Evaluation of the effects of alpha-phenyl-N-tert-butyl nitrone pretreatment on the neurobehavioral effects of methamphetamine

A relationship between formation of reactive oxygen species (ROS) and energy depletion has been proposed to play an important role in mediating methamphetamine (METH)-induced neurotoxicity. To evaluate this relationship, we examined the effect of the spin-trap agent, alpha-phenyl-N-tert-butyl nitrone (PBN) on hyperthermia and self-injurious behavior (SIB) and striatal dopamine (DA) depletion produced by METH (4 injections of 4 mg/kg, 2 hr intervals, s.c.) in BALB/c mice. Repeated administration of METH induced hyperthermia, incidence of SIB and striatal DA depletion (84% after 3 days). Pretreatment with PBN (4 injections of 60 or 120 mg/kg, i.p.) reduced METH-induced hyperthermia, but did not significantly attenuate METH-induced SIB or the striatal DA depletion. On the other hand, pretreatment with high doses of PBN (4 injections of 180 or 240 mg/kg, i.p.) protected against METH-induced hyperthermia and SIB, and PBN (180 mg/kg) also completely protected against the acute striatal DA depletion 60 min after the last injection of the drug. However, the long-lasting striatal DA depletion was only attenuated by 52 or 56%, respectively. These results indicate that METH-induced hyperthermia contributes to, but is not solely responsible for METH-induced neurotoxicity, and supports a role for formation of ROS and other mechanisms in the generation of METH-induced striatal dopaminergic neurotoxicity. In addition, the difference in the efficacy of PBN to protect against the acute or long-lasting striatal DA depletion induced by METH may indicate that both ROS formation and other mechanisms are required for METH-induced neurotoxicity to develop.     

Transient depletion of serotonin in the nervous system of Helisoma

The present study shows that the drug 5,7-dihydroxytryptamine (5,7-diHT) can be used reliably to deplete the neurotransmitter serotonin (5-HT) from the nervous system of the snail Helisoma. The depletion is more effective in axonal and synaptic regions (85-90%) than in the somata (55%), is reasonably specific for serotonin (dopamine is affected to a much lesser extent), and is transient, with normal levels of neurotransmitter being restored by 2 months. A physiological correlate of 5-HT depletion has been shown in that an EPSP elicited by a cerebral serotonergic neuron (C1) onto a buccal motoneuron (B19) is much smaller during depletion and also recovers with time as 5-HT regains normal concentration. Despite the severe 5-HT depletion and physiological impairment, the gross morphology of neuron C1 remains indistinguishable from controls. Serotonergic depletion is not accompanied by development of receptor supersensitivity nor by the production of serotonin in extraneuronal sources.     

Cardiovascular regulation after destruction of the C1 cell group of the rostral ventrolateral medulla in rats

To evaluate the role of C1 neurons in the rostral ventrolateral medulla (RVLM) in cardiovascular regulation, we studied rats in which this cell group was destroyed by the injection of anti-dopamine-beta-hydroxylase-saporin into the RVLM. These immunotoxin injections resulted in 32-99% depletion of the RVLM-C1 neurons and approximately 50% depletion of the A5 cell population. In conscious rats with large (>80%) depletion of the RVLM-C1 cell population, resting arterial pressure was approximately 10 mmHg lower than in control injected rats, although heart rate was not significantly different. Similar results were observed when arterial pressure was recorded in urethan-anesthetized rats, although under anesthesia, heart rate was also reduced in rats with large (>80%) depletion of the RVLM-C1 neuronal population. Sympathoexcitatory responses to baroreceptor unloading, chemoreceptor activation, and electrical stimulation of sciatic nerve afferent fibers were attenuated in rats with >80% depletion of the RVLM-C1 cell population. These effects of RVLM-C1 plus A5 cell populations were not mimicked by either smaller lesions of the RVLM-C1 population or by selective destruction of the A5 cell population with 6-hydroxydopamine. Sympathoinhibitory responses such as decreases in arterial pressure and heart rate evoked by injection of GABA into the RVLM or by intravenous phenylbiguanide administration were not altered by RVLM-C1 plus A5 cell depletion. These data suggest that RVLM-C1 cells contribute to the maintenance of baseline arterial pressure and play an integral role in sympathoexcitatory responses.     

