A dual‐tracer study of extrastriatal 6‐[18F]fluoro‐m‐tyrosine and 6‐[18F]‐fluoro‐l‐dopa Uptake in Parkinson's disease

6‐[¹⁸F]‐Fluoro‐l ‐dopa (FDOPA) has been widely used as a biomarker for catecholamine synthesis, storage, and metabolism—its intense uptake in the striatum, and fainter uptake in other brain regions, is correlated with the symptoms and pathophysiology of Parkinson's disease (PD). 6‐[¹⁸F]fluoro‐m‐tyrosine (FMT), which also targets l ‐amino acid decarboxylase, has potential advantages over FDOPA as a radiotracer because it does not form catechol‐O‐methyltransferase (COMT) metabolites. The purpose of the present study was to compare the regional distribution of these radiotracers in the brains of PD patients. Fifteen Parkinson's patients were studied with FMT and FDOPA positron emission tomography (PET) as well as high‐resolution structural magnetic resonance imaging (MRI). MRI's were automatically parcellated into neuroanatomical regions of interest (ROIs) in Freesurfer ( http://surfer.nmr.mgh.harvard.edu ); region‐specific uptake rate constants (K_occ) were generated from coregistered PET using a Patlak graphical approach. The essential findings were as follows: (1) regional K_occ were highly correlated between the radiotracers and in agreement with a previous FDOPA studies that used different ROI selection techniques; (2) FMT K_occ were higher in extrastriatal regions of relatively large uptake such as amygdala, pallidum, brainstem, hippocampus, entorhinal cortex, and thalamus, whereas cortical K_occ were similar between radiotracers; (3) while subcortical uptake of both radiotracers was related to disease duration and severity, cortical uptake was not. These results suggest that FMT may have advantages for examining pathologic changes within allocortical loop structures, which may contribute to cognitive and emotional symptoms of PD. Synapse 68:325–331, 2014 . © 2014 Wiley Periodicals, Inc.     

The place of cladribine in the treatment of chronic lymphocytic leukemia: a 10-year experience in Poland

Cladribine (2-CdA) is structurally similar to another purine analog, fludarabine (FA), recently accepted in several centers as the first-line treatment in chronic lymphocytic leukemia (CLL). Unfortunately, there is less experience with the use of 2-CdA than with FA in patients with CLL in the majority of Western countries. In the last decade we performed several phase II studies and two phase III randomized trials to evaluate the activity and toxicity of 2-CdA in previously treated and untreated patients with CLL. We have also compared the results of Polish studies with the data presented by other investigators. Similarly to FA this agent has been found to be more effective in previously untreated CLL than in patients refractory to or relapsed after conventional therapy with alkylating agents. In different studies the overall response (OR) rate ranged from 70 to 85% and complete response (CR) from 10 to 47%. Higher CR and OR rates in CLL patients treated with 2-CdA and prednisone than with chlorambucil and prednisone were confirmed in our multicenter, randomized study. Subsequently, we performed a multicenter, randomized study comparing 2-CdA alone with a combination of 2-CdA and cyclophosphamide (CC) or cyclophosphamide and mitoxantrone (CMC). Our updated results seem to indicate that the CC program used as a first-line therapy in CLL gives higher CR and OR and better elimination of minimal residual disease (MRD) than 2-CdA alone. CC is also less myelotoxic than CMC. More recently, we have undertaken a phase II study to determine the efficacy and toxicity of 2-CdA combined with the anti-CD20 monoclonal antibody rituximab in CLL and other refractory or relapsed indolent lymphoproliferative disorders. The preliminary results seem to be better than in similar patients previously treated in our institution with 2-CdA alone. In conclusion, the studies performed in the last decade in Poland and other countries have shown that 2-CdA used alone or in combination with other agents is, similarly to FA, a highly active and relatively safe agent in previously treated and untreated patients with CLL.     

Efficacy of Combined Treatment with Lanreotide and Cabergoline in Selected Therapy-Resistant Acromegalic Patients

