Determination of genomic copy number with quantitative microsphere hybridization

We developed a novel quantitative microsphere suspension hybridization (QMH) assay for determination of genomic copy number by flow cytometry. Single copy (sc) products ranging in length from 62 to 2,304 nucleotides [Rogan et al., 2001; Knoll and Rogan, 2004] from ABL1 (chromosome 9q34), TEKT3 (17p12), PMP22 (17p12), and HOXB1 (17q21) were conjugated to spectrally distinct polystyrene microspheres. These conjugated probes were used in multiplex hybridization to detect homologous target sequences in biotinylated genomic DNA extracted from fixed cell pellets obtained for cytogenetic studies. Hybridized targets were bound to phycoerythrin-labeled streptavidin; then the spectral emissions of both target and conjugated microsphere were codetected by flow cytometry. Prior amplification of locus-specific target DNA was not required because sc probes provide adequate specificity and sensitivity for accurate copy number determination. Copy number differences were distinguishable by comparing the mean fluorescence intensities (MFI) of test probes with a biallelic reference probe in genomic DNA of patient samples and abnormal cell lines. Concerted 5' ABL1 deletions in patient samples with a chromosome 9;22 translocation and chronic myelogenous leukemia were confirmed by comparison of the mean fluorescence intensities of ABL1 test probes with a HOXB1 reference probe. The relative intensities of the ABL1 probes were reduced to 0.59+/-0.02 fold in three different deletion patients and increased 1.42+/-0.01 fold in three trisomic 9 cell lines. TEKT3 and PMP22 probes detected proportionate copy number increases in five patients with Charcot-Marie-Tooth Type 1a disease and chromosome 17p12 duplications. Thus, the assay is capable of distinguishing one allele and three alleles from a biallelic reference sequence, regardless of chromosomal context.     

Axonal damage and demyelination in long-term dorsal root ganglia cultures from a rat model of Charcot-Marie-Tooth type 1A disease

Clinical progression in hereditary and acquired demyelinating disorders of both the central and peripheral nervous system is mainly due to a time-dependent axonal impairment. We established 90-day dorsal root ganglia (DRG) cultures from a rat model of Charcot-Marie-Tooth type 1A (CMT1A) neuropathy to evaluate the structure of myelin and axons, and the expression of myelin-related proteins and cytoskeletal components, by morphological and molecular techniques. Both wild-type and CMT1A cultures were rich in myelinated fibres. Affected cultures showed dysmyelinated internodes and focal myelin swellings. Furthermore, uncompacted myelin and smaller axons with increased neurofilament (NF) density were found by electron microscopy, and Western blots showed higher levels of nonphosphorylated NF. Confocal microscopy demonstrated an abnormal distribution of the myelin-associated glycoprotein which, instead of being expressed at the noncompact myelin level, showed focal accumulation along the internodes while other myelin proteins were normally distributed. These findings suggest that CMT1A DRG cultures, similarly to the animal model and human disease, undergo axonal atrophy over a period of time. This model may be utilized to study the molecular changes underlying demyelination and secondary axonal impairment. As axonal damage may occur after just 3 months and tissue cultures represent a strictly controlled environment, this model may be ideal for testing neuroprotective therapies.     

Nerve ultrasound findings differentiate Charcot-Marie-Tooth disease (CMT) 1A from other demyelinating CMTs

Objective

Ulnar/median motor nerve conduction velocity (MNCV) is ≤38?m/s in demyelinating Charcot-Marie-Tooth disease (CMT). Previous nerve high resolution ultrasound (HRUS) studies explored demyelinating CMT assuming it as a homogeneous genetic/pathological entity or focused on CMT1A.

Vitamin B-6 metabolism in the brains of young adult and senescent mice

The steady state levels of pyridoxal-P and pyridoxamine-P, the activities of pyridoxal (pyridoxine) kinase and pyridoxamine-(pyridoxine)-P oxidase, and the metabolism of [³H]pyridoxine were determined in the brains of C57B1/6J mice of selected ages. The steady state concentratioons of the coenzymes and the activities of the enzymes required for pyridoxal-P synthesis did not change significantly as a function of age. The uptake and metabolism of vitamin B-6 by the brain was studied by injecting [³H]pyridoxine in the tail vein of young adult and senescent mice, killing the mice after 15 or 30 min, and separating the B-6 metabolites by ion exchange chromatography. More total radioactivity was accumulated in 15 min in the brains of the senescent mice than the brains of the young mice. The brains from both age groups rapidly synthesized pyridoxal-P from pyridoxine. However, less radioactive pyridoxamine-P and more radioactive pyridoxal were formed in the brains of the senescent mice than in the young mice killed 15 min after injection. These results are similar to those obtained for the metabolim of [³H]-pyridoxine in the liver of these senescent mice. The senescent mice appear to be vitamin B-6 deficient, have decreased brain amino acid transaminase activity, and either increased pyridoxal-P phosphatase activity or decreased protection of brain pyridoxal-P.     

A novel<Emphasis Type="Italic"> PMP22</Emphasis> mutation Ser22Phe in a family with hereditary neuropathy with liability to pressure palsies and CMT1A phenotypes

We describe a Cypriot family in which some family members presented with episodes of pressure palsies, while other family members had a slowly progressive chronic polyneuropathy typical of the Charcot-Marie-Tooth type 1 phenotype. All family members were evaluated clinically, with nerve conduction studies, and with genetic testing. In all affected individuals there was clinical and electrophysiological evidence of diffuse demyelinating sensorimotor polyneuropathy and a novel point mutation in the PMP22 gene (Ser22Phe) was identified.     