The depletion of starch from the sapwood of the ash (Fraxinus excelsior) and its relation to attack by powder-post beetles (Lyctus spp.)

The depletion of starch in the living sapwood of ash was examined as a possible means of rendering converted timber immune from attack by Lyctus (powder-post beetle).
Observations on disks of timber kept under controlled conditions showed that depletion is conditioned by access of oxygen; thus although in the standing tree depletion proceeds from without inwards, it can be induced in any part of the sapwood, and in any direction, by permitting access of oxygen, i.e. there is no polarity in depletion. The optimum temperature range for depletion in ash is from 31 to 36 C.: above 45 C. death of the cells may interrupt depletion.
The presence of β-indoly acetic acid does not influence rate of depletion. Reformation of the starch in the depleted wood in the presence of cane sugar could not be induced. The enzyme concerned in mobilization of the starch appears to be a labile one with an optimum in the neighbourhood of 40 C. and to be produced during the active respiration of the cells, starch depletion ceasing when oxygen is withdrawn.
In transversely cut disks the rate of respiration at 33 C. ceases to be proportional to the volume of tissue after a thickness of about 6 mm. has been attained. At 20 C. disks 10 mm. thick may be evenly depleted. Infestation experiments upon timber undergoing depletion showed that the attack by Lyctus is circumscribed by starch-level and not by total nitrogen or soluble sugars.
Under correct conditions of kilning, 1 in. sapwood plank can be rendered starch-free in about 20 days: with larger sizes depletion is uncertain and probably uneconomic.
The methods of starch and sugar analysis used in the work are appended.     

Curcumin-induced degradation of ErbB2: A role for the E3 ubiquitin ligase CHIP and the Michael reaction acceptor activity of curcumin

We investigated the molecular mechanism underlying curcumin depletion of ErbB2 protein. Curcumin induced ErbB2 ubiquitination but pretreatment with proteasome inhibitors neither prevented curcumin depletion of ErbB2 protein nor further accumulated ubiquitinated ErbB2. Curcumin increased association of endogenous and ectopically expressed CHIP, a chaperone-dependent ubiquitin ligase, with ErbB2. In COS7 cells cotransfected with ErbB2 and various CHIP plasmids followed by curcumin treatment, CHIP-H260Q (a mutant lacking ubiquitin ligase activity) promoted less curcumin-induced ErbB2 ubiquitination than did wild type CHIP, and CHIP-K30A (a mutant incapable of binding Hsp90 and Hsp70) neither associated with ErbB2 nor promoted its ubiquitination. ErbB2 mutants lacking the kinase domain failed to associate with CHIP and were completely resistant to ubiquitination and depletion induced by curcumin. Finally, curcumin's Michael reaction acceptor functionality was required for both covalent association of curcumin with ErbB2 and curcumin-mediated ErbB2 depletion. These data suggest (1) that CHIP-dependent ErbB2 ubiquitination is implicated in curcumin-stimulated ErbB2 depletion, and (2) that covalent modification of ErbB2 by curcumin is the proximal signal which initiates this process.     

Depletion of nicotinamide adenine dinucleotide in normal and xeroderma pigmentosum fibroblast cells by the antitumor drug CC-1065

CC-1065 is an extremely potent antitumor antibiotic that forms a well-defined adduct with DNA in which the molecule lies within the minor groove and is covalently attached through N3 of adenine. Addition of CC-1065 to human fibroblast cells produced a prolonged depletion of the nicotinamide adenine dinucleotide (NAD) pool even at extremely low drug concentrations (0.01 microgram/mL). The depletion of NAD by CC-1065 was blocked by 3-aminobenzamide, which is consistent with a NAD depletion mechanism involving poly-(ADP-ribose) synthesis in response to a repair-induced DNA strand breakage event. Significantly, similar extents of NAD depletion were also evident in xeroderma pigmentosum cells of complementation groups A and D following exposure to CC-1065. Since this NAD depletion is presumably associated with repair-induced incision, the repair of CC-1065-DNA adducts can probably take place by a pathway distinct from that involved in repair of more conventional bulky DNA adducts. The prolonged depletion of NAD, even at low doses of drug, suggests that CC-1065 causes DNA damage that results in a delay or block in DNA excision repair between the excision and ligation steps.     

