Redox active or thiol reactive? Optimization of rapid screens to identify less evident nuisance compounds

Compounds that exhibit assay interference or undesirable mechanisms of bioactivity are routinely encountered in assays at various stages of drug discovery. We observed that assays for the investigation of thiol-reactive and redox-active compounds have not been collected in a comprehensive review. Here, we review these assays and subject them to experimental optimization to improve their reliability. We demonstrate the usefulness of our assay cascade by assaying a library of bioactive compounds, chemical probes, and a set of approved drugs. These high-throughput assays should complement the array of wet-lab and in silico assays during the initial stages of hit discovery campaigns to pursue only hit compounds with tractable mechanisms of action.     

晕可宁颗粒对急、慢性梅尼埃病模型的影响

目的　探讨晕可宁颗粒的主要药效学 ,为临床提供药效学资料及治疗学基础。方法　采用三氯甲烷破坏豚鼠一侧膜迷路感受器模型 ,探讨受试药对眼震颤、摆头及旋转的影响 ;采用内淋巴囊和内淋巴管阻塞手术复制豚鼠膜迷路实验性膜迷路积水模型 ,研究内耳组织平均中阶面积 (SMA)增加率及形态学的变化。结果　抑眩宁阳性对照组、晕可宁颗粒 (8、16g/kg)模型给药组豚鼠眼球震颤次数减少 ,差异有显著意义 (P <0 .0 5 )。成功复制了不同程度膜迷路积水豚鼠模型 ,表现为前庭膜重度膨出 ,前庭阶缩小 ,膜蜗管增大 ,SMA增加率变大 ,差异有显著意义 (P <0 .0 1) ;晕可宁颗粒灌胃后可减轻豚鼠实验性膜迷路积水的程度 ,差异有显著意义 (P <0 .0 1) ;但与空白对照组比较SMA增加率差异无显著意义 (P >0 .0 5 )。结论　晕可宁颗粒可以减轻内淋巴囊积水程度 ,对梅尼埃病症状有对抗治疗作用。     

Gastro-duodenal digestion products of the major peanut allergen Ara h 1 retain an allergenic potential

BACKGROUND: The process of gastro-duodenal digestion may play a role in determining the allergenic properties of food proteins. The sensitizing and allergenic potential of digestion products of highly degraded allergens, such as the major peanut allergen Ara h 1, is currently under debate. We evaluated the effect of in vitro gastro-duodenal digestion of Ara h 1 on T cell reactivity and basophil histamine release. METHODS: An in vitro model of gastro-duodenal digestion was used to investigate changes in the allergenic properties of Ara h 1 using in vitro assays monitoring T cell reactivity (proliferation, cytokine production) and histamine release of basophils from peanut allergic individuals. The digestion process was monitored using an SDS-PAGE gel. RESULTS: In vitro gastric digestion led to rapid degradation of Ara h 1 into small fragments M(r) L5600. Gastric digestion did not affect the ability of Ara h 1 to stimulate cellular proliferation. Gastro-duodenal digestion significantly reduced its ability to stimulate clonal expansion (P<0,05; Wilxocon's signed rank test). The Th-2 type cytokine polarization of T cells from peanut allergic donors (IFN-gamma/IL-13 ratio and IFN-gamma/IL-4 ratio of CFSE(low) CD4(+) T cells) remained unchanged regardless of the level of digestion. Histamine release of basophils from peanut allergic individuals was induced to the same extent by native Ara h 1 and its digestion products. CONCLUSION: Gastro-duodenal digestion fragments of Ara h 1 retain T cell stimulatory and IgE-binding and cross-linking properties of the intact protein.     

Differing time courses between Δlactate and mitochondrial respiration during coronary occlusion and after reperfusion in canine hearts

Summary The present study was designed to clarify whether or not a difference between arterial and venous lactate (lactate) levels is useful for evaluation of mitochondrial function in ischemia-reperfused myocardium. In the first experiment, 12 dogs were divided into 2 groups: 10-min occlusion of the left anterior descending coronary artery (LAD) followed by 10-min reperfusion, or 30-min occlusion followed by 40-min reperfusion, were performed. The lactate levels in the femoral artery and the great cardiac vein were measured enzymatically. Lactate was reversed immediately after occlusion. Ten min and 20 min were required for the recovery of lactate in the 10-min-occlusion with 10-min-reperfusion, and 30-min-occlusion with 40-min-reperfusion groups, respectively. In the second experiment, 36 dogs were divided into 6 groups: 10-min occlusion of LAD; 10-min occlusion with 10-min reperfusion; 30-min occlusion; and 30-min occlusion with 10-, 20-, or 40-min reperfusion were performed. Mitochondria from normal and occluded or reperfused areas were prepared, and the respiratory function of the mitochondria was measured polarographically. No significant decreases in the mitochondrial function were observed in the 10-min-occlusion, and 10-min-occlusion with 10-min-reperfusion groups. On the other hand, respiratory function of mitochondria was impaired by 30-min occlusion and was not improved by 10- or 20-min reperfusion. Significant recovery in the mitochondrial function was observed after 40-min reperfusion. That is, differing recovery time courses between lactate and the mitochondrial function were observed.     

Fatal intoxications in Denmark following intake of morphine from opium poppies

Summary In Denmark it is legal to grow opium poppies for the production of poppy seeds and until 1986 for decoration purposes, too. Danish poppy capsules contain 0.3–5 mg morphine per capsule and the content of morphine in opium exuded from the capsules may amount to 24%. This has resulted in misuse as both fresh and dried poppy capsules have been used for the production of opium tea. During the period 1982–1985, seven casualties occurred among drug addicts in Denmark which were solely or partly caused by these opium poppies.     