The aim of this study was to evaluate the efficacy of a 6-month treatment with lanreotide (LAN) (60–90 mg/month) alone and combined with cabergoline (CAB) (1.5-3 mg/week) in 10 acromegalic patients previously demonstrated to be poor responders to octreotide (OCT) (0.6 mg/day) alone and combined with quinagolide (CV) (0.6 mg/day).All patients had previously undergone unsuccessful surgery and none of them received radiotherapy. Immunohistochemistry showed intense positive GH staining in all adenomas, positive PRL staining in 5 adenomas and faint ACTH or FSH/LH positive staining in other 2 adenomas. Moderately elevated serum PRL levels (35 and 47 ng/ml) were recorded in two patients. Fasting plasma IGF-I and serum GH levels were assayed at baseline and 30, 60, 90 and 120 days after each treatment. Gallbladder ultrasonography and sellar MRI were performed before and after 6 months of OCT and LAN treatments.After OCT treatment circulating GH and IGF-I levels remained elevated in all patients, while after 3 months of combined OCT+CV treatment, serum GH levels were suppressed (below 2.5 ng/ml) in only 1 patient. Significant increase of the percent GH (83.9±4.3 vs. 70.3±5.6%, p<0.01) and IGF-I suppression (54±4.4 vs. 45.3±5.7, p<0.01) and decrease of the nadir of GH (8.5±1.2 vs. 14.6±1.9 ng/ml, p<0.01) and IGF-I (400.9±32.8 vs. 462.1±45.1 ng/ml) were obtained with the combined treatment when compared to OCT treatment alone. After a 15–30 days wash-out, circulating GH and IGF-I levels significantly increased up to pretreatment level in all patients. After 6 months of treatment with LAN, suppression of serum GH was achieved in 1 patient, but no difference in GH (66.3±6.3%) and IGF-I (43.9±4.6%) suppression was recorded in comparison to OCT treatment. After 3 months of treatment with LAN combined with CAB, suppression of serum GH and normalization of plasma IGF-I levels was achieved in 4 and 5 patients, respectively. Percent suppression of GH (88.1±2.1%) and IGF-I (57.5±2.8%) was significantly greater with the combined treatment than with LAN treatment alone. In the 7 patients with evident residual mass no change was documented by magnetic resonance imaging (MRI). None of the patients withdrew LAN+CAB treatment for poor tolerance, one patient had mild hypotension. Sludge was shown after 6 months of LAN treatment in one patient without notable change after 3 months of LAN+CAB treatment.In conclusion, the treatment with dopaminergic drugs such as CV and CAB, significantly increased the efficacy of somatostatin analogs, and can be used in combined therapy in poorly responsive patients.     

江苏徐州地区核苷类似物疗效不佳的慢性乙型肝炎621例血清乙型肝炎病毒耐药突变分析

目的 探讨江苏徐州地区经核苷类似物治疗疗效不佳的慢性乙型肝炎患者HBV耐药基因突变分布特征.方法 收集2010年8月～2013年5月在我院就诊的门诊及住院慢性乙型肝炎患者血清标本621份,均为经核苷类似物治疗3个月以上且血清HBV DNA阳性者,采用核酸扩增技术结合荧光标记探针杂交方法对乙型肝炎病毒DNA进行定量检测,其产物通过测序分析进行耐药突变的检测,率的比较采用x2检验.结果 621例患者HBV耐药总变异检出率为75.8％（471/621）,其中拉米夫定、阿德福韦酯、替比夫定和恩替卡韦分别为88.9％ （144/162）、53.7％（108/201）、89.0％（47/53）和88.0％ （22/25）,服用过2种或者2种以上抗病毒药物者为83.3％（150/180）,以上5组间变异率总检出率比较差异有统计学意义（P＜0.05）,其中阿德福韦酯组与其他各组分别比较差异均有统计学意义（P＜0.05）,其他各组两两比较差异均无统计学意义（P＜0.05）.耐药变异病例数构成比服用过2种或者2种以上抗病毒药物者（31.8％）最高,拉米夫定、阿德福韦酯、替比夫定和恩替卡韦分别为30.6％、22.9％、10.0％和4.7％,以上5组构成比比较总差异有统计学意义（P＜0.05）.除拉米夫定组与服用过2种或者2种以上抗病毒药物组之间差异无统计学意义（P＞0.05）,其他各组两两比较差异均有统计学意义（P＜0.05）.比较各位点变异率,所检测的25个位点的总差异有统计学意义（P＜0.05）,204位点耐药突变率最高（243例次,39.1％）,其次是180位点（184例次,29.6％）、181位点（123例次,19.8％）,这3个位点两两比较或者分别与其他各位点比较差异均有统计学意义（P＜0.05）.结论 204位点、180位点、181位点耐药突变检出率高,恩替卡韦及替比夫定经治患者较少进行HBV耐药突变检测.     