Effects of lethal exposure to hyperoxia and to hydrogen peroxide on NAD(H) and ATP pools in Chinese hamster ovary cells

Cell death by oxidative stress has been proposed to be based on suicidal NAD depletion, typically followed by ATP depletion, caused by the NAD-consuming enzyme poly(ADP)ribose polymerase, which becomes activated by the presence of excessive DNA-strand breaks. In this study NAD+, NADH and ATP levels as well as DNA-strand breaks (assayed by alkaline elution) were determined in Chinese hamster ovary (CHO) cells treated with either H2O2 or hyperoxia to a level of more than 80% clonogenic cell killing. With H2O2 extensive DNA damage and NAD depletion were observed, while at a higher H2O2 dosage ATP also became depleted. In agreement with results of others, the poly(ADP)ribose polymerase inhibitor 3-aminobenzamide completely prevented NAD depletion. However, both H2O2-induced ATP depletion and cell killing were unaffected by the inhibitor, suggesting that ATP depletion may be a more critical factor than NAD depletion in H2O2-induced killing of CHO cells. With hyperoxia, only moderate DNA damage (2 X background) and no NAD depletion were observed, whereas ATP became largely (70%) depleted. We conclude that (1) there is no direct relation between ATP and NAD depletion in CHO cells subjected to toxic doses of H2O2 or hyperoxia; (2) H2O2-induced NAD depletion is not by itself sufficient to kill CHO cells; (3) killing of CHO cells by hyperoxia is not due to NAD depletion, but may be due to depletion of ATP.     

Effects of serotonin depletion on local interneurons in the developing olfactory pathway of lobsters

During embryonic life, the growth of the olfactory and accessory lobes of the lobster brain is retarded by serotonin depletion using 5,7-dihydroxytryptamine (5,7-DHT) (Benton et al., 1997). The local and projection interneurons that synapse with chemosensory cells in the olfactory lobes are potential targets of this depletion. This study documents proliferation and survival in the local interneuron cell clusters, and examines the differentiation of a prominent local interneuron, the serotonergic dorsal giant neuron (DGN), following serotonin depletion. An increase in dye coupling between the DGN and nearby cells is seen after serotonin depletion. However, morphometric analyses of individual DGNs in normal, sham-injected, and 5,7-DHT-treated embryos show that the general morphology and size of the DGNs are not significantly altered by serotonin depletion. Thus, the DGN axonal arbor occupies a greater proportion of the reduced olfactory lobes in the 5,7-DHT-treated embryos than in normal and sham-injected groups. The paired olfactory globular tract neutrophils (OGTNs), where olfactory interneurons synapse onto the DGNs, are 75% smaller in volume than the comparable region in either sham-injected or normal embryos. In vivo experiments using bromodeoxyuridine (BrdU) show that proliferation in the local interneuron soma clusters is reduced by 5,7-DHT treatment and that survival of newly proliferated local interneurons is also compromised. Our data suggest that alterations in the growth of the DGNs do not contribute to the dramatic reduction in size of the olfactory neutrophils following serotonin depletion, but that cell proliferation and survival among the local interneurons are regulated by serotonin during development. Reduced numbers of local interneurons are therefore one likely reason for the growth reduction observed after serotonin depletion.     

B cell depletion reduces the number of autoreactive T helper cells and prevents glucose-6-phosphate isomerase-induced arthritis

The therapeutic benefit of B cell depletion in patients with rheumatoid arthritis has provided proof of concept that B cells are relevant for the pathogenesis of arthritis. It remains unknown which B cell effector functions contribute to the induction or chronification of arthritis. We studied the clinical and immunological effects of B cell depletion in glucose-6-phosphate isomerase-induced arthritis. We targeted CD22 to deplete B cells. Mice were depleted of B cells before or after immunization with glucose-6-phosphate isomerase (G6PI). The clinical and histological effects were studied. G6PI-specific antibody responses were measured by ELISA. G6PI-specific T helper (Th) cell responses were assayed by polychromatic flow cytometry. B cell depletion prior to G6PI-immunization prevented arthritis. B cell depletion after immunization ameliorated arthritis, whereas B cell depletion in arthritic mice was ineffective. Transfer of antibodies from arthritic mice into B cell depleted recipients did not reconstitute arthritis. B cell depleted mice harbored much fewer G6PI-specific Th cells than control animals. B cell depletion prevents but does not cure G6PI-induced arthritis. Arthritis prevention upon B cell depletion is associated with a drastic reduction in the number of G6PI-specific effector Th cells.     

