Search

When CCRF-CEM cells were incubated with 5-40 microM CdCl(2,) apoptosis was observed most clearly at 10 microM. Prior to the development of apoptosis, mitogen-activated protein kinases (MAPKs), i.e. extracellular signal-regulated protein kinase (ERK), c-Jun N-terminal kinase (JNK), and p38 MAPK, were activated with different sensitivity to CdCl(2) exposure. ERK and p38 MAPK were phosphorylated with incubation of 1 microM CdCl(2,) but higher than 20 microM CdCl(2) was required for the clear phosphorylation of JNK. In the time-course study, ERK and p38 MAPK were phosphorylated earlier than JNK after CdCl(2) exposure. The in vitro activities of MAPKs also increased in response to CdCl(2) exposure. Pretreatment with an intracellular Ca(2+) chelator, 1, 2-bis(o-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid tetra(acetoxymethyl) ester (BAPTA/AM), suppressed almost completely CdCl(2)-induced phosphorylation of JNK and p38 MAPK, but not ERK phosphorylation, indicating that the activation of JNK and p38 MAPK depends on the intracellular Ca(2+) but that of ERK does not. On the other hand, treatment with a MAPK/ERK kinase (MEK) inhibitor, U0126 (1,4-diamino-2,3-dicyano-1,4-bis[2-aminophenylthio]butadiene ), suppressed CdCl(2)-induced ERK activation and the apoptosis as well. The inhibition of p38 MAPK activity with SB203580 (4-[4-fluorophenyl]-2-[4-methylsulfinylphenyl]-5-[4-pyridyl]1H- imidaz ole) did not protect cells from apoptosis. The present results showed that the activation of ERK, JNK, and p38 MAPK is differently regulated in CCRF-CEM cells exposed to CdCl(2,) and that the ERK pathway seems to be responsible for the induction of apoptosis by CdCl(2) exposure in this human T cell line.

Rat brain was examined with 4.7 T proton magnetic resonance (MR). On administering hexachlorophene (HCP) 30 mg/kg/day for 5 days, myelin-rich structures stood out in T2-weighted images. Apparent diffusion coefficient (ADC) was markedly suppressed in all regions examined except for cerebral cortex. Seven days after terminating the exposure to HCP, without enhancement in T2-weighted images, ADC was still decreased in corpus callosum, optic nerve and trigeminal nerve. Rat administered with HCP and followed with high magnetic field proton MR seems to provide a good model for cytotoxic brain edema, and it may also be useful to visualize heavily myelinated structures.

The level of phosphorylated c-Jun NH2-terminal kinase (JNK) in LLC-PK1cells treated with CdCl2increased after 30 min and remained elevated even at 8 hr. And the activity of JNK assayed using glutathioneS-transferase-c-Jun as substrate increased dose-dependently. Consistent with the JNK activation, marked increases in the levels of c-Jun and c-Jun phosphorylated on Ser63 and Ser73 were observed in cells treated with CdCl2. The pretreatment with an intracellular Ca2+chelator, 1,2-bis(o-aminophenoxy)ethane-N,N,N′,N′-tetraacetic acid tetra(acetoxymethyl) ester (BAPTA/AM), abolished cadmium-induced JNK phosphorylation. However, pretreatment with a cell permeable chelator of heavy metals,N,N,N′,N′-tetrakis(2-pyridylmethyl)ethylenediamine (TPEN), did not. The present results showed that cadmium induces persistent activation of JNK pathway in a renal epithelial cell line, and that intracellular Ca2+is necessary for the activation.

The c-fos, a member of the immediate early genes, has been reported to be expressed in the renal proximal tubule in response to ischemic and toxic injury. In the present study, effects of mercury chloride (HgCl2) on the expression of c-fos were examined in LLC-PK1 cells. The reverse transcription polymerase chain reaction (RT-PCR) analysis for the semi-quantification of mRNA showed that the treatment of 20 microM HgCl2, markedly increased c-fos mRNA levels. The level of c-fos mRNA began to increase after a 30-min exposure, peaked at 1 h and then returned to the control level at 8 h. The HgCl2-induced c-fos expression was abolished completely by actinomycin-D, indicating it was due to transcriptional activation of the gene. Western blotting immunodetection revealed accumulation of c-Fos protein after 1 h exposure to 20 microM HgCl2. The cytotoxicity of HgCl2 as assayed by mitochondrial dehydrogenase activity (MTT conversion) was observed after 18 h exposure but not at 0.5-8 h. Also, the decrease in cell viability was accompanied with DNA fragmentation, which is characteristic of apoptosis. The present results showed that HgCl2 could induce the early expression of c-fos gene in a renal epithelial cell line.

The effects of tributyltin on the expression of immediate early genes, c-fos and c-jun, were examined in PC12 cells using reverse transcription polymerase chain reaction analysis. Tributyltin at concentrations of more than 0.4 μM induced the expression of c-fos after 15 min exposure. The induction of c-fos was accompanied with c-jun expression. Tributyltin-induced c-fos expression was abolished completely by actinomycin D, indicating it was due to transcriptional activation of the gene. Chelation of intracellular Ca(2+) suppressed the expression of c-fos markedly, while removal of external Ca(2+) did not. These results suggest that Ca(2+) mobilized from intracellular stores played a major role in the tributyltin-induced transcriptional activation of c-fos in PC12 cells.

