Episomal and stable expression of the luciferase reporter gene for quantifying Leishmania spp. infections in macrophages and in animal models

We have expressed the reporter firefly luciferase gene (LUC) in Leishmania donovani and Leishmania major either as part of episomal vectors or integrated into the parasite genome under the control of their respective ribosomal promoter regions. An excellent linear correlation between parasite number and luciferase activity was observed with all the transfectants. LUC-expressing recombinant parasites were useful to monitor Leishmania spp. infections in macrophages or in animal models. For prolonged growth in absence of drug selection, such as within animal models, quantitation of parasites is more reliable when the reporter gene LUC is stably integrated in the parasite genome. These recombinant strains should be useful tools to monitor Leishmania growth under a number of conditions.     

How self-initiated memorized movements become automatic: a functional MRI study

We used functional magnetic resonance imaging (fMRI) and dual tasks to investigate the physiology of how movements become automatic. Normal subjects were asked to practice some self-initiated, self-paced, memorized sequential finger movements with different complexity until they could perform the tasks automatically. Automaticity was evaluated by having subjects perform a secondary task simultaneously with the sequential movements. Our secondary task was a letter-counting task where subjects were asked to identify the number of times a target letter from the letter sequences was seen. Only the performances that achieved high accuracy in both single and dual tasks were considered automatic. The fMRI results before and after automaticity was achieved were compared. Our data showed that for both conditions, sequential movements activated similar brain regions. No additional activity was observed in the automatic condition. There was less activity in bilateral cerebellum, presupplementary motor area, cingulate cortex, left caudate nucleus, premotor cortex, parietal cortex, and prefrontal cortex during the automatic stage. These findings suggest that most of the motor network participates in executing automatic movements and that it becomes more efficient as movements become more automatic. Our results do not provide evidence for any area to become more activated for automatic movements.     

Linkage Disequilibrium and Haplotype Architecture for two ABC Transporter Genes (ABCC1 and ABCG2) in Chinese Population: Implications for Pharmacogenomic Association Studies

Information about linkage disequilibrium (LD) patterns and haplotype structures for candidate genes is instructive for the design and analysis of genetic association studies for complex diseases and drug response. ABCC1 and ABCG2 are genes coding for two multidrug resistance (MDR) associated transporters; they are also related to some pathophysiological traits. To pinpoint the LD profiles of these MDR genes in Chinese, we systemically screened 27 unrelated individuals for single nucleotide polymorphisms (SNPs) in the coding and regulatory regions of these genes, and thereby characterized their haplotype structures. Despite marked variations in haplotype diversity, LD pattern and intragenic recombination intensity between the two genes, both loci could be partitioned into several LD blocks, in which a modest number of haplotypes accounted for a high fraction of the sampled chromosomes. We concluded that each locus has its own genomic LD profile, but that they still share a common segmental LD architecture with low haplotype diversity. Our data will benefit genetic association studies of complex traits and drug response possibly related to these genes.     

Altered sensitivity to sodium channel-specific neurotoxins in cultured neurons from temperature-sensitive paralytic mutants of Drosophila

In vitro culture of central nervous system neurons from Drosophila larvae enables direct examination of effects of neurological mutations at a single-cell level not readily amenable to in vivo experimentation. Using this system, we examined the cytotoxic effect of veratridine, which selectively causes persistent activation of sodium channels, on the mutants parats1 and napts known to have a temperature-dependent block in propagation of nerve action potentials. Even at a permissive temperature (22 degrees C) for the mutant flies, the veratridine-induced neuronal lethality was significantly lower in both parats1 and napts cultures than in normal cultures. At a temperature (35 degrees C) causing paralysis of mutant flies, napts neurons showed the same high degree of resistance to veratridine; while parats1 neurons showed an increased resistance to a level similar to that of napts neurons. A similar reduction in the veratridine-induced neuronal death was also observed in normal cultures that were pretreated with the sodium channel blocker tetrodotoxin. These results support the idea that both parats and napts affect sodium channel functions at the level of isolated single neurons. It was also found that parats1 and napts mutations, like the sodium channel blocker tetrodotoxin, do not affect the morphological differentiation and survival of central nervous system neurons in culture. These findings indicate that functional sodium channels are not required for neurite outgrowth and survival of neurons at this developmental stage.     

Breakpoint clusters of the PML gene in acute promyelocytic leukemia: Primary structure of the reciprocal products of the PML-RARA gene in a patient with t(15;17)

DNA studies of the translocation t(15;17) in acute promyelocytic leukemia (APL) have shown that the retinoic acid receptor alpha (RARA) gene on chromosome 17 is juxtaposed to the promyelocytic leukemia (PML) gene on chromosome 15. The PML breakpoints have been mapped to 3 clusters: bcr1, bcr2, and bcr3. We have examined the PML breakpoint distribution in a series of 33 Chinese patients with APL Twenty-two patients fell within bcr1, 2 within bcr2, and 9 within bcr3. The primary structure of the reciprocal chromosome translocation joints of one patient and that of their normal counterparts have been determined and compared to those of 2 previously reported cases. These studies revealed possible topoisomerase II cleavage sites close to the breakpoints and suggested implications of DNA attachment sites to nuclear matrix. We propose that these features are relevant to the process of illegitimate recombination generating the translocation. © 1993 Wiley-Liss, Inc.     