RNA interference-mediated silencing of the PAR gene inhibits the growth of PC3 cells via the induction of G2/M cell cycle arrest and apoptosis

BACKGROUND: The prostate androgen-regulated (PAR) gene is ubiquitously overexpressed in prostate cancer (PCa) cells and is involved in proliferation of PCa. However, the mechanism by which the modulation of PAR gene expression elicits its biological effects on PCa cells is not well documented. Here, we investigate the mechanism of PAR depletion inhibiting PCa cell growth. METHODS: PAR expression was depleted by small interfering RNA (siRNA) and its subsequent effects on proliferation of PC3 cells were determined by the trypan blue exclusion assay. Flow cytometric analysis provided the evidence for the progression of cell cycle and the induction of apoptosis which was further confirmed by the observation of cleavage of poly(ADP-ribose) polymerase. Western blot analysis was performed to investigate the involvement of critical molecular events known to regulate the cell cycle and the apoptotic machinery. RESULTS: siRNA transfection results in a dose-dependent inhibition of cell growth in PC3 cells by causing G2/M phase cell cycle arrest and apoptosis. The G2/M arrest by PAR depletion was associated with decreased levels of cyclin B1, pCdc2 (Tyr15), Cdc2 and Cdc25C. PAR depletion also was found to result in inhibition of procaspases 9, 8, 6 and 3 with significant increase in the ratio of Bax : Bcl-2. CONCLUSIONS: Our data indicate that PAR depletion induces G2/M arrest via the Cdc25C-Cdc2/cyclin B1 pathway. Furthermore, the results of the present study point toward involvement of pathways mediated by both caspase 8 and caspase 9 in apoptosis induction by PAR depletion.     

The effect of tryptophan depletion on brain activation measured by functional magnetic resonance imaging during the Stroop test in healthy subjects

We investigated the role of serotonin in cognitive activation of the frontal cortex. The serotonergic system was affected by the administration of an amino acids mixture without tryptophan (tryptophan depletion). In a placebo-controlled double-blind cross-over study with 20 healthy volunteers, we tested the hypothesis that a tryptophan (serotonin) decrease affects the activation of prefrontal cortex by the Stroop test. Cognitive brain activation was evaluated by functional magnetic resonance imaging (fMRI). Tryptophan depletion decreased the plasma tryptophan level up to 90 % for five hours after the tryptophan-free drink had been consumed when compared with the same mixture with tryptophan (p?0.0001). Tryptophan depletion did not affect the Stroop test performance. We compared fMRI activation in both conditions (tryptophan depletion and placebo) with plasma tryptophan levels as the covariates. The tryptophan depletion increased the activation (fMRI signal) in the bilateral mediofrontal cortex, anterior cingulate and left dorsolateral prefrontal cortex. The present findings allow the postulate that serotonergic medial forebrain and cingulum bundle pathways play a role in the activity of cortical structures involved in Stroop test processing.     

The role of calcium in the recall of stored morphogenetic information by plants

Flax seedlings grown in the absence of environmental stimuli, stresses and injuries do not form epidermal meristems in their hypocotyls. Such meristems do form when the stimuli are combined with a transient depletion of calcium. These stimuli include the "manipulation stimulus" resulting from transferring the seedlings from germination to growth conditions. If, after a stimulus, calcium depletion is delayed, meristem production is also delayed; in other words, the meristem-production instruction can be memorised. Memorisation includes both storage and recall of information. Here, we focus on information recall. We show that if the first transient calcium depletion is followed by a second transient depletion there is a new round of meristem production. We also show that if an excess of calcium follows calcium depletion, meristem production is blocked; but if the excess of calcium is in turn followed by another calcium depletion, again there is a new round of meristem production. The same stored information can thus be recalled repeatedly (at least twice). We describe a conceptual model that takes into account these findings.     

