Inhibition of translocation of nascent apolipoprotein B across the endoplasmic reticulum membrane is associated with selective inhibition of the synthesis of apolipoprotein B.
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In HepG2 cells, inhibition of apolipoprotein B100 (apoB) translocation across the endoplasmic reticulum by an microsomal triglyceride transfer protein (MTP) inhibitor (CP-10447) in the presence of N-acetyl-leucinyl-norleucinal, a proteasomal inhibitor, results in accumulation of newly synthesized apoB in the translocation channel. Here we demonstrated that such accumulation led to a specific reduction of apoB synthesis. ApoB mRNA levels remained unchanged, but we observed reduced rates of elongation of nascent apoB in puromycin-synchronized cells pretreated with MTP inhibitor. This observation was consistent with a longer half-ribosome transit time for the synthesis of apoB in MTP-inhibited cells. Initiation of translation of apoB mRNA was not impaired by MTP inhibition. Overall, these findings suggest that translocation arrest of apoB in the endoplasmic reticulum channel can exert a selective and negative effect on the synthesis of apoB at the stage of elongation. (+info)
Thin-layer chromatographic detection and identification of methaqualone metabolites in urine.
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A procedure for detecting methaqualone and identifying methaqualone metabolites in urine by thin-layer chromatography is described and evaluated. Urine is hydrolyzed with HCl or NaIO4, adjusted to pH 9.5, and extracted with chloroform. The chloroform extract is evaporated, reconstituted in methanol, applied to fluorescent silica-gel plates, and developed with ethyl acetate:methanol:ammonium hydroxide (28%) (85:10:5 by vol). Methaqualone use is detected by a pattern of four metabolites, which can be seen under ultraviolet light or are made visible by acidified iodoplatinate reagent. Synthetic methaqualone metabolites are used for identification and to compensate for procedural variables. More than 250 positive urine specimens were correctly identified by this method. Hydrolyzed natural and synthetic metabolites were identical by several criteria. (+info)
The late addition of core lipids to nascent apolipoprotein B100, resulting in the assembly and secretion of triglyceride-rich lipoproteins, is independent of both microsomal triglyceride transfer protein activity and new triglyceride synthesis.
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Although microsomal triglyceride transfer protein (MTP) and newly synthesized triglyceride (TG) are critical for co-translational targeting of apolipoprotein B (apoB100) to lipoprotein assembly in hepatoma cell lines, their roles in the later stages of lipoprotein assembly remain unclear. Using N-acetyl-Leu-Leu-norleucinal to prevent proteasomal degradation, HepG2 cells were radiolabeled and chased for 0-90 min (chase I). The medium was changed and cells chased for another 150 min (chase II) in the absence (control) or presence of Pfizer MTP inhibitor CP-10447 (CP). As chase I was extended, inhibition of apoB100 secretion by CP during chase II decreased from 75.9% to only 15% of control (no CP during chase II). Additional studies were conducted in which chase I was either 0 or 90 min, and chase II was in the presence of [(3)H]glycerol and either BSA (control), CP (inhibits both MTP activity and TG synthesis),BMS-1976360-1) (BMS) (inhibits only MTP activity), or triacsin C (TC) (inhibits only TG synthesis). When chase I was 0 min, CP, BMS, and TC reduced apoB100 secretion during chase II by 75.3, 73.9, and 53.9%. However, when chase I was 90 min, those agents reduced apoB100 secretion during chase II by only 16.0, 19.2, and 13.9%. Of note, all three inhibited secretion of newly synthesized TG during chase II by 80, 80, and 40%, whether chase I was 0 or 90 min. In both HepG2 cells and McA-RH7777 cells, if chase I was at least 60 min, inhibition of TG synthesis and/or MTP activity did not affect the density of secreted apoB100-lipoproteins under basal conditions. Oleic acid increased secretion of TG-enriched apoB100-lipoproteins similarly in the absence or presence of either of CP, BMS, or TC. We conclude that neither MTP nor newly synthesized TG is necessary for the later stages of apoB100-lipoprotein assembly and secretion in either HepG2 or McA-RH7777 cells. (+info)
Radioimmunoassay of methaqualone and its monohydroxy metabolites in urine.
