Diversified expression patterns of autotaxin,a gene for phospholipid‐generating enzyme during mouse and chicken development

Autotaxin (ATX), or nucleotide pyrophosphatase‐phosphodiesterase 2, is a secreted lysophospholipase D that generates bioactive phospholipids that act on G protein–coupled receptors. Here we show the expression patterns of the ATX gene in mouse and chicken embryos. ATX has a dynamic spatial and temporal expression pattern in both species and the expression domains during neural development are quite distinct from each other. Murine ATX (mATX) is expressed immediately rostral to the midbrain‐hindbrain boundary, whereas chicken ATX (cATX) is expressed in the diencephalon and later in the parencephalon‐synencephalon boundary. In the neural tube, cATX is expressed in the alar plate in contrast to mATX in the floor plate. ATX is also expressed in the hindbrain and various organ primordia such as face anlagen and skin appendages of the mouse and chicken. These results suggest conserved and non‐conserved roles for ATX during neural development and organogenesis in these species. Developmental Dynamics 236:1134–1143, 2007. © 2007 Wiley‐Liss, Inc.     

Evaluation of a chromogenic commercial assay using VWF-73 peptide for ADAMTS13 activity measurement

Introduction

Thrombotic thrombocytopenic purpura (TTP) is a thrombotic microangiopathy (TMA), related to a severe functional deficiency of ADAMTS13 activity (< 10% of normal). ADAMTS13 activity is thus crucial to confirm the clinical suspicion of TTP, to distinguish it from other TMAs, and to perform the follow-up of TTP patients.

Material and methods

We compared the performance of the commercial chromogenic assay Technozym® ADAMTS13 Activity ELISA (chromogenic VWF73 substrate, Chr-VWF73, Technoclone, Vienna, Austria), to that of our in-house FRETS-VWF73 used as reference method. A large group of 247 subjects (30 healthy volunteers and 217 patients with miscellaneaous TMAs) was studied.

Results

The lower limit of detection of the Chr-VWF73 was 3%, which is well adapted to the clinically relevant threshold for TTP diagnosis (10%). Our results showed a reasonable agreement between FRETS-VWF73 and Chr-VWF73 assays to distinguish samples with an ADAMTS13 activity < 10% from those with an ADAMTS13 activity > 10%. However, Chr-VWF73 assay provided false negative results in ~ 12% of acute TTP patients. Inversely, the Chr-VWF73 assay globally underestimated ADAMTS13 activity in detectable values ranging from 11 to 100% (with a great variability compared to FRETS-VWF73), which may be a concern for the follow-up of TTP patients in remission.

Conclusion

In-house assays developed and performed by expert laboratories remain the reference methods that should be used without limitation to control values provided by commercial assays when needed. Also, the development of an international reference preparation will be crucial to improve standardization.     

神经病理性疼痛发病机制及多学科综合治疗研究进展

神经病理性疼痛是神经系统损伤引起的慢性疼痛。单纯镇痛药物很难满意控制疼痛,如何有效控制疼痛是临床治疗的难点,针对神经痛的机制及对疼痛病人进行药物和多学科综合治疗被认为是目前研究热点。本文就该病的发病机制和药物及多学科综合治疗的研究进展进行综述。     

Effects of Different Variants in the ENPP1 Gene on the Functional Properties of Ectonucleotide Pyrophosphatase/Phosphodiesterase Family Member 1

Ectonucleotide pyrophosphatase/phosphodiesterase family member 1 (E‐NPP1), encoded by ENPP1, is a plasma membrane protein that generates inorganic pyrophosphate (PP_i), a physiologic inhibitor of hydroxyapatite formation. In humans, variants in ENPP1 are associated with generalized arterial calcification of infancy, an autosomal‐recessive condition causing premature onset of arterial calcification and intimal proliferation resulting in stenoses. ENPP1 variants also cause pseudoxanthoma elasticum characterized by ectopic calcification of soft connective tissues. To determine the functional impact of ENPP1 missense variants, we analyzed 13 putative pathogenic variants in vitro regarding their functional properties, that is, activity, localization, and PP_i generation. Transfection of eight of the 13 variants led to complete loss of NPP activity, whereas four mutants (c.1412A > G, p.Tyr471Cys; c.1510A > C, p.Ser504Arg; c.1976A > G, p.Tyr659Cys; c.2330A > G, p.His777Arg) showed residual activity compared with wild‐type E‐NPP1. One putative pathologic variant (c.2462 G > A, p.Arg821His) showed normal activity. The five mutants with normal or residual E‐NPP1 enzyme activity were still able to generate PP_i and localized in the plasma membrane. In this study, we identified a functional ENPP1 polymorphism, which was expected to be pathogenic till now. Furthermore, we identified four mutants (p.Tyr471Cys, p.Ser504Arg, p.Tyr659Cys, p.His777Arg) with residual E‐NPP1 function, which would be potential therapeutical targets for conformational‐stabilizing agents.     

