Andrew Chen

Modulation of benzodiazepine receptor binding: insight into pharmacological efficacy.

The effects of GABA on the binding of analogues of benzodiazepines, triazolopyridazines, beta-carbolines and imidazodiazepines were examined in ligand/[3H] flunitrazepam competition experiments. GABA increased the potency of anxiolytics, like flunitrazepam, whereas the potency of benzodiazepine antagonists, like Ro15-1788, was largely insensitive to the influence of GABA. Several other agents including pyrazolopyridines, barbiturates and etomidate caused a chloride dependent enhancement of [3H] flunitrazepam binding but not an enhancement of [3H] propyl-beta-carboline-3-carboxylate binding.

I. High performance liquid chromatography of pharmacologically active amines and peptides in biological materials

Precise and quantitative reversed-phase high performance liquid chromatographic (HPLC) procedures are described which can be used in biogenic amine and neuropeptide research. The amine procedure was applied to various pharmacological matrices including plasma, heart tissue and brain. The use of peptide HPLC as an analytical tool for various neuropeptides is illustrated by studies on des-tyrosine-gamma-endorphin (DT gamma E) metabolism in the brain and the stability of an ACTH (ORG-2766) analogue during a chronic infusion in rats. The power of HPLC as a research tool in peptide pharmacology is described, discussed and demonstrated as an aid in the understanding of the pharmacological effects of exogenous peptides and the function of the brain.

Identification of β-endorphin-(6–17) as the principal metabolite of des-tyrosine-γ-endorphin (DTγE) in vitro and assessment of its activity in neurotransmitter receptor binding assays

Abstract Des-tyrosine-γ-endorphin (β-endorphin-(2–17); DTγE) lacks direct in vitro activity at dopaminergic receptors, but does inhibit in vivo [ 3 H]spiperone binding in various rat brain areas. The principal objective of these studies was to test the hypothesis that DTγE may exert its selective, neuroleptic-like activity through an active metabolite. Accordingly, DTγE was incubated at 37°C in a whole rat brain homogenate of neutral pH after which samples were prepared for HPLC analysis. The major, heat-stable metabolite of DTγE was identified as the clinically active, β-endorphin related fragment, β-endorphin-(6–17). The β-endorphin sequences 4–17, 5–17, 10–17, 12–17 and 2–16 were also present but in minor amounts. Identical results were obtained studying DTγE metabolism using rat striatal tissue slices. Neurotransmitter receptor binding experiments showed that β-endorphin-(6–17) was inactive at central dopaminergic, serotonergic, muscarinic, benzodiazepine and opiate receptors measured in vitro. Thus, like DTγE, β-endorphin-(6–17) differs from classical neuroleptics in that it does not inhibit in vitro [ 3 H]spiperone binding in the corpus striatum, frontal cortex or mesolimbic areas of the rat brain. It may be that DTγE and β-endorphin-(6–17) exert their selective neuroleptic-like activity through an indirect inhibition of central dopaminergic activity, possibly in combination with an in vivo antagonism of the postsynaptic dopamine receptor.

Metabolic clearance rate and half-time disappearance rate of human N-terminal and adrenocorticotropin of pro-opiomelanocortin in the rat: A comparative study

