Engineered zinc-binding sites confirm proximity and orientation of transmembrane helices I and III in the human serotonin transporter

The human serotonin transporter (hSERT) regulates neurotransmission by removing released serotonin (5-HT) from the synapse. Previous studies identified residues in SERT transmembrane helices (TMHs) I and III as interaction sites for substrates and antagonists. Despite an abundance of data supporting a 12-TMH topology, the arrangement of the TMHs in SERT and other biogenic amine transporters remains undetermined. A high-resolution structure of a bacterial leucine transporter that demonstrates homology with SERT has been reported, thus providing the basis for the development of a SERT model. Zn2+-binding sites have been utilized in transporters and receptors to define experimentally TMH proximity. Focusing on residues near the extracellular ends of hSERT TMHs I and III, we engineered potential Zn2+-binding sites between V102 or W103 (TMH I) and I179-L184 (TMH III). Residues were mutated to either histidine or cysteine. TMH I/III double mutants were constructed from functional TMH I mutants, and Zn2+ sensitivity was assessed. Dose-response assays suggest an approximately twofold increase in sensitivity to Zn2+ inhibition at the hSERT V102C/M180C and approximately fourfold at the V102C/I179C mutant compared to the hSERT V102C single mutant. We propose that the increased sensitivity to Zn2+ confirms the proximity and the orientation of TMHs I and III in the membrane. Homology modeling of the proposed Zn2+-binding sites using the coordinates of the Aquifex aeolicus leucine transporter structure provided a structural basis for interpreting the results and developing conclusions.     

Bacteria divert resources from growth for magellanic penguin chicks

The influence of bacteria on the growth of their wild avian hosts is unknown. We tested experimentally whether administration of a wide‐spectrum antibiotic (cephalosporine) during early development of magellanic penguin (Spheniscus magellanicus) chicks had any effect on their growth rates in the wild. Chicks that were injected in two occasions with cephalosporine grew faster than control untreated chicks. The positive effect of medication on nestling growth disappeared after the treatment ceased, did not alter haematological indices indicative of health status, had no influence on chick survival until near independence and was related to a changed bacterial composition of the faecal microbiota of treated chicks when compared with that from control chicks. These results were similar to those obtained for poultry with antimicrobials promoting growth and chick nutrient assimilation rates. Gram‐positive bacilli in the diphtheroid genus Corynebacterium are likely candidates to cause decreased growth rates in magellanic penguin chicks.     

Cardiovascular Risk of Essential Hypertension: Influence of Class,Number, and Treatment-Time Regimen of Hypertension Medications

A number of observational studies have found that treated hypertensive patients, even those with controlled clinic blood pressure (BP), might have poorer prognosis than untreated hypertensives. Different trials have also shown that relatively low cardiovascular disease (CVD) risk cannot be achieved in high-risk hypertensive patients, leading to the belief they have a “residual CVD risk” that cannot be attenuated by conventional treatment. All these conclusions disregard the facts that the correlation between BP level and CVD risk is stronger for ambulatory than clinic BP and that the BP-lowering efficacy and effects on the 24-h BP pattern of different classes of hypertension medications exhibit statistically and clinically significant treatment-time (morning versus evening) differences. Accordingly, we evaluated the potential differential administration-time-dependent effects on CVD risk of the various classes of hypertension medications and the number of them used for therapy in the MAPEC (Monitorización Ambulatoria para Predicción de Eventos Cardiovasculares, i.e., Ambulatory Blood Pressure Monitoring for Prediction of Cardiovascular Events) study, a prospective, open-label, blinded-endpoint trial on 2156 hypertensive patients (1044 men/1112 women), 55.6?±?13.6 (mean?±?SD) yrs of age, randomized to ingest all prescribed once-a-day hypertension medications upon awakening or the entire daily dose of ≥1 of them at bedtime. Ambulatory BP was measured for 48?h at baseline, and again annually or more frequently (quarterly) when adjustment of treatment was necessary to achieve ambulatory, i.e., awake and asleep, BP control. CVD risk according to the number and classes of medications used at the final evaluation was calculated by comparison with that of 734 normotensive subjects who were identically followed and remained untreated. After a median follow-up of 5.6 yrs, CVD risk of hypertensive patients randomized to ingest all medications upon awakening was progressively higher with increase in the number of medications (adjusted hazard ratio [HR]: 1.75, 2.26, 3.02, and 4.18 in patients treated with 1, 2, 3, and ≥4 medications daily, respectively; p?<?.001 compared with normotensive subjects). CVD risk was markedly lower in patients ingesting ≥1 medications at bedtime (HR: .35, 1.45, .94, and 2.28 with 1, 2, 3, and ≥4 medications daily, respectively), and even lower in patients ingesting all medications at bedtime (HR: .35, .39, .87, and .79 with 1, 2, 3, and ≥4 medications daily, respectively). Patients ingesting ≥1 medications at bedtime evidenced significantly lower CVD risk than those ingesting all medications upon awakening, independent of class. Greater benefits were observed for bedtime compared with awakening treatment with angiotensin-II receptor blockers (ARBs) (HR: .29 [95% confidence interval, CI .17–.51]; p?<?.001) and calcium channel blockers (HR: .46 [95% CI: .31–.69]; p?<?.001). CVD risk was similar for all six classes of tested hypertension medications in patients randomized to ingest all of them upon awakening. Among patients randomized to ingest ≥1 medications at bedtime, however, ARBs were associated with significantly lower HR of CVD events than ingestion of any other class of medication also at bedtime (p?<?.017). We document significantly reduced CVD risk among hypertensive patients ingesting medications at bedtime, independent of the number of hypertension medications required to achieve proper ambulatory BP control. These findings challenge the current belief of “residual CVD risk,” as a bedtime-treatment regimen of current hypertension medications, even in risk-high patients, can reduce such risk. (Author correspondence: rhermida@uvigo.es)     

