Efficient gene transduction of neurons by lentivirus with enhanced neuron-specific promoters.

In the field of basic and clinical neurosciences, it is important to develop a method for easy delivery and persistent expression of transgene in central neurons. We firstly generated lentiviral vectors with five kinds of neuron-specific promoters, such as synapsin I (SYN), calcium/calmodulin-dependent protein kinase II, tubulin alpha I, neuron-specific enolase and platelet-derived growth factor beta chain promoters and then novel hybrid promoters by fusing cytomegalovirus enhancer (E) to those neuron-specific promoters. Neuron-specific expression of green fluorescent protein (GFP) with those promoters was examined in vivo by injecting the lentiviral vectors into the rat neostriatum, thalamus and neocortex. Among all the promoters, SYN promoter displayed the highest specificity for neuronal expression in all the regions examined (more than 96%). Although GFP production by the hybrid promoters was about 2-4 times larger than the non-enhanced promoters, the neuronal specificity was significantly decreased in most cases. However, the neuronal specificity of E/SYN hybrid promoter exhibited the least decrease only in the thalamus. Furthermore, the transcriptional activity and neuronal specificity of E/SYN promoter were sustained for up to 8 weeks. Thus, lentivirus with E/SYN promoter is the best vector for strong persistent expression in neurons.

Differential distribution of vesicular glutamate transporters in the rat cerebellar cortex.

The chemical organization of excitatory axon terminals in the rat cerebellar cortex was examined by immunocytochemistry and in situ hybridization histochemistry of vesicular glutamate transporters 1 and 2 (VGluT1 and VGluT2). Chemical depletion of the inferior olivary complex neurons by 3-acetylpyridine treatment almost completely removed VGluT2 immunoreactivity from the molecular layer, leaving VGluT1 immunoreactivity apparently intact. On the other hand, neuronal deprivation of the cerebellar cortex by kainic acid injection induced a large loss of VGluT1 immunoreactivity in the molecular layer. In the cerebellar granular layer, both VGluT1 and VGluT2 immunoreactivities were found in mossy fiber terminals, and the two immunoreactivities were mostly colocalized in single-axon terminals. Signals for mRNA encoding VGluT2 were found in the inferior olivary complex, and those for VGluT1 and VGluT2 mRNAs were observed in most brainstem precerebellar nuclei sending mossy fibers, such as the pontine, pontine tegmental reticular, lateral reticular and external cuneate nuclei. These results indicate that climbing and parallel fibers selectively use VGluT2 and VGluT1, respectively, whereas mossy fibers apply both VGluT1 and VGluT2 together to accumulate glutamate into synaptic vesicles. Since climbing-fiber and parallel-fiber terminals are known to make depressing and facilitating synapses, respectively, VGluT1 and VGluT2 might have distinct properties associated with those synaptic characteristics. Thus, it would be the next interesting issue to determine whether mossy-fiber terminals co-expressing VGluT1 and VGluT2 show synaptic facilitation or depression.

Acute effects of cigarette smoking on platelet-dependent thrombin generation.

AIMS: Thrombin is an important factor in the pathogenesis of thrombotic diseases. To clarify whether smoking has an effect in platelet-dependent thrombogenesis, we studied the acute effects of smoking on platelet-dependent thrombin level in smokers. METHODS AND RESULTS: Subjects consisted of ten smokers and nine non-smokers. Platelet-dependent thrombin level measured after overnight fasting was greater in smokers than in non-smokers (smokers vs non-smokers, 121 +/- 47 vs 56 +/- 5 mIU. ml(-1), P < 0.01). When subjects in the smokers group smoked two cigarettes containing 0.9 mg of nicotine per cigarette, platelet-dependent thrombin levels showed a transient three-fold increase in blood samples obtained immediately after smoking (365+/-76 mIU. ml(-1), P < 0.001). Thrombin levels in the blood samples obtained 10 min and 30 min after smoking were less than that in the samples obtained immediately after smoking ceased, but were not significantly different from those in the samples obtained before smoking. Blood nicotine level increased significantly immediately after smoking (P < 0.001), and plasma protein C activity decreased significantly 30 min after smoking (P < 0.05). When nicotine or cotinine was added to the platelet-rich plasma of non-smokers ex vivo, the platelet-dependent thrombin level increased significantly (P < 0.002). CONCLUSION: Platelet-dependent thrombin level is enhanced in smokers, even when not smoking, when compared with non-smokers and increases immediately after smoking. Increases in nicotine and cotinine levels caused by smoking induced a prothrombotic state in smokers via increased platelet-dependent thrombogenesis.

[Dental findings in a case of idiopathic hypoparathyroidism].

Idiopathic hypoparathyroidism is characterized by a deficiency of the parathyroid hormone, origin unknown, and causes a low serum calcium and a high serum inorganic phosphorus. This report concerns a clinical observation of a case of idiopathic hypoparathyroidism. This case was examined from the dental point of view. The findings were as follows: 1. The roentgenographic findings revealed that there was no demonstrable abnormality concerning bone age indicated between the ages of 5 and 6 years. 2. Severe dental caries were noted in the deciduous teeth and the permanent teeth. 3. Hypoplasia and hypocalcemia were recognized in most of the permanent teeth. 4. Large pulp chambers were observed in the deciduous teeth and the permanent teeth, and thickening of the lamina dura was observed in the permanent teeth. 5. The histological findings of the extracted deciduous teeth and the upper right first molar were such that: (1) there was hypoplasia and hypocalcemia in the dentin of deciduous teeth, and there were multiple resorptions in the cementum. (2) there was hypoplasia in the enamel of the upper right first molar. 6. Electron probe microanalysis of the upper left second molar revealed that the degree of mineralization and concentration of Ca, P and Mg in the dentin of the root showed a lower intensity than in the dentin of the normal one. 7. It was assumed from the dental point of view that this hypoparathyroid condition first appeared at about 11'months approximately 18'months of age.

[Immediate postoperative enteral feeding with an elemental diet (Survimed) using a new application form of the so-called fine needle catheter jejunostomy. A prospective study]. / Unmittelbare postoperative enterale Ernährung mit der Elementardiät (Survimed) mittels neuer Applikationsform einer sogenannten Feinnadel-Katheter-Jejunostomie (FNKJ). Eine prospektive Studie.

In a prospective study (n = 50) the clinical usefulness of a fine-needle-catheter-jejunostomy based on the method of Delany et al. [1] was studied. In all cases the jejunostomy was inserted intraoperatively without difficulty and without complication. The set used (Intestofix) consisted of one polyurethan-catheter (75 cm length), 2.1 mm inner diameter) and of two hollow needles constructed as split-cannulas. This system has been proved in our study to be simple, rapid and safe. Patient's tolerance of the feeding was, however, unsatisfactory; diarrhea occurred on the 5th postoperative day in 35% of all patients, in addition we observed inconvenient belching (42.5%) and sensation of repletion (25%). There may be different causes for these uncomfortable symptoms; for example application schedule or the composition of food. We think that the operation itself exerts an important influence upon patient's tolerance because patients without operation but under the same conditions tolerate the nutrition via jejunostomy better than those in our study (with concomitant operation).