Mitochondrial fusion regulates proliferation and differentiation in the type II neuroblast lineage in Drosophila

Optimal mitochondrial function determined by mitochondrial dynamics, morphology and activity is coupled to stem cell differentiation and organism development. However, the mechanisms of interaction of signaling pathways with mitochondrial morphology and activity are not completely understood. We assessed the role of mitochondrial fusion and fission in the differentiation of neural stem cells called neuroblasts (NB) in the Drosophila brain. Depleting mitochondrial inner membrane fusion protein Opa1 and mitochondrial outer membrane fusion protein Marf in the Drosophila type II NB lineage led to mitochondrial fragmentation and loss of activity. Opa1 and Marf depletion did not affect the numbers of type II NBs but led to a decrease in differentiated progeny. Opa1 depletion decreased the mature intermediate precursor cells (INPs), ganglion mother cells (GMCs) and neurons by the decreased proliferation of the type II NBs and mature INPs. Marf depletion led to a decrease in neurons by a depletion of proliferation of GMCs. On the contrary, loss of mitochondrial fission protein Drp1 led to mitochondrial clustering but did not show defects in differentiation. Depletion of Drp1 along with Opa1 or Marf also led to mitochondrial clustering and suppressed the loss of mitochondrial activity and defects in proliferation and differentiation in the type II NB lineage. Opa1 depletion led to decreased Notch signaling in the type II NB lineage. Further, Notch signaling depletion via the canonical pathway showed mitochondrial fragmentation and loss of differentiation similar to Opa1 depletion. An increase in Notch signaling showed mitochondrial clustering similar to Drp1 mutants. Further, Drp1 mutant overexpression combined with Notch depletion showed mitochondrial fusion and drove differentiation in the lineage, suggesting that fused mitochondria can influence differentiation in the type II NB lineage. Our results implicate crosstalk between proliferation, Notch signaling, mitochondrial activity and fusion as an essential step in differentiation in the type II NB lineage.     

Molecular weight dependence of the depletion attraction and its effects on the competitive adsorption of lung surfactant

Albumin competes with lung surfactant for the air-water interface, resulting in decreased surfactant adsorption and increased surface tension. Polyethylene glycol (PEG) and other hydrophilic polymers restore the normal rate of surfactant adsorption to the interface, which re-establishes low surface tensions on compression. PEG does so by generating an entropic depletion attraction between the surfactant aggregates and interface, reducing the energy barrier to adsorption imposed by the albumin. For a fixed composition of 10 g/L (1% wt.), surfactant adsorption increases with the 0.1 power of PEG molecular weight from 6 kDa-35 kDa as predicted by simple excluded volume models of the depletion attraction. The range of the depletion attraction for PEG with a molecular weight below 6 kDa is less than the dimensions of albumin and there is no effect on surfactant adsorption. PEG greater than 35 kDa reaches the overlap concentration at 1% wt. resulting in both decreased depletion attraction and decreased surfactant adsorption. Fluorescence images reveal that the depletion attraction causes the surfactant to break through the albumin film at the air-water interface to spread as a monolayer. During this transition, there is a coexistence of immiscible albumin and surfactant domains. Surface pressures well above the normal equilibrium surface pressure of albumin are necessary to force the albumin from the interface during film compression.     

The critical role of the proximal calcium ion in the structural properties of horseradish peroxidase

The extent to which the structural Ca(2+) ions of horseradish peroxidase (HRPC) are a determinant in defining the heme pocket architecture is investigated by electronic absorption and resonance Raman spectroscopy upon removal of one Ca(2+) ion. The Fe(III) heme states are modified upon Ca(2+) depletion, with an uncommon quantum mechanically mixed spin state becoming the dominant species. Ca(2+)-depleted HRPC forms complexes with benzohydroxamic acid and CO which display spectra very similar to those of native HRPC, indicating that any changes to the distal cavity structural properties upon Ca(2+) depletion are easily reversed. Contrary to the native protein, the Ca(2+)-depleted ferrous form displays a low-spin bis-histidyl heme state and a small proportion of high-spin heme. Furthermore, the nu(Fe-Im) stretching mode downshifts 27 cm(-1) upon Ca(2+) depletion revealing a significant structural perturbation of the proximal cavity near the histidine ligand. The specific activity of the Ca(2+)-depleted enzyme is 50% that of the native form. The effects on enzyme activity and spectral features observed upon Ca(2+) depletion are reversible upon reconstitution. Evaluation of the present and previous data firmly favors the proximal Ca(2+) ion as that which is lost upon Ca(2+) depletion and which likely plays the more critical role in regulating the heme pocket structural and catalytic properties.     

