Dystonia induced by combined treatment with L-dopa and MK-801 in parkinsonian monkeys.

We examined whether the N-methyl-D-aspartate antagonist MK-801 (dizocilpine) would reverse parkinsonism or potentiate the effects of L-dopa in primates treated with 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP). In contrast to its effect in rodent models, treatment with MK-801 (0.1 mg/kg) caused bradykinesia and ataxia in parkinsonian primates, but no locomotor stimulation. Coadministration of MK-801 (0.1 mg/kg) with L-dopa (20 mg/kg) induced marked dystonia accompanied by bradykinesia and ataxia. Dystonia was not induced by either treatment given alone. These findings indicate that MK-801 should not be advocated as an adjunct to dopamine agonist therapy in Parkinson's disease.     

乳酸脱氢酶实验检测细胞新陈代谢的实验研究

目的 探索LDH实验检测细胞活力的可行性。方法 原代培养骨髓细胞和软骨细胞，用LDH实验测定上述两组细胞的活力，并与镜下活体观察到细胞的生长状况相比较。与目前比较成熟的测定细胞活力的MTS实验的测得的值相比较。结果 LDH实验对上述两组细胞的活力的测定结果与镜下活体观察到的结果相符合。与MTS实验的测得的结果经统计学处理无显著差异。结论 LDH实验可用于细胞活力的直接测定，而对活细胞的生存、繁殖无影响。     

Mutations in the Ca(2+)-sensing receptor gene cause autosomal dominant and sporadic hypoparathyroidism

Parathyroid hormone secretion is negatively regulated by a 7- transmembrane domain, G-protein coupled Ca(2+)-sensing receptor. We hypothesized that activating mutations in this receptor might cause autosomal dominant hypoparathyroidism (ADHP). Consistent with this hypothesis, we identified, in two families with ADHP, heterozygous missense mutations in the Ca(2+)-sensing receptor gene that cosegregated with the disorder. None of 50 normal controls had either mutation. We also identified a de novo, missense Ca(2+)-sensing receptor mutation in a child with severe sporadic hypoparathyroidism. The amino acid substitution in one ADHP family affected the N-terminal, extracellular domain of the receptor. The other mutations involved the transmembrane region. Unlike patients with acquired hypoparathyroidism, patients with these mutations had hypercalciuria even at low serum calcium concentrations. Their greater hypercalciuria presumably reflected activation of Ca(2+)-sensing receptors in kidney cells, where the receptor negatively regulates calcium reabsorption. This augmented hypercalciuria increases the risk of renal complications and thus has implications for the choice of therapy.      

Thalidomide-induced neuropathy and genetic differences in drug metabolism

A pharmacogenetic predisposition to thalidomide-induced neuropathy has been investigated. Differences of drug metabolism were examined in 16 patients with severe orogenital ulceration, who were treated with thalidomide (200 mg/day) for 0.3–5.0 years. Eight had evidence of early peripheral neuropathy according to nerve conduction studies. Rates of C-hydroxylation, N-acetylation, and conjugation reactions with sulphate, glucuronide and glycine, were tested with the probe compounds debrisoquine, sulphadimidine, paracetamol and aspirin, respectively. Urinary drug metabolites were analysed by high pressure liquid chromatography. Results were compared with 16 healthy age- and sex-matched volunteers.Of the patients 6.25% and 13.3% of the controls had a poor Debrisoquine Hydroxylator Ratio (DMR); none of the patients with neuropathy had a poor DMR as compared to 12.5% without neuropathy. Of the patients 40.0% and 35.7% of the controls were slow acetylators; 28.6% with neuropathy were slow acetylators as opposed to 50% without neuropathy. Similarly, there were no significant differences in rates of conjugation between groups. All unaffected patients were active smokers, whereas only two of those with neuropathy smoked. Cumulative dose or duration of therapy were unrelated to risk of neuropathy.In conclusion, changes of nerve conductivity are a frequent and unpredictable adverse effect of thalidomide (200 mg/day), although smoking may have a protective action against their development. Nerve conduction studies are required before and during treatment, irrespective of the prescribed dose.     