Autotaxin inhibitors: a perspective on initial medicinal chemistry efforts

The lysophospholipase D enzyme, autotaxin (ATX), has been linked to numerous human diseases including cancer, neurophatic pain, obesity and Alzheimer's disease. Although the ATX protein was initially purified and characterized in 1992, a link to bioactive lipid metabolism was not made until 2002. In the past decade, metal chelators, lysophospholipid product analogs, and more recently, small non-lipid inhibitors of the enzyme were successfully identified. The majority of these inhibitors have been characterized using recombinant purified ATX in vitro, with very few examples studied in more complex systems. Translation of ATX inhibitors from the hands of medicinal chemists to clinical use will require substantially expanded characterization of ATX inhibitors in vivo.     

Two different modes of enzymatic changes in serum with progression of Duchenne muscular dystrophy

Nineteen serum enzymes from patients with Duchenne muscular dystrophy and asthma, and normal subjects were studied. These enzymes include aminopeptidases, cathepsin C, angiotensin-converting enzyme, serine proteinase, sulphatase, phosphatase, esterases and ribonuclease. The enzymatic changes in dystrophic patients were related to two parameters: severity of the disease as judged from symptomatology, and duration of the disease. Most of the enzyme levels tested were increased in milder cases, but they tended to decrease with severity of the disease. On the other hand, there was a group of enzymes showing just opposite tendencies: serine proteinase, cathepsin C and ribonuclease. Even when viewed from the relationship to duration of the disease, the above mentioned grouping of enzymes was generally valid. Most of the enzyme levels, including those routinely applied as clinical parameters, tended to decrease, logarithmically, with an increase in duration of the disease. On the contrary, some others, including serine proteinase, cathepsin C and ribonuclease, tended to increase toward their control levels. Such tendencies were not found in the patients with asthma. The discrepancy between the above two groups of enzymes may have some implications for the process of protein degradation in dystrophic patients.     

Metformin (Glucophage) inhibits tyrosine phosphatase activity to stimulate the insulin receptor tyrosine kinase

Metformin is a commonly used anti-diabetic but whether its mechanism involves action on the insulin receptor or on downstream events is still controversial. With a time course that was slow compared with insulin action, metformin increased tyrosine phosphorylation of the regulatory domain of the insulin receptor (specifically, tyrosine residues 1150 and 1151). In a direct action, therapeutic levels of metformin stimulated the tyrosine kinase activity of the soluble intracellular portion of the beta subunit of the human insulin receptor toward a substrate derived from the insulin receptor regulatory domain. However, metformin did not alter the order of substrate phosphorylation by the insulin receptor kinase. Using a Xenopus oocyte preparation, we simultaneously recorded tyrosine kinase and phosphatase activities that regulate the insulin receptor by measuring the tyrosine phosphorylation and dephosphorylation of peptides derived from the regulatory domain of the human insulin receptor. In an indirect stimulation of the insulin receptor, metformin inhibited endogenous tyrosine phosphatases and purified human protein tyrosine phosphatase 1B that dephosphorylate and inhibit the insulin receptor kinase. Thus, there was evidence that metformin acted directly upon the insulin receptor and indirectly through inhibition of tyrosine phosphatases.     

Basal ganglia and other afferent projections to the peribrachial region in the rat: A study using retrograde and anterograde transport of horseradish peroxidase

The afferent projections to the peribrachial region in the rat were studied using retrograde and anterograde transport of horseradish peroxidase. Particular attention was paid to descending projections from the basal ganglia and related nuclei to the region of nucleus tegmenti pedunculopontinus. Following injection of peroxidase into nucleus tegmenti pedunculopontinus, few retrogradely-labelled neurons were found in the entopeduncular nucleus proper, but larger numbers were found with a wide distribution within the boundaries of the internal capsule and cerebral peduncle. Labelled cells were also consistently observed in the amygdala, the caudal globus pallidus, the subthalamus including zona incerta and subthalamic nucleus, the hypothalamus, the substantia nigra and the ventral tegmental area. Following iontophoretic injections of horseradish peroxidase into the entopeduncular nucleus, lateral hypothalamus, subthalamic nucleus or ventral tegmental area, terminal labelling was observed in and around the branchium conjunctivum in an area apparently corresponding to nucleus tegmenti pedunculopontinus in the rat.     

Membrane-associated nucleotide pyrophosphatase activity on leucocytes from different compartments

The distribution of membrane-associated nucleotide pyrophosphatase activity has been investigated on mouse leucocytes of different origin. It is shown that enzyme-specific activity is higher on bone-marrow cells than on cells derived from peripheral organs. Since thymus lymphocytes show the lowest activity, it is obvious that the thymus environment can influence the enzyme activity on bone-marrow stem cells which have entered this organ. Spleen cells from thymus-deficient nude mice displayed more enzyme activity than spleen cells from normal mice, although the percentage of lymphocytes was similar in both donor-cell populations.     

美西律经利多卡因筛选后在治疗神经病理性痛中的应用研究

目的:评价利多卡因筛选后美西律在治疗神经病理性痛(NPP)中有效性和安全性.方法:选择NPP患者89例,男40例,女49例,年龄18～63岁.所有患者分为加巴喷丁组(A组,29例)、美西律组(B组,29例)、利多卡因筛选后美西律组(C组,31例).治疗四周后观察各组疗效和副作用.结果:治疗后各组症状均改善,但C组改善程度和有效率高于A组和B组,且C组副作用发生率小于A组和B组.结论:经利多卡因筛选可以明显提高美西律治疗NPP有效性和安全性,为临床治疗NPP提供一种较好的选择.