核苷和核苷酸类药物治疗慢性乙型肝炎的长期性

核苷和核苷酸类药物(NAs)已成功用于慢性乙型肝炎(CHB)治疗。目前一致认为,HBV复制是肝损伤和疾病进展的关键因素,因此CHB治疗的主要目的是最大限度地持续抑制HBV复制。现已证明,应用NAs长期治疗CHB可明显改善肝脏组织学、逆转肝纤维化或肝硬化,以及减少肝细胞癌的发生。本文对CHB长期治疗的必要性、临床获益及管理进行了综述。     

α-Fluorophosphonates reveal how a phosphomutase conserves transition state conformation over hexose recognition in its two-step reaction

β-Phosphoglucomutase (βPGM) catalyzes isomerization of β-d-glucose 1-phosphate (βG1P) into d-glucose 6-phosphate (G6P) via sequential phosphoryl transfer steps using a β-d-glucose 1,6-bisphosphate (βG16BP) intermediate. Synthetic fluoromethylenephosphonate and methylenephosphonate analogs of βG1P deliver novel step 1 transition state analog (TSA) complexes for βPGM, incorporating trifluoromagnesate and tetrafluoroaluminate surrogates of the phosphoryl group. Within an invariant protein conformation, the β-d-glucopyranose ring in the βG1P TSA complexes (step 1) is flipped over and shifted relative to the G6P TSA complexes (step 2). Its equatorial hydroxyl groups are hydrogen-bonded directly to the enzyme rather than indirectly via water molecules as in step 2. The (C)O–P bond orientation for binding the phosphate in the inert phosphate site differs by ∼30° between steps 1 and 2. By contrast, the orientations for the axial O–Mg–O alignment for the TSA of the phosphoryl group in the catalytic site differ by only ∼5°, and the atoms representing the five phosphorus-bonded oxygens in the two transition states (TSs) are virtually superimposable. The conformation of βG16BP in step 1 does not fit into the same invariant active site for step 2 by simple positional interchange of the phosphates: the TS alignment is achieved by conformational change of the hexose rather than the protein.Efficient enzyme catalysis of the manipulation of phosphates is one of the great achievements of evolution (1). Enzymes that operate on phosphate monoesters and anhydrides transfer the phosphoryl moiety, PO₃⁻, with rate accelerations approaching 10²¹ for monoesters, placing them among the most proficient of all enzymes (1). Phosphomutases, including α-phosphoglucomutase (αPGM) (2, 3) and β-phosphoglucomutase (βPGM) (4–6), phosphoglycerate mutase (7), α-phosphomannomutase (αPMM/PGM) (8), and N-acetylglucosamine-phosphate mutase (9), merit special attention because these enzymes have to be effective in donating a phosphoryl group to either of two hydroxyl groups that have intrinsically different reactivity. Only when both half-reactions of a phosphomutase are accessible to mechanistic analysis can the problem of how an enzyme accommodates two distinct chemistries within a single active site be resolved. Hexose 1-phosphate mutases, including enzymes central to glycolysis and other metabolic pathways, are well characterized (10, 11). They are generally activated by phosphorylation to form a covalent phosphoenzyme, which then donates its PO₃⁻ group to either of its substrates to deliver a common, transient, hexose 1,6-bisphosphate intermediate species. However, structural studies on phosphomutases are complicated by the rapid and often imbalanced equilibrium position between the substrates, and kinetic studies are problematic because of competitive, parallel pathways of enzyme activation and substrate inhibition (12, 13). As a result, transition states (TSs) for both half-reactions have not hitherto been accessible for mechanistic analysis.βPGM is the best-characterized hexose 1-phosphate mutase and is a member of the haloacid dehalogenase (HAD) superfamily (14), which has 58 HAD homologs in Homo sapiens (11). The key cellular role for βPGM is to support growth on maltose (14), which demands isomerization of β-d-glucose 1-phosphate (βG1P) via β-d-glucose 1,6-bisphosphate (βG16BP) into d-glucose 6-phosphate (G6P), a universal source of cellular energy. This interconversion is achieved via a transient, covalent phosphoenzyme intermediate involving an essential aspartic acid, Asp8, to conserve the phosphoryl group that migrates intermolecularly (Fig. 1). Mechanistically, this pathway demands the architecture of the catalytic site to be effective in promoting phosphoryl transfer from phospho-Asp8 to the 6-OH group of βG1P (step 1), followed by reverse phosphoryl transfer from 1β-OH of βG16BP to Asp8 (step 2).Open in a separate windowFig. 1.Reaction scheme and free energy profile for the conversion of βG1P into G6P via βG16BP catalyzed by βPGM. The phosphoryl transfer reaction between βG1P and the phosphoenzyme (βPGM^P) is step 1 (transferring phosphate is shown in blue), and the equivalent reaction between G6P and the phosphoenzyme is step 2 (transferring phosphate is shown in red). The two intermediate complexes are labeled βG16BP and βG61BP to indicate the two orientations of bound β-bisphosphoglucose. Intramolecular hydrogen bonds within the glucose phosphates are indicated in green. The PDB ID codes (shown in brown) for the structures of metal fluoride ground state analog (GSA) and TSA complexes are listed next to the corresponding steps. G6P is ca. 8 kJ⋅mol⁻¹ lower in free energy than βG1P at equilibrium (12). βG1P binds fivefold less tightly than G6P in an AlF₄⁻ TSA complex, corresponding to a binding energy difference of ca. 4 kJ⋅mol⁻¹. This places the TSA for step 1 (blue) ca. 12 kJ⋅mol⁻¹ (4 kJ⋅mol⁻¹ + 8 kJ⋅mol⁻¹) higher in free energy than the TSA for step 2 (red). The free energy levels of TS1 and TS2 are placed only approximately, using the assumption that the free energy difference (wavy arrows) between the TSA complex and the true TS is similar for both step 1 and step 2. The approximate relative free energy levels for the intermediate enzyme-bound states denoted with βG16BP and βG61BP are based on published data (13).Step 2 has been studied intensively, with analyses focused on structural studies of trifluoromagnesate (MgF₃⁻) and tetrafluoroaluminate (AlF₄⁻) transition state analogs (TSAs) and trifluoroberyllate ground state analogs for G6P complexes (4–6, 15). ¹⁹F NMR resonances for these complexes additionally have provided in situ probes for the electronic and protonic environment of the phosphate moiety in the active site (4–6, 15, 16). Such studies have confirmed a trigonal bipyramidal (tbp) TS associated with inline stereochemistry and general acid–base catalysis, following the rearrangement of near-attack conformers (6). By contrast, step 1, involving phosphorylation of the 6-OH group of βG1P, is not well understood. The corresponding TSA complexes hitherto have proved inaccessible; attempted crystallization of the mutase using βG1P with magnesium and fluoride provides the same MgF₃⁻ TSA complex as is formed directly with G6P because residual enzyme activity catalyzes mutation of βG1P into G6P at a rate competitive with crystallization of the complex (17). Similarly, although ¹⁹F NMR studies have identified a transient TSA complex for an AlF₄⁻ complex of βG1P, it readily isomerizes into the corresponding TSA complex of G6P (SI Appendix, Fig. S1). This impasse is resolved here by the synthesis and use of stable analogs of βG1P that resist mutase-catalyzed isomerization. Because it has been established that α-fluorination of 6-phosphonomethyl-6-deoxy-glucose (G6CP) can enhance or impair analog binding to glucose 6-phosphate dehydrogenase, depending on the stereochemistry of the α-fluorine substituent (18), we have synthesized both diastereoisomeric α-monofluoromethylenephosphonate analogs of βG1P, its methylenephosphonate analog, and the three corresponding phosphonate 1α-hydroxyl analogs. We have identified the two best-binding analogs by ¹⁹F NMR and measured their affinities with βPGM in TSA complexes using fluorescence titration. We have thereby obtained three high-resolution crystal structures of TSA complexes for step 1 of the mutase catalytic reaction. Their comparison with TSA complexes for step 2 establishes the substantially different binding modes for βG1P and G6P in their respective reactions.     