Family-based association study between autism and glutamate receptor 6 gene in Chinese Han trios.

The glutamate pathways are involved in diverse processes such as learning and memory, epilepsy, and they play important roles in neural plasticity, neural development, and neurodegeneration. It has been proposed that autism could be a hypoglutamatergic disorder. Recently, Jamain et al. reported that the glutamate receptor 6 (GluR6 or GRIK2) is in linkage disequilibrium with autism. In the present study, the transmission disequilibrium test (TDT) and the haplotype transmission were performed to analyze the four SNPs (SNP1: rs995640; SNP2: rs2227281; SNP3: rs2227283; SNP4: rs2235076) of GluR6 in 174 Chinese Han parent-offspring trios. The TDT demonstrated that the two SNPs (SNP2 and SNP3) showed preferential transmission (TDT P = 0.032). The global chi(2) test for haplotype transmission also revealed an association between GluR6 and autism (chi(2) = 10.78, df = 3, P = 0.013). Our results suggested that GluR6 is in linkage disequilibrium with autism.     

纤维蛋白原与吸附白蛋白、肝素的新型血管支架材料氧化钛的血液相容性

探索纤维蛋白原(Fibrinogen,FIG)与吸附白蛋白、肝素的新型血管支架材料氧化钛(Titanium Oxide,Ti-O)的血液相容性。(1)研制Ti-O,切割成薄膜;(2)Ti-O薄膜涂层白蛋白和肝素;(3)血小板(platelet,PL)吸附试验;(4)酶联免疫试验测FIG吸附量;(5)动物犬股动脉内植入涂层的Ti-O薄膜与对照试片Ti-O和不锈钢(Stainless steel,SS)薄膜。结果发现:Ti-O完全具有固定白蛋白和肝素的结构与性能,比未涂层的Ti-O能更一步减少PL和FIG的吸附,实验动物体内薄膜6个月后取出扫描电镜观察黏附的PL少,形态无改变,血管内无血栓,优于未涂层的Ti-O,更明显优于SS。Ti-O为N型半导体,不易接受FIG的电荷,并且与血细胞有相似的界面张力,决定生物材料Ti-O有较好的血液相容性。Ti-O对白蛋白、肝素有极好的亲和力是因以化学键相结合,在血中进一步减少FIG和PL被涂层的Ti-O吸附。实验证明涂层的Ti-O有持久和稳定的抗凝血性能。     

The effect of a levonorgestrel-releasing intrauterine device on human endometrial oestrogen and progesterone receptors after one year of use

Thirty-four women bearing a levonorgestrel-releasing intrauterine device, 20 micrograms/day (LNG-IUD-20), for 12-15 months were recruited. Endometrial biopsies were collected during the late proliferative phase of the cycle (on cycle days 10-12) before (control) and after the use of the IUD for 12 months, and assayed for oestrogen receptors (ER) and progesterone receptors (PR). An immunohistochemical technique with the peroxidase-antiperoxidase detection system (PAP method) was employed. D75 and JZB39 were the primary antibodies for ER and PR respectively. The immunostaining semiquantitative analysis was performed with a computerized microscope image processor, and expressed as 'grey value'. Both endometrial ER and PR populations were significantly lower after insertion of the IUD (P < 0.01) than in control biopsies. The intensity of nuclear staining and the percentage of positively stained cells for ER and PR in women with LNG-IUD were each about 50% of those in control biopsies. The results suggested that LNG released locally from the IUD has a depressive action on the ER and PR, which may contribute to the contraceptive effectiveness of this type of IUD and also to the possible causes of LNG-IUD-induced irregular bleeding and amenorrhoea.     

The nNOS/cGMP signal transducing system is involved in the cardiovascular responses induced by activation of NMDA receptors in the rostral ventrolateral medulla of cats

The potential neuroprotective effects of the novel nitro-derivate of aspirin (NCX4016) on permanent focal cerebral ischemia in spontaneously hypertensive rats (SHRs) was investigated. Reference compounds were acetylsalicilic acid (ASA) and FK506 (tacrolimus). Ten minutes after surgery, SHRs were randomly divided into four groups of ten, pharmacologically treated and sacrificed 24 h after treatment. Brains were removed and processed to measure infarct volume, 70 kDa heat shock protein (hsp70), glial fibrillary acidic protein (GFAP) and vimentin (Vim) immunoreactivity (IR), and apoptosis using terminal deoxynucleotidyl transferase (TdT)-mediated dUTP-digoxigenin nick end-labeling (TUNEL) assay. NCX-4016 significantly reduced total infarct volume compared to ASA (-20%, P < 0.05), FK506 (-18%, P < 0.05) and vehicle treatment (-20%, P < 0.05). Experimental groups did not differ in hsp70-IR and GFAP-IR. Conversely, hyperplastic astrocytes, measured by Vim-IR, were significantly lower in NCX-4016 than in the vehicle group (-36%, P<0.01). TUNEL assay indicated a significantly lower degree of apoptosis in NCX-4016 group than vehicle in both the homolateral (-27%, P < 0.01) and contralateral hemisphere (-29%, P < 0.05). These findings indicate that NO release associated with aspirin confers neuroprotective effects against ischemic injury.