A Biotin–Avidin-Based Enzyme Immunoassay for βh-Endorphin

An avidin–biotin enzyme-linked immunosorbent assay (ELISA) is described for _h-endorphin (_h-EP). Microtiter plates coated with commercially available antibodies were used together with _h-EP tracer derivatives that were biotinylated in positions 24, 28, and 29 via a C₆ spacer arm. Nonspecific binding of biotinylated derivatives to the microtiter plates was blocked with a mixture of 1% casein and 10% ethanolamine in 0.1 M NaHCO₃. A sequential saturation procedure using a high-affinity antiserum in combination with an avidin–alkaline phosphatase complex matched the sensitivity of reported radioimmunoassays (RIAs), with a detection limit of 0.5 fmol/assay. The intra- and interassay coefficients of variation were 5 and 12%, respectively. Results obtained by ELISA and RIA showed good correlations (r = 0.95). The -EP concentration in extracted rat plasma after high-performance liquid chromatographic (HPLC) fractionation was determined by this method to be 1600 fmol/ml.     

Brain kinetics of 11 C-labelled L-tryptophan and 5-hydroxy-L-tryptophan in the Rhesus monkey

Summary 5-Hydroxy-L-tryptophan labelled with 11 C is introduced as a tracer for the in vivo assessment of brain serotonin synthesis in the Rhesus monkey using positron emission tomography, PET. Increasing radioactivities were seen in the striatal area in contrast to that seen in other brain regions. Following 11 C-labelled L-tryptophan an even spread of brain radioactivity was seen. This selective increase most probably results from the decarboxylation of tracer and retention of formed products since no striatal increase of radioactivity was seen when 5-hydroxy-L-tryptophan labelled with 11 C in the carboxy-position was administered. Furthermore, pretreatment of the monkey with a centrally active decarboxylase inhibitor (NSD 1015,10 mg/kg) did not lead to increased striatal radioactivities after the administration of 5-hydroxy-(-11C)-L-tryptophan. The selective utilization of the radiotracer in the striatal area increased with a rate constant calculated to be 0.0055 ± 0.0015 min^–1 (n = 5) using the surrounding brain as reference area. A non-significant influence of radiolabelled metabolites to the rate constants measured was shown after pretreatment of the monkeys with selective and non-selective monoamine oxidase inhibitors, respectively. These results may give a basis for the use of the new tracer 5-hydroxy-(-11 C)-L-tryptophan in PET-studies of brain serotonin metabolism in health and disease.     

Neuropathy in igMλ paraproteinemia

Summary In axonal neuropathies associated with IgM paraproteinemia, reports of antigen specificity of the M-protein are few. A patient with IgM paraproteinemia presented with progressive mononeuritis multiplex. IgM was found deposited in striking amounts in endoneurium and shown to bind specifically to neural proteins and chondroitin sulfates. Direct immune mechanisms, as well as the physical effects of IgM deposition, likely contributed to the development of the neuropathy.     

Diffuse type of senile plaques in the cerebellum of Alzheimer-type dementia demonstrated byβ protein immunostain

Summary We studied senile plaques (SP) in the cerebella of six autopsied subjects with Alzheimer-type dementia (ATD) and ten non-ATD autopsied subjects between the ages of 78 and 90. Neither SP nor amyloid angiopathy (AA) was observed in any of the non-ATD subjects. In the four of the six ATD subjects, diffuse plaques in the molecular layer were seen as ill-defined areas of fine fibrillar materials by protein immunostaining with formic acid pretreatment, the modified Bielschowsky stain, and periodic acid-methenamine silver (PAM) stain. The plaques were not visible with Bodian, Congo red, or periodic acid-Schiff stains. Compact plaques in the Purkinje cell or in the granular cell layers were found in three of the six subjects. Their amyloid core was often surrounded by areolar amyloid deposits. AA was observed in three of the six subjects. The argyrophilia of the diffuse and compact plaques, demonstrated by the modified Bielschowsky and PAM stains, became undetectable when the sections were first treated with formic acid. Such treatment made the plaques immunoreactive with protein antiserum. The findings suggested that cerebellar diffuse plaques and compact plaques consist mainly of an amyloid component, and are characteristic of ATD.     

The reduction of water intake in rats caused by a low dose of apomorphine is unaltered by α-methyl-p-tyrosine: are autoreceptors not involved?

Summary Low doses of the dopamine (DA) agonist apomorphine (APO) induces a behavioural syndrome characterized by reduced spontaneous activity, reduced food and water intake and induction of yawning and penile erections. Traditionally these effects of APO have been considered to be caused by a preferential stimulation of DA autoreceptors, causing a decreased amount of transmitter at the postsynaptic receptors. If this is so, it could be hypothesized that 1) the same behavioural effects should be obtained if DA transmission is decreased by some other means, for example by synthesis inhibition, and that 2) the response to APO should be altered if DA transmission is already lowered.It was found that high doses of -methyl-p-tyrosine (-MPT; 50–200 mg/ kg) did not reduce water intake in thirsty rats, which low doses of APO do. It was further found that pretreatment with -MPT did not alter the response to APO. These results are difficult to reconcile with the DA autoreceptor hypothesis claiming that behavioural effects of low doses of APO are caused by a decreased release of DA. An alternative interpretation is that low doses of APO stimulates a certain population of sensitive postsynaptic D-2 receptors.