Hypoglycemia and hyperglycemia were induced in mice by fasting and by injecting with glucose, respectively. These and normally fed (normoglycemic) animals were exposed to 0.5% carbon monoxide (CO) for 10 min. This altered concentrations of energy metabolites in the brain, including decreases in phosphocreatine (PCr) and increases in creatine and lactate. The only difference between normoglycemic and hypoglycemic mice was lower lactate in the latter. In hyperglycemic mice, PCr and ATP were better preserved during CO exposure and lactate was lower than in normoglycemic mice. Blood glucose concentrations correlated well with glucose but not with lactate in the brain. Thus, moderate hypo- or hyperglycemia seems not to exacerbate CO-induced alterations of brain energy metabolism.

After the rat lung was flushed with 40 mmol/L phosphate-buffered saline solution containing 160 mEq/L of sodium and the heart-lung block was immersed in the same solution for 6 hours, the lung was reperfused with diluted autologous blood and ventilated for 30 minutes. Pulmonary artery pressure, airway pressure, difference in oxygen tension between inflow and outflow perfusate, and wet to dry weight ratio of the tissue were determined. Lungs treated at pH 7.75 showed a significantly lower pulmonary artery pressure and wet to dry weight ratio than those treated at pH 7.26 or 7.96. Organs preserved at 10 degrees C showed a significantly lower pulmonary artery pressure than those preserved at 5 degrees or 15 degrees C. When the effect of smaller temperature variations was examined, the 12 degrees C group showed a significantly lower pulmonary artery pressure than the 10 degrees C group. Thus, the isolated rat lung reperfusion model appears to provide an efficient screening system to examine the preservation solution or other conditions for lung preservation. Under the present experimental conditions, the optimal pH and temperature for rat lung preservation seem to be 7.75 and 12 degrees C, respectively.

Although l -carnitine has been reported to have protective effects against ammonia toxicity, conflicting results have also been presented and the mechanisms underlying the protection, if any, are not clear. In the present study, we examined the effects of l -carnitine, d -carnitine and acetyl- l -carnitine on the neurotoxicity of ammonia. Administration of ammonium acetate (15 mmol/kg) to mice caused Seizures, elevation of blood ammonia and urea concentrations, and marked alterations of brain energy metabolites. Pretreatment with either l -carnitine, d -carnitine or acetyl- l -carnitine reduced the frequency of the Seizures, prolonged the time until the first fit, lowered the levels of ammonia in the blood and brain, and suppressed the alterations of brain energy metabolites caused by hyperammonemia. There was no significant difference between l - and d -carnitine in the potency to inhibit the Seizures. In addition, there was no difference between the two chemicals in the potency to decrease the ammonia contents in the blood and brain, or to suppress the alterations of energy metabolites in the brain. When compared with l -carnitine, however, acetyl- l -carnitine better preserved ATP in the brain, while it lowered ammonia in the blood and brain less markedly. These results show that l -carnitine and its analogues do have the potential to suppress the neurotoxicity of ammonia. Moreover, the results suggest that the protective effects of carnitine against the toxicity of ammonia are systemic, that the action of acetyl- l -carnitine may differ from that of l - or d -carnitine, and that the “classical” function of carnitine is not the sole mechanism underlying the suppression of the neurotoxicity of ammonia.

The effects of ethylene oxide (EO), acrylamide, N,N′ -methylene-bis-acrylamide (bis-acrylamide), and methyl mercury chloride (MMC) on brain creatine kinase (CK) activity were examined in vivo . EO and acrylamide, both of which cause central–peripheral distal axonopathy, inhibited CK activity in the brain and spinal cord. On the other hand, neither bis-acrylamide, a nonneurotoxic analogue, nor MMC which causes neuronopathy affected brain CK activity significantly. The inhibition of CK may play a role in the pathogenesis of distal axonal degeneration in the central and peripheral nervous systems.

Administration of ammonium acetate to mice caused seizures and alterations of brain energy metabolites. Pretreatment of animals with L-carnitine suppressed the frequency of the seizures and prolonged the latency to the first fit. When examined using the 'freeze clamp' method, brain energy metabolites were well preserved and the elevation of ammonia was less marked on administration of L-carnitine. Thus, L-carnitine suppresses ammonia-induced seizures and biochemical alterations of the brain in mice.

c-Jun NH 2 -terminal kinase (JNK), also known as stress-activated protein kinase (SAPK), is activated primarily by inflammatory cytokines and environmental stresses including toxic metal exposure. To reveal the upstream kinase responsible for JNK activation by toxic metals, the phosphorylation status and the activity of JNK were examined in mouse embryonic stem (ES) cells lacking MKK4 or MKK7 following exposure to CdCl 2 or HgCl 2 . Treatment with CdCl 2 or HgCl 2 induced the phosphorylation of JNK in a dose- and time-dependent manner in wild-type ES cells. In both mkk4 −/− and mkk7 −/− ES cells, CdCl 2 - or HgCl 2 -induced phosphorylation and activation of JNK were suppressed significantly. However, in mkk7 −/− ES cells treated with CdCl 2 and HgCl 2 , JNK activation was not abolished (suppressed by 56% and 78%, respectively). These findings suggest that the full activation of JNK by toxic metal exposure requires both MKK4 and MKK7, and these upstream kinases might contribute differentially in JNK activation between mouse ES cells exposed to CdCl 2 and HgCl 2 .

Pentachlorophenol (PCP) inhibited acetylcholinesterase (AchE) activity in human erythrocyte membranes with high cooperativity. The Hill coefficient for the inhibition was 4-5 in “untreated” membranes. Differences in the temperature (13, 25 and 37°) or treatment with 1% Triton X-100 did not clearly affect the cooperativity which, however, increased after the erythrocyte membranes were treated with 2-mercaptoethanol and iodoacetatic acid, suggesting that higher cooperativity in the inhibition of AchE by PCP may reflect conformational changes of AchE. Thus, PCP may be useful for the study of AchE in human erythrocytes.

Ischemia (decapitation) caused marked changes of energy metabolites in the mouse brain. However, the changes were clearly less severe, when the mouse was pre-treated with l -carnitine. This suggests that l -carnitine may protect the brain from the ischemic attack. Since d -carnitine showed a similar effect, the effects may be due to mechanism(s) other than the ‘classical’ function of carnitine.

In vitro, both acrylamide and N,N′-methylene-bis-acrylamide inhibited creatine kinase (CK) activity from brain or sciatic nerve with almost the same potency. In vivo, only acrylamide (50 mg/kg b. wt./day, 8 days) caused paralysis of hind limbs and suppressed CK activity in cerebrum, cerebellum, spinal cord and muscle in rats. Bis-acrylamide (50 mg or 100 mg/kg b. wt./day, 8 days) caused no paralysis nor inhibition of CK activity in any tissue examined. Definite inhibition of CK was not found in sciatic nerve from rats given either chemical. The inhibition of CK may play a role in the genesis of toxicity of acrylamide especially in the central nervous system.

Erythrocyte membrane in myotonic dystrophy (MyD) was studied with respect to acetylcholinesterase (AchE), an enzyme localized on the external surface of the membrane. The activity was determined over the temperature range of 7–41 °C (in hemolysates) and 25–41 °C (in ghosts). The activity, the transition temperature in the Arrhenius plots, and the activation energy either above or below the transition temperature did not differ between MyD and controls. The Hill coefficient for the inhibition by fluoride was approximately 1 in MyD and in controls at 13, 25 and 37°C. The dose of fluoride for 50% inhibition of the enzyme activity differed between different temperatures but not between MyD and controls. There seems to be no gross abnormality in AchE or its environment in erythrocyte membrane in MyD.

Seven biopsied sural nerves from patients with beriberi were morphometrically evaluated. In teased fiber analysis the mean frequency of myelinated fibers showing axonal degeneration and segmental demyelination was 37.5 and 5.3%, respectively. In two cases with frequency of segmental demyelination higher than 5%, segmental demyelination was shown by statistical criteria to have occurred on certain fibers in a clustered fashion. Therefore, the segmental demyelination in beriberi may be secondary to axonal degeneration. Electromyographic findings and slow improvement of muscle weakness were compatible with axonal degeneration of motor fibers. Determinations of fiber densities revealed preferential decrease of the density of large myelinated fibers with the preservation of the density of small myelinated and unmyelinated fibers. The preferential nerve fiber involvement in beriberi was not associated with pain in the lower limbs and this fact is contrary to the expectation of the proponents of the gate control theory.

Pi uptake and acetoacetate formation were suppressed by psychosine (galactosylsphingosine) in rat liver mitochondria. Besides, reduced form of cytochrome c increased in the reaction mixture which contained psychosine. Using reduced form of cytochrome c as substrate, less than 5 μM of psychosine (0.1 μmoles/mg of mitochondrial protein) inhibited cytochrome c oxidase by more than 50%. The inhibition was completely reversed by 1% human serum albumin. Thus, a lipid which is produced in the brain has a powerful and yet reversible inhibitory effect on an enzyme of cellular respiration.

The kidney tissue of the twitcher mice, a neurological mutant caused by a genetic deficiency of galactosylceramidase, contains enormously increased amounts, up to 50 times normal, of galactosylceramide. The finding is in sharp contrast with those in the enzymatically equivalent human disease, globoid cell leukodystrophy (Krabbe disease), in which no specific abnormal accumulation of galactosylceramide occurs despite the same genetic block in the catabolic pathway. This indicates that the same genetic defect can result in entirely different consequences in different species. Caution must be exercised even when "authentic animal models" are utilized for studies of human diseases.