In metabolic clearance rate (MCR) and plasma half-time disappearance rate (t 1/2) of human N-terminal (1-76) and adrenocorticotropin(hACTH 1-39) of pro-opiomelanocortin were compared after intravenous bolus injection of both peptides simultaneously into rat. The level of immunoreactive (IR) hNT and IR-ACTH in plasma and urine samples were measured by specific and homologous radioimmunoassays (RIAs). The MCR and hNT and hACTH were 3.01 +/- 0.20 ml/min (M +/- S.D., N = 4) and 2.04 +/- 0.06 ml/min, respectively (p less than 0.05), The curve for the disappearance rate of IR-hNT was triphasic (rapid t 1/2 = 0.96 +/- 0.39 min, intermediate t 1/2 = 6.7 +/- 2.25 min, and slow t 1/2 = 74 +/- 15.8 min), while that of IR-ACTH was biphasic (rapid t 1/2 = 3.3 +/- 0.68 min, and slow t 1/2 = 41.5 +/- 3.03 min) as analyzed by the non-linear least-squares methods. Statistically significant difference (p less than 0.01) was found between IR-hNT and IR-hACTH in the rapid t 1/2 and in the slow t 1/2. Subsequent analysis of pooled plasma sample (30 min post-injection) by molecular sieve chromatography on Sephadex G-50 superfine column revealed that the majority of IR-hNT (90-95%) and IR-ACTH (60-70%) are co-chromatographed with [125I]iodo hNT and [125I]iodo ACTH respectively. Similarly, gel filtration of pooled urine sample (120 min post-injection) on Sephadex G-50 superfine revealed that 80-90% of IR-hNT and less than 50% of IR-ACTH co-eluted with [125I]iodo hNT and [125I]iodo ACTH, respectively. Smaller molecular forms of IR-hNT and IR-ACTH were definitely apparent in the urine sample. In conclusion, hNT has a larger MCR and a longer half-time disappearance rate (t 1/2) than IR-hACTH in rat plasma and it appears that hNT is more resistant to degradation by plasma and by kidney than hACTH.

A non-equilibrium 24-hour vasopressin radioimmunoassay: development and basal levels in the rat brain

Immunocytochemical studies, localizing vasopressin (AVP) within the neurohypophyseal system and in the CNS, paved the way for quantification of this peptide 2,3,23,25. Subsequently, concentrations of arginine vasopressin (AVP) in neural tissue have been measured by radioimmunoassay ( R I A ) 6,83°-12,15,2°,21. Earlier studies reported AVP levels in plasma, urine and cerebrospinal f l u i d 1,7,1236-18,22. Sensitivity of the RIAs utilized in these studies ranged from 0.1 to 4.0 pg of AVP 12,15, while the incubation times varied from 2 days (using a double antibody technique) to 5 and 7 days 12,15-28. While these techniques are generally useful for measuring low levels of AVP, we found it difficult to produce equilibrium assays with a detectable level below 1.0 pg AVP. We are interested in investigating extrahypothalamic vasopressin which is present in femtomole to picomole quantities 6,8,1°,JI,2~. In at least two studies 3,18, it was reported that AVP levels were below the detection of presently utilized AVP assays. Therefore, we examined the possibility that a rapid, non-equilibrium RIA, which would increase the sensitivity of d e t e c t i o n 9,19,22, could be applied to measure this psychologically relevant neuropeptide 5,~3,~4,24. In this paper we report a highly sensitive nonequilibrium RIA which can be performed within 24 h. To demonstrate the sensitivity of this RIA, brain regions from rat were examined for vasopressin content. Known amounts of synthetic vasopressin (Calbiochem-Behring) were serially diluted from 100 pg to 0.1 pg in 50 mM Na+/K + phosphate buffer containing 0.1% bovine serum albumin and 0.05% sodium azide, pH 7.4. A 50/~1 aliquot of a 1:7500 dilution of vasopressin antiserum (AVP-Ab; Calbiochem-Behring) or a 50/~1 aliquot ofa 1:75,000 dilution of AVP-Ab (gift of D. A. Fisher) was added to each 10 × 75 mm disposable glass tube. Following the preincubation period, approximately 10,000 cpm of [12q]AVP (50/~1, Calbiochem-Behring; spec. act. 2200 Ci/mmol) was added to each tube. Two control tubes, one containing antiserum and [1251]AVP and the other containing only [125I]AVP were included for estimates of maximum binding achieved and for separation effectiveness, respectively. Final assay volume was 300/~1. All assays were performed in duplicate. Bound [125I]AVP was separated from free [I25I]AVP by the addition of 500/~1 of dextrancoated charcoal. Assay tubes were then vortexed, incubated at 0-4 °C for 15 min and centrifuged at 1500 × g for 15 rain (Beckman TJ6). The supernatant was counted in a gamma counter (Tracor Model 1197 gamma counter; Eft. = 85%). The percent of maximum bound (bound/max bound) [I25I]AVP was plotted against

Chronic Migraineurs Form Carboxyhemefibrinogen and Iron-Bound Fibrinogen.

