SVP bound on the immobilized Ni(2+) ions. A dominant factor was identified as being chicken heat shock protein 90 (Hsp90) (cHsp90) by mass spectrometry. Results of biotinylation experiments and indirect fluorescence assays indicated that cHsp90 is located on the surface of DF-1 cells. Virus overlay protein binding assays and far-Western assays also concluded that cHsp90 interacts with IBDV and SVP, respectively. Finally, both Hsp90 and anti-Hsp90 can inhibit the infection of DF-1 cells by IBDV. Taken together, for the first time, our results suggest that cHsp90 is part of the putative cellular receptor complex essential for IBDV entry into DF-1 cells.

Characterization of central axon terminals of putative stretch receptors in leeches.

Sensory feedback from stretch receptors, neurons that detect position or tension, is crucial for generating normal, robust locomotion. Among the eight pairs of putative stretch receptors associated with longitudinal muscles in midbody segments of medicinal leeches, only the ventral stretch receptor has been characterized in detail. To achieve the identification of all such receptors, we penetrated large axons in the nerve roots of nerve cords from adult leeches with dye-filled (Alexa Fluor hydrazide) electrodes. We identified the terminal arborizations of two additional putative stretch receptors with axons in anterior nerve roots and four more such receptors with axons in posterior roots of midbody ganglia. The axons are nonspiking and are individually identifiable by their entry point into the CNS; their projections within the neuropile; the pattern, extent, and orientation of their terminal branches; and the characteristics of small "spike-like" events. At least two of these axons undergo membrane potential oscillations that are phase locked to the swimming rhythm expressed in nerve cord-body wall preparations and, at a different phase angle, also in isolated nerve cords. Thus the membrane potentials of at least two axons are phasically modulated by the periphery and hence could provide cycle-by-cycle sensory input to coordinate swimming activity. One of these neurons has a soma associated with the dorsal body wall and hence is a putative stretch receptor in dorsal longitudinal muscle. Thus the traveling body wave expressed by swimming leeches may be regulated by sensory feedback from both ventral and dorsal longitudinal muscles.

A dye mixture (Neurobiotin and Alexa 488) reveals extensive dye-coupling among neurons in leeches; physiology confirms the connections.

Although the neuronal circuits that generate leech movements have been studied for over 30 years, the list of interneurons (INs) in these circuits remains incomplete. Previous studies showed that some motor neurons (MNs) are electrically coupled to swim-related INs, e.g., rectifying junctions connect IN 28 to MN DI-1 (dorsal inhibitor), so we searched for additional neurons in these behavioral circuits by co-injecting Neurobiotin and Alexa Fluor 488 into segmental MNs DI-1, VI-2, DE-3 and VE-4. The high molecular weight Alexa dye is confined to the injected cell, whereas the smaller Neurobiotin molecules diffuse through gap junctions to reveal electrical coupling. We found that MNs were each dye-coupled to approximately 25 neurons, about half of which are likely to be INs. We also found that (1) dye-coupling was reliably correlated with physiologically confirmed electrical connections, (2) dye-coupling is unidirectional between MNs that are linked by rectifying connections, and (3) there are novel electrical connections between excitatory and inhibitory MNs, e.g. between excitatory MN VE-4 and inhibitory MN DI-1. The INs found in this study provide a pool of novel candidate neurons for future studies of behavioral circuits, including those underlying swimming, crawling, shortening, and bending movements.

A quantitative method for assessing of the affective component of the pain: conditioned response associated with CO2 laser-induced nocifensive reaction.

Sensory and affective components are included in the overall behavioral manifestation in a nocifensive reaction. We have developed a behavioral model using classical conditioning to differentiate the affective component from the sensory responses following a thermal noxious stimulus. In laser-pain conditioning, free moving rats were trained to associate a tone (conditioned stimulus, CS) and short CO2 laser pulsation (unconditioned stimulus, US). A monotonous tone (800 Hz, 0.6 s) was delivered through a loudspeaker as the CS. CO(2) laser pulses (5 W at 100 ms in duration) applied to the hind paw were adopted as the US. The CS-US interval was 0.5 s. The conditioned responses as quantitatively measured by their body movement were developed over a period of 40 CS-US pairings. These conditioned responses were found retained when the rats were tested by presenting CS alone, immediate to and 24 h subsequent to training. The conditioned responses however diminished significantly following both morphine and buspirone treatment. This method demonstrated that neutral auditory stimuli could form association with unlearned nocifensive responses evoked by noxious CO2 laser pulses stimuli. Thus, the assessment of conditioned response may be a valuable tool for the measurement of the affective component of nociception.

Contribution of the anterior cingulate cortex to laser-pain conditioning in rats.

The emotional component of nociception is seldom distinguished from pain behavioral testing. The aim of the present study was to develop a behavioral test that indicates the emotional pain responses using the classical conditioning paradigm. The role of the anterior cingulate cortex (ACC) in the process of this pain conditioning response was also evaluated. In laser-pain conditioning, free moving rats were trained to associate a tone (conditioned stimulus, CS) and short CO(2) laser pulsation (unconditioned stimulus, US). Monotonous tone (800 Hz, 0.6 s) was delivered through a loud-speaker as CS. CO(2) laser pulses (5 W at 50 or 100 ms in duration) applied to the hind paw was adopted as US. The CS-US interval was 0.5 s. Laser-pain conditioning was developed during 40 CS-US pairings. CS and US pairing with 100-ms laser pulse stimuli was more effective in establishing conditioning responses than that of 50-ms stimuli. The conditioning responses remained, tested by presenting CS alone, immediate to and 24 h subsequent to training. The performance of laser-pain conditioning was significantly reduced after bilateral lesioning of the ACC. Similar results were also obtained by bilateral lesions of the amygdala. The conditioning responses were also diminished following morphine treatment. The association between a neutral stimulus and a noxious stimulus could be demonstrated in a Pavlovian conditioning test in free moving rats. Thus, the conditioned response may be employed as a measure of the emotional component of the nociception. It is also suggested that the ACC may play an important role in mediating this conditioning effect.