Behavioural profiling of a murine Charcot-Marie-Tooth disease type 1A model

Different features of motor behaviour were studied on a transgenic mouse model of Charcot-Marie-Tooth's disease (CMT). Mutants with 4 or 7 copies of the human PMP22 gene leading to a phenotype significantly close to CMT's disease type 1A were compared with control animals. The aim of the study was to validate this transgenic model and to characterise the impairments occurring in the various lines. Three main types of analysis were performed in 2-month-old mice without any peculiar visible deficit: (i) a study of standardised clinical tests (SHIRPA protocol) demonstrated that only a few motor deficits were expressed; (ii) a measurement of general spontaneous activity by means of a commercial video-tracking system was performed and revealed that the main spontaneous activities were identical in the three lines with, however, some slight localised modifications; and, (iii) by contrast, the three lines respond very differently to the footprints, grip strength, splay test and rotarod test. Even in lines with a significantly limited copy number of the transgene, we observed and quantified impairments. In conclusion, mutants of CMT1A seem to be a very pertinent model of this human pathology and will certainly be useful for therapeutic procedures and for theoretical studies on this disease.     

除害专业队伍实施城市灭蚊的研究

目的比较3个在用人、培训、技术措施和管理方面不同的实施单位在城市灭蚊工作中灭蚊效果的差异。方法2002年6~9月,3个实施单位分别承包了武汉7个灭蚊区的灭蚊试点工作。其中城区灭蚊项目组将其负责的灭蚊区内下岗人员训练为除害员,开展了蚊虫孳生地普查,然后利用普查资料和地图搜索、处理蚊虫孳生地。除害员对灭蚊区市民进行面对面的灭蚊宣传。项目组建立防止遗漏蚊虫孳生地的管理制度。而另外两个实施单位在这几方面与项目组不同。试点结束前由专家对灭蚊区进行问卷调查和达标考核。结果在项目组负责的灭蚊区有12个蚊密度测试点,其中10个点的RPI值为0。普查后每小时能在灭蚊区找到的蚊虫孳生地数量显著下降。项目组的问卷调查和达标考核结果均优于另外2个实施单位。结论蚊虫孳生地普查,训练灭蚊区当地的下岗人员为除害员,建立合理的管理制度能有效防止蚊虫孳生地的遗漏,提高除害专业队伍实施城市灭蚊的工作质量。     

Vitamin B-6 metabolism in the livers of young adult and senescent mice

The steady state level of pyridoxal-P and the activities of pyridoxal (pyridoxine) kinase and pyridoxine-P oxidase were determined in the livers of C57B1/6J mice of selected ages. The concentration of pyridoxal-P and the activities of these two enzymes did not change significantly as a function of age. In addition, the uptake and metabolism in the liver of [³H]pyridoxine injected in the tail vein of young adult and senescent mice was studied. More total radioactivity was accumulated in 15 and 30 min in the livers of the senescent mice than the livers of the young mice. The livers from both age groups rapidly synthesized the coenzyme, pyridoxal-P, from pyridoxine. However, a smaller percentage of the total radioactivity appeared in the other coenzyme form, pyridoxamine-P, and a larger percentage appeared in the hydrolysis product, pyridoxal, in the livers of the senescent mice than the livers of the young mice. These results may indicate that the senescent mice were B-6 deficient, that they had decreased total amino acid transaminase activity, and that they had increased pyridoxal-P phosphatase activity or decreased protection of liver pyridoxal-P.     

Glucocorticosteroids stimulate the activity of the promoters of peripheral myelin protein-22 and protein zero genes in Schwann cells

To better understand the mechanism by which glucocorticosteroids (GLUC) could enhance myelination in the PNS, cultured rat Schwann cells were transiently transfected with reporter constructs in which luciferase expression was controlled by the promoter region of either the peripheral myelin protein-22 (PMP22) or the protein zero (P(0)) genes. GLUC stimulated the activity of the P(0) promoter and the PMP22 promoters 1 and 2. The effect of GLUC was specific as estradiol and testosterone did not activate the promoters. The antagonist RU486 did not abolish the effect of GLUC, but instead stimulated promoter activities by itself. In the mammary carcinoma cell line 34i, which expresses GLUC receptors, GLUC did not stimulate the P(0) and PMP22 promoters while the promoter of the mouse mammary tumor virus was strongly activated. Thus, the activation by GLUC of the promoter activities of two peripheral myelin protein genes is Schwann cell-specific.     

不同浓度血小板激活剂激活血小板微粒膜表达PAC-1、CD62p的比较

目的　比较不同浓度的激活剂激活血小板形成的血小板微粒 (PMP)膜表达PAC 1、CD6 2p的差异。方法　抽取 10例健康人静脉血 ,以枸橼酸钠抗凝 ,离心得富血小板血浆 ,分别以不同浓度的ADP(5、10、2 0 μmol/L)、凝血酶 (0 .1、0 .5、1.0U/ml)、胶原 (5、10、2 0 μg/ml)作诱导剂 ,激活同一标本中的血小板 ,比较其PMP表达PAC 1和CD6 2p的情况。 结果　随着激活剂浓度的增加 ,CD6 2p PMP、PAC 1 PMP的百分率都逐渐增加 ,同一诱导剂各浓度间有显著性差异 (P <0 .0 1)。结论　不同刺激强度的血小板诱导剂ADP、凝血酶、胶原 ,随浓度的增加 ,其诱导血小板所形成的CD6 2p PMP、PAC 1 PMP的百分率逐渐增加。