Chronic migraine (CM) is a disabling painful condition that is associated with dementia and thrombotic disease. It has been proposed that carbon monoxide (CO) and iron may play a role in CM, and CO and iron are products of the heme oxygenase system which is widespread within the brain. Further, CO and iron enhance plasmatic coagulation in part via a fibrinogen-dependent mechanism. Thus, our goal was to determine whether patients with CM had experienced carboxyhemefibrinogen formation, iron bound fibrinogen formation and plasmatic hypercoagulability. Nonsmokers with CM were recruited after informed, written consent. Blood was collected, anticoagulated with sodium citrate, and then centrifuged with plasma stored at -80ºC. Carboxyhemefibrinogen formation, iron bound fibrinogen formation and coagulation kinetics were determined via thrombelastographic methods. Patient results were compared with laboratory values generated from normal control plasmas. Incidence (95% confidence intervals) of the various parameters was determined using the Clopper-Pearson method. Twenty-six CM patients (24 female) were recruited; they were 46±12 years old. With regard to fibrinogen modification, 88.5% (69.8%-97.6%) of CM patients had formation of carboxyhemefibrinogen, iron bound fibrinogen, or both. With regard to coagulation, 42.3% (23.4%-63.1%) of patients had abnormally decreased time to clot initiation, 80.8% (60.6%-93.4%) had abnormally large velocity of clot formation, and 46.2% (26.6%-66.7%) had abnormally strong clot strength. Patients with CM have a large incidence of carboxyhemefibrinogen and iron bound fibrinogen formation and hypercoagulability. Confirmatory and potential therapeutic clinical trials targeting CO and iron modified hypercoagulation as a source of pain and vascular disease in CM patients are indicated.

Variable effects of gender and Western diet on lipid and glucose homeostasis in aged PCSK9-deficient C57BL/6 mice 性别与西方饮食对CSK9基因缺陷的PC57BL/6老龄小鼠的脂质以及葡萄糖内稳态的不同影响: Aged PCSK9-null mice: gender and diet effects

Proprotein convertase subtilisin/kexin‐type 9 (PCSK9) downregulates clearance of plasma cholesterol by liver. Its inactivation increases this clearance, reducing cardiovascular risk. However, a lack of PCSK9 could also lead to cholesterol accumulation in pancreatic islet beta cells, impairing insulin secretion. We reported earlier that 4‐month‐old male PCSK9‐deficient (KO) C57BL/6 mice were hyperglycemic and insulin‐insufficient relative to their wild‐type (WT) counterparts. Here, we examined how gender and diet affect lipid and glucose homeostasis in these mice at 8 months of age.

Structural and biochemical investigation of heptad repeat derived peptides of human SARS corona virus (hSARS-CoV) spike protein.

hSARS-CoV is the causative agent for SARS infection. Its spike glycoprotein (S) is processed by host furin enzyme to produce S1 and S2 fragments, the latter being crucial for fusion with the host membrane. This takes place via formation of a coiled coil 6-helix bundle involving N and C-terminal heptad repeat domains (HR-N and HR-C) of S2. Several fluorescent and non-fluorescent peptides from these domains were synthesized to examine their interactions by circular dichroism, thermal denaturation, native-page, mass spectrometry and fluorescence spectroscopy studies. Data revealed that HR-C domains (1153-1189), (1153-1172) and (1164-1184) all exhibit potent binding interactions with HR-N(892-931) domain. These peptides may find useful therapeutic applications in SARS intervention.