Polykystose rénale autosomique dominante : le traitement est-il pour demain ?

Autosomal dominant polycystic kidney disease (ADPKD) is the most frequent Mendelian inherited disorder. It covers 6.1% of incident ESRD patients in France in 2011. Long left untreated, this disease will soon benefit from targeted therapies currently under evaluation. Several molecules have already reached the stage of clinical trials: the evaluation of mTOR inhibitors yielded deceiving results and, more recently, 2 different molecules demonstrated a slight impact on the progression of total kidney volume (TKV): tolvaptan, vasopressin receptor-V2 inhibitor and somatostatin analogues; both of these molecules acting throughout the decrease of intracellular AMPc. The purpose of this review is to briefly describe the signaling pathways involved, then to present both the published and ongoing clinical trials and the promising molecules evaluated in murine models.     

高效液相色谱法结合主成分分析法分析云芝胞内糖肽中核苷和核碱的组成

目的:本实验利用超声提取-高效液相色谱测定技术,建立了云芝胞内糖肽原料和制剂中核苷和核碱组成的检测方法.方法:样品以水为溶剂超声提取,以月旭AQ-C18柱(4.6 mm×250 mm,5μm)为填充柱,流动相为水、甲醇梯度洗脱,检测波长为260nm,流速为1.0mL·min-1,进样体积为20μL.结果:云芝胞内糖肽由...