Dibenzo[a,l]pyrene-induced DNA adduction, tumorigenicity, and Ki-ras oncogene mutations in strain A/J mouse lung

Dibenzo[a,l]pyrene (DB[a,l]P), an environmental polycyclic aromatic hydrocarbon, is the most potent carcinogen ever tested in mouse skin and rat mammary gland. In this study, DB[a,l]P was examined for DNA adduction, tumorigenicity, and induction of Ki-ras oncogene mutations in tumor DNA in strain A/J mouse lung. Groups of mice received a single i.p. injection of 0.3, 1.5, 3.0, or 6.0 mg/kg DB[a,l]P in tricaprylin. Following treatment, DNA adducts were measured at times between 1 and 28 days, while tumors were counted at 250 days and analyzed for the occurrence of point mutations in codons 12 and 61 of the Ki-ras oncogene. DB[a,l]P in strain A/J mouse lung induced six major and four minor DNA adducts. Maximal levels of adduction occurred between 5 and 10 days after injection followed by a gradual decrease. DB[a,l]P-DNA adducts in lung tissue were derived from both anti- and syn-11,12- dihydroxy-13,14-epoxy- 11,12,13,14-tetrahydrodibenzo[a,l]pyrene (DB[a,l]PDE) and both deoxyadenosine (dAdo) and deoxyguanosine (dGuo) residues in DNA as revealed by cochromatography. The major adduct was identified as a product of the reaction of an anti-DB[a,l]PDE with dAdo in DNA. DB[a,l]P induced significant numbers of lung adenomas in a dose- dependent manner, with the highest dose (6.0 mg/kg) yielding 16.1 adenomas/mouse. In tricaprylin-treated control animals, there were 0.67 adenomas/mouse. Based on the administered dose, DB[a,l]P was more active than other environmental carcinogens including benzo[a]pyrene. As a function of time-integrated DNA adduct levels, DB[a,l]P induced lung adenomas with about the same potency as other PAHs, suggesting that the adducts formed by DB[a,l]P are similar in carcinogenic potency to other PAHs in the strain A/J mouse lung model. Analysis of the Ki- ras mutation spectrum in DB[a,l]P-induced lung tumors revealed the predominant mutations to be G-->T transversions in the first base of codon 12, A-->G transitions in the second base of codon 12, and A-->T transversions in the second or third base of codon 61, concordant with the DNA adduct profile.      

Cancer‐associated somatic DICER1 hotspot mutations cause defective miRNA processing and reverse‐strand expression bias to predominantly mature 3p strands through loss of 5p strand cleavage

Our group recently described recurrent somatic mutations of the miRNA processing gene DICER1 in non‐epithelial ovarian cancer. Mutations appeared to be clustered around each of four critical metal‐binding residues in the RNase IIIB domain of DICER1. This domain is responsible for cleavage of the 3′ end of the 5p miRNA strand of a pre‐mRNA hairpin. To investigate the effects of these cancer‐associated 'hotspot' mutations, we engineered mouse DICER1‐deficient ES cells to express wild‐type and an allelic series of the mutant DICER1 variants. Global miRNA and mRNA profiles from cells carrying the metal‐binding site mutations were compared to each other and to wild‐type DICER1. The miRNA and mRNA profiles generated through the expression of the hotspot mutations were virtually identical, and the DICER1 hotspot mutation‐carrying cells were distinct from both wild‐type and DICER1‐deficient cells. Further, miRNA profiles showed that mutant DICER1 results in a dramatic loss in processing of mature 5p miRNA strands but were still able to create 3p strand miRNAs. Messenger RNA (mRNA) profile changes were consistent with the loss of 5p strand miRNAs and showed enriched expression for predicted targets of the lost 5p‐derived miRNAs. We therefore conclude that cancer‐associated somatic hotspot mutations of DICER1, affecting any one of four metal‐binding residues in the RNase IIIB domain, are functionally equivalent with respect to miRNA processing and are hypomorphic alleles, yielding a global loss in processing of mature 5p strand miRNA. We further propose that this resulting 3p strand bias in mature miRNA expression likely underpins the oncogenic potential of these hotspot mutations. Copyright © 2012 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd.