S-adenosylmethionine metabolism and DNA methylation in hydrazine-treated rats

The treatment of rats with hepatotoxic doses of hydrazine (NH2-NH2) induces the rapid formation of 7-methylguanine and O6-methylguanine in liver DNA. The methyl moiety in these reactions might be derived from the cellular S-adenosylmethionine pool because radioactivity administered to these rats as methionine rapidly appears in the DNA as methylated guanine. An increased incorporation of radioactivity into 5-methylcytosine was previously reported followed by subsequent suppression. This increased radiolabeling of 5-methylcytosine coincided with time of maximal DNA guanine methylation. To determine the nature of S-adenosylmethionine metabolism during the period of DNA methylation induced by hydrazine treatment, and to determine if the increased radiolabeling of 5-methylcytosine at this time reflected an actual increase in 5-methylcytosine synthesis, liver DNA synthesis and S-adenosylmethionine levels and turnover were assayed. Liver S-adenosylmethionine concentrations varied slightly between control rats and hydrazinetreated rats during the first five hours after hydrazine administration, and no difference was detectable in the incorporation of administered [3H]methionine into S-adenosylmethionine. Because S-adenosylmethionine specific radioactivity in hydrazine-treated rats was not different from control rats, the previously observed increased radiolabeling of 5-methylcytosine appeared to represent an actual increase in synthesis. This conclusion was supported by finding that incorporation of radioactive thymidine into DNA was also accelerated immediately following hydrazine administration, again followed by a decrease. 5-Methylcytosine sythesis, therefore, appears to follow DNA synthesis during hydrazine toxicity, and formation of 7-methylguanine and O6-methylguanine in liver DNA of hydrazine-treated rats occurs during a short period of increased DNA sythesis and 5-methylcytosine formation very early in hydrazine toxicity.     

Substrate phosphorylation in the protein kinase Cgamma knockout mouse

The phosphorylation state of three identified neural-specific protein kinase C substrates (RC3, GAP-43/B-50, and MARCKS) was monitored in hippocampal slices of mice lacking the gamma-subtype of protein kinase C and wild-type controls by quantitative immunoprecipitation following 32Pi labeling. Depolarization with potassium, activation of glutamate receptors with glutamate, or direct stimulation of protein kinase C with a phorbol ester increased RC3 phosphorylation in wild-type animals but failed to affect RC3 phosphorylation in mice lacking the gamma-subtype of protein kinase C. Our results suggests the following biochemical pathway: activation of a postsynaptic (metabotropic) glutamate receptor stimulates the gamma-subtype of protein kinase C, which in turn phosphorylates RC3. The inability to increase RC3 phosphorylation in mice lacking the gamma-subtype of protein kinase C by membrane depolarization or glutamate receptor activation may contribute to the spatial learning deficits and impaired hippocampal LTP observed in these mice.     

Exercise training, postprandial hypertriglyceridemia, and LDL subfraction distribution

The purpose of this study was to examine differences in postprandial hypertriglyceridemia (PP-HTG) and low density lipoprotein (LDL) subfraction distribution among groups of men and women with different fitness levels. Fifty-four men and women (ages 30-53 yr) were recruited based on their previous two-year activity level: sedentary (S), recreational exercisers (R), and endurance trained (T). After a 24-h dietary preparation, blood was collected, and LDL subfractions were separated and analyzed for cholesterol (C) and apoprotein B100. Plasma triglyceride (TG) concentration was assessed before and at 2, 4, 6, and 8 h after fat meal. PP-HTG was significantly higher for the S group compared with the two activity groups. LDL3-C and LDL3-apoprotein B100 were significantly higher for the S group compared with the T group and for men compared with women. These findings suggest that both recreational and competitive aerobic training are associated with a lower TG response after a fatty meal. However, higher volume aerobic training may be necessary to reduce the number of dense LDL molecules and their cholesterol content.     