Binding of glucose to the D-galactose/D-glucose-binding protein from Escherichia coli restores the native protein secondary structure and thermostability that are lost upon calcium depletion

The effect of the depletion of calcium on the structure and thermal stability of the D-galactose/D-glucose-binding protein (GGBP) from Escherichia coli was studied by fluorescence spectroscopy and Fourier-transform infrared spectroscopy. The calcium-depleted protein (GGBP-Ca) was also studied in the presence of glucose (GGBP-Ca/Glc). The results show that calcium depletion has a small effect on the secondary structure of GGBP, and, in particular it affects a population of alpha-helices with a low exposure to solvent. Alternatively, glucose-binding to GGBP-Ca eliminates the effect induced by calcium depletion by restoring a secondary structure similar to that of the native protein. In addition, the infrared and fluorescence data obtained reveal that calcium depletion markedly reduces the thermal stability of GGBP. In particular, the spectroscopic experiments show that the depletion of calcium mainly affects the stability of the C-terminal domain of the protein. However, the binding of glucose restores the thermal stability of GGBP-Ca. The thermostability of GGBP and GGBP-Ca was also studied by molecular dynamics simulations. The simulation data support the spectroscopic results. New insights into the role of calcium in the thermal stability of GGBP contribute to a better understanding of the protein function and constitute important information for the development of biotechnological applications of this protein. Mutations and/or labelling of amino acid residues located in the protein C-terminal domain may affect the stability of the whole protein structure.     

Arachidonic acid converts the glutathione depletion-induced apoptosis to necrosis by promoting lipid peroxidation and reducing caspase-3 activity in rat glioma cells

Intracellular glutathione (GSH) depletion induced by buthionine sulfoximine (BSO) caused cell death that seemed to be apoptosis in C6 rat glioma cells. Arachidonic acid (AA) promoted BSO-induced cell death by accumulating reactive oxygen species (ROS) or hydroperoxides. AA inhibited caspase-3 activation and internucleosomal DNA fragmentation during the BSO-induced GSH depletion. Furthermore, AA reduced intracellular ATP content, induced dysfunction of mitochondrial membrane and enhanced 8-hydroxy-2'-deoxyguanosine (8-OH-dG) production. There was significant increase of 12-lipoxygenase activity in the presence of AA under the BSO-induced GSH depletion in C6 cells. These results suggest that AA promotes cell death by changing to necrosis from apoptosis through lipid peroxidation initiated by lipid hydroperoxides produced by 12-lipoxygenase under the GSH depletion in C6 cells. Some ROS such as hydroperoxide produced by unknown pathway make hydroxy radicals and induce 8-OH-dG formation in the cells. The conversion of apoptosis to necrosis may be a possible event under GSH depleted conditions.     

2-deoxy-d-ribose induces apoptosis by inhibiting the synthesis and increasing the efflux of glutathione

Oxidative stress is caused by imbalance between the production of reactive oxygen species (ROS) and biological system ability to readily detoxify the reactive intermediates or repair the resulting damage. 2-deoxy-D-ribose (dRib) is known to induce apoptosis by provoking an oxidative stress by depleting glutathione (GSH). In this paper, we elucidate the mechanisms underlying GSH depletion in response to dRib treatment. We demonstrated that the observed GSH depletion is not only due to inhibition of synthesis, by inhibiting gamma-glutamyl-cysteine synthetase, but also due to its increased efflux, by the activity of multidrug resistance associated proteins transporters. We conclude that dRib interferes with GSH homeostasis and that likely cellular oxidative stress is a consequence of GSH depletion. Various GSH fates, such as direct oxidation, lack of synthesis or of storage, characterize different kinds of oxidative stress. In the light of our observations we conclude that dRib does not induce GSH oxidation but interferes with GSH synthesis and storage. Lack of GSH allows accumulation of ROS and cells, disarmed against oxidative insults, undergo apoptosis.