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A commercial radioimmunoassay kit was evaluated for efficacy in detecting methaqualone or its metabolites in urine of persons receiving this drug. The drug and its unconjugated 3'- and 4'-monohydroxy metabolites could be detected equally well. The unconjugated alpha-monohydroxy metabolite was about 80% as reactive and the unconjugated 6-monohydroxy metabolite reacted only very weakly. Quantitation of the conjugated metabolites was less sensitive than of unconjugated. Nineteen urine specimens which reacted positively to radioimmunoassay and which thin-layer chromatography had shown to contain methaqualone and its metabolites were also examined by gas-liquid chromatography. Those specimens that reacted strongly to radioimmunoassay contained high concentrations of the drug or its metabolites. In the specimens examined by gas-liquid chromatography, the apparent concentrations of the metabolites were generally higher than those of the drug itself. Methaqualone in combination with its unconjugated metabolites reacted additively with the radioimmunoassay, resembling the same concentration of parent drug alone. Detection limits were between 10-200 mug/liter. (+info)
Cannabis and other drug use among trauma patients in three South African cities, 1999-2001.
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OBJECTIVE: To assess the extent of cannabis and other drug use among patients presenting with recent injuries at trauma units in Cape Town, Port Elizabeth and Durban from 1999 to 2001. DESIGN: Cross-sectional surveys were conducted during a 4-week period at each of the above sites in 1999, 2000 and 2001. The concept of an idealised week was used to render representative samples. OUTCOME MEASURES: Cause of injury and biological markers to assess use of cannabis, methaqualone (Mandrax), opiates, cocaine, amphetamine, and methamphetamine. RESULTS: Over half of all patients tested experienced violent injuries. Excluding opiates, across sites and over time between 33% and 62% of patients tested positive for at least one drug (N = 1565). In most cases the drugs were cannabis and/or methaqualone. While no inter-city differences were found, male patients were typically more likely to test positive for drugs in general and specific drugs such as cannabis and the cannabis/methaqualone ('white pipe') combination than female patients. Drug positivity was higher in 2001 than in the previous 2 years in Cape Town, and patients injured as a result of violence in Cape Town and Durban were more likely to test positive for drugs than patients with certain other types of injuries. CONCLUSIONS: Drug use among trauma patients has remained consistently high for each of the 3 study periods. Efforts to combat the abuse of drugs such as cannabis and methaqualone would appear to be paramount in reducing the burden of injuries on health care services. The study has raised numerous issues requiring further research. (+info)
Cross-physical dependence of several drugs in methaqualone-dependent rats.
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We investigated the characteristics of physical dependence on methaqualone. Rats were made physically dependent on methaqualone by the use of the drug-admixed food (DAF) method for 33 days. Pentobarbital, barbital, ethanol and diazepam were cross-administered against methaqualone to evaluate the degree of suppression of methaqualone withdrawal signs as an index for the cross-physical dependence liability of these drugs to methaqualone. To evaluate the cross-physical dependence liability, we used AUC of body weight loss and withdrawal scores between the first cross-administration (9 hr after the withdrawal) and 27 hr after the withdrawal. AUC of weight loss was significantly suppressed by the four test drugs as compared to each control. Withdrawal scores were also significantly inhibited by the cross-administration of barbital, ethanol and diazepam. Considering that the rats given barbital or ethanol fell asleep after the cross-administration, diazepam seems to cause the strongest suppression of methaqualone withdrawal signs among the four test drugs. Thus, physical dependence on methaqualone may be similar by nature to that on benzodiazepines rather than barbiturates and alcohol. (+info)
Gas-liquid chromatography of undervatized drugs after chromatographic extraction from blood.
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We have developed an integrated method that overcomes the two main procedural difficulties of gas-liquid chromatography, namely, solvent-solvent extraction and chemical derivatization. Drugs are extracted from serum by column chromatography on granular diatomaceous earth (kieselguhr). Subsequent gas-liquid chromatography of underivatized samples can be performed on either of two liquid phases. A mixed liquid phase, used for quantitative gas-chromatographic assay on patients with a known therapeutic regimen, has enabled quantitation of 12 drugs in serum. Alternatively, a single liquid phase, used with the mixed liquid phase, permits the gas-chromatographic identification of unknown drugs on the basis of the characteristic pattern of the two relative retention times; by this approach more than 40 drugs have been identified in cases of suspected intoxication, both in serum and in gastric aspirate. Besides providing ease of performance and wide applicability, the proposed procedure offers a degree of precision and accuracy that compares favorably with established methods. (+info)
A comparison of sleep patterns in natural and mandrax- and tuinal-induced sleep.
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A within-subject comparison of the effects on the overnight sleep EEG of 1 tablet of Mandrax (containing methaqualone base 250 mg. and diphenhydramine hydrochloride 25 mg.) and 200 mg. Tuinal (equal parts of quinalbarbitone sodium and amylobarbitone sodium) in 14 normal subjects is reported.Mandrax-induced sleep was not significantly different from natural sleep in the duration of light, moderate, deep and REM phases. Tuinal produced a significant reduction in REM sleep (P < 0.01) compared with natural sleep and with Mandrax-induced sleep. (+info)