Routine portable chest radiographs in the medical intensive care unit: effects and costs

OBJECTIVE: To determine the effects and net costs of routine chest radiographs in a medical intensive care unit (ICU). DESIGN: A prospective, cohort study. A survey of experts in critical care and pulmonary diseases was undertaken to assess the effect of routine radiographs on patient management. SETTING: Medical ICU of a university hospital. PATIENTS: Eighty randomly selected patients admitted to a medical ICU. Two hundred fourteen experts were surveyed; 118 (55%)/214 responded. MEASUREMENTS AND MAIN RESULTS: Daily interviews with medical ICU clinicians were conducted to assess the radiographic findings in the routine radiographs and actions taken based on these findings. Experts evaluated the findings, their importance, the actions taken, and the probability of complications if the actions had not been taken at that time. Experts also predicted increases in length of stay associated with these complications. Presence of radiographic findings, changes in management because of the findings, net costs of routine chest radiographs, cost per finding that prompted an action, and expected changes in length of stay resulting from the actions were also assessed. Seventy-two (33%) of 221 routine radiographs (95% confidence interval: 25% to 39%) had findings, of which 44 (61%) were judged important, and 18 (8%, 95% confidence interval: 5% to 12%) prompted actions. Experts predicted that each action averted, on average, 2.1 +/- 1.7 days (SD) in the medical ICU. Mean savings per routine radiograph was $98. Net savings from routine chest radiographs remained after sensitivity analysis for expected change in length of stay, percentage of patients with routine radiographs, and percentage of routine radiographs that produce changes in management. CONCLUSION: The policy of obtaining routine chest radiographs in the medical ICU is effective and results in net savings.     

Nonclinical model for assessing gastric effects of bisphosphonates

Mechanisms by which ketones potentiate manganese-bilirubin (Mn-BR)-induced cholestasis are unknown. The purpose of the present study was to investigate the effect of methyl isobutyl ketone (MiBK), a widely used ketonic solvent, at the level of the bile canalicular membrane (BCM) and to verify if altered membrane lipid dynamics could be involved in MiBK-potentiated Mn-BR cholestasis. Male Sprague-Dawley rats were exposed 4 hr/day for 3 days to MiBK vapors (200 or 600 ppm). Eighteen hours after the last exposure, manganese (Mn, 4.5 mg/kg) was given i.v. followed 15 min later by bilirubin (BR, 25 mg/kg). Rats were killed 30 min after BR; liver cell plasma membranes (bile canalicular and sinusoidal), microsomes, mitochondria, and cytosol were isolated by differential centrifugation. Lipids were extracted and cholesterol was measured in each fraction. After Mn-BR and MiBK exposure (600 ppm), results indicated a marked increase in BCM cholesterol content compared to rats exposed to air only. This increase was greater than that due to Mn-BR or MiBK given alone. Also, results indicated that cholesterol increased in a dose-related fashion in BCM after MiBK exposure, whereas PM cholesterol remained unaltered. To identify the source of the increased BCM cholesterol and to permit distinction between de novo cholesterol synthesis and subcellular shifts, the hepatic lipid pool was labeled in vivo with [3H]-cholesterol and [2-14C]-mevalonic acid, a cholesterol synthesis precursor. Results showed that after 600 ppm MiBK exposure, 14C-labeled cholesterol was greater than 3H-labeled cholesterol, indicating that the contribution of de novo cholesterol synthesis to the total cholesterol content of the various isolated hepatocellular fractions was more important than the contribution of intracellular pools. Therefore, increased BCM cholesterol content and enhanced accumulation of newly synthesized cholesterol appear to be involved in MiBK potentiation of Mn-BR-induced cholestasis.     

A novel small conductance Ca2+-activated K+ channel blocker from Oxyuranus scutellatus taipan venom. Re-evaluation of taicatoxin as a selective Ca2+ channel probe

Taicatoxin, isolated from the venom of the Australian taipan snake Oxyuranus scutellatus, has been previously regarded as a specific blocker of high threshold Ca2+ channels in heart. Here we show that taicatoxin (in contrast to a range of other Ca2+ channel blockers) interacts with apamin-sensitive, small conductance, Ca2+-activated potassium channels on both chromaffin cells and in the brain. Taicatoxin displays high affinity recognition of 125I-apamin acceptor-binding sites, present on rat synaptosomal membranes (Ki = 1.45 +/- 0.22 nM) and also specifically blocks affinity-labeling of a 33-kDa 125I-apamin-binding polypeptide on rat brain membranes. Taicatoxin (50 nM) completely blocks apamin-sensitive after-hyperpolarizing slow tail K+ currents generated in rat chromaffin cells (mean block 97 +/- 3%, n = 12) while only partially reducing total voltage-dependent Ca2+ currents (mean block 12 +/- 4%, n = 6). In view of these findings, the use of taicatoxin as a specific ligand for Ca2+ channels should now be reconsidered.