Synthesis, enantiomeric resolution, and selective C-11 methylation of a highly selective radioligand for imaging the norepinephrine transporter with positron emission tomography

Reboxetine, 2-[alpha-(2-ethoxyphenoxy)benzyl]morpholine, is a highly selective norepinephrine transporter (NET) blocker that has been used for the treatment of depression. Its methyl analogue, 2-[alpha-(2-methoxyphenoxy)benzyl]morpholine (MRB), has been radiolabeled with C-11 for studies of the NET system with positron emission tomography (PET). The normethyl precursor, 2-[alpha-(2-hydroxyphenoxy)benzyl]morpholine (desethylreboxetine), was synthesized in 6% overall yield via a multi-step regio- and stereo-specific synthesis, starting from a mono-O-protected catechol. The resulting racemic mixture of desethylreboxetine was resolved by chiral HPLC to provide the (2S,3S) and (2R,3R) enantiomers in >98% enantiomeric excess. These enantiomers were then used as precursors for radiosynthesis to prepare enantiomerically pure individual 11C-labeled MRB enantiomers for comparative PET studies in baboons. Selective C-11 methylation at the phenolic oxygen with [11C]CH3I was achieved in the presence of excess base. After HPLC purification, racemic ((2S,3S)/(2R,3R)) or enantiomerically pure ((2S,3S) or (2R,3R)) [11C]MRB was obtained in 61-74% decay-corrected radiochemical yields from [11C]CH3I in a synthesis time of 40 min with a radiochemical purity of >96% and a specific activity of 1.7-2.3 Ci/micromol (63-85 GBq/micromol) corrected from the end of bombardment (EOB).     

Semicarbazide-Sensitive Amine Oxidase (SSAO) in the Brain

Semicarbazide-sensitive amine oxidase (SSAO) is widely distributed in almost tissues. However, its presence in brain microvessels is still controversial. The affinity of SSAO towards benzylamine (Bz) is considerably higher than that of monoamine oxidase (MAO). SSAO plays a role in the toxicity of several environmental and endogenous amines. SSAO-mediated production of toxic aldehydes has been proposed to be related to pathophysiological conditions. The most potent of inhibition of SSAO in monkey brain was observed by tricyclic antidepressant drug imipramine, as compared to tetracyclic drug maprotiline or non-cyclic drug nomifensine. An endogenous SSAO modulator in rat brain cytosol after immobilization stress (IMMO) was found and that this inhibitor could be induced by IMMO. SSAO activity in rat brain might be regulated by the level of this inhibitor. Semicarbazide, a SSAO inhibitor, enhances the formation of OH products of efflux/oxidation due to 1-methyl-4-phenylpyridinium ion (MPP⁺). The precise physiological functions of SSAO could play an important role in the control of energy balance in adipose tissue. SSAO could play an important role in the regulation of adipocyte homeostasis.     

Purification and characterization of dihydropyrimidine dehydrogenase enzyme from sheep liver and determination of the effects of some anaesthetic and antidepressant drugs on the enzyme activity

Dihydropyrimidine dehydrogenase (DPD, E.C. 1.3.1.2) was purified from sheep liver with a yield of 16.7%, purification fold of 407.5 and specific activity of 0.705?EU/mg proteins. The purification procedure consisted of ammonium sulphate fractionation, DEAE ion exchange chromatography and 2′,5′-ADP Sepharose-4B affinity chromatography. The molecular weight determined by SDS-PAGE and was found 111?kDa. Optimum pH, ionic strength temperature and stable pH were determined as 8.0, 0.9?mM, 50?°C and 6.0, respectively. The kinetic parameters (K_m and V_max) of the enzyme were determined with NADPH as 22.97?μM and 0.17?EU/mL, respectively. The same parameters were determined with uracil as 17.46?μM and 0.14?EU/mL, respectively. Additionally, in vitro inhibitory effects of some antidepressant drugs including escitalopram, fluoxetine, mirtazapine, haloperidol and some anaesthetic drugs including propofol and lidocaine were investigated against DPD. In addition, IC₅₀ values for each active drug obtained for escitalopram, fluoxetine, mirtazapine, haloperidol, propofol and lidocaine were determined as 1736.11, 13.24, 86.65, 99.03, 0.21 and 15.07?μM, respectively.     

贯叶连翘抗抑郁研究新进展

贯叶连翘,又名圣约翰草,是传统中药材之一,圣约翰草在德国用于抗抑郁症已有几百年的历史。贯叶连翘提取物对轻度和中度抑郁症患者和动物模型都有很好疗效,最新药理学研究表明贯叶连翘提取物中抗抑郁的主要成分是贯叶金丝桃素。贯叶金丝桃素是神经递质5-羟色胺(5-HT)、多巴胺(DA)、去甲肾上腺素(NE)的非竞争性重吸收抑制剂,贯叶金丝桃素还可以抑制突触体对γ氨基丁酸(GABA)和L-谷氨酸(L-glu)的重吸收,其作用机理至今还不甚明了,研究表明它的作用很可能是通过提高突触体细胞内钠离子浓度或通过降低突触体内突触小泡的跨膜pH梯度实现的。