Will the new cytogenetics replace the old cytogenetics?

With the advent of array-based comparative genomic hybridization technology, the analog cytogenetic analysis that has been used for the past 100 years could be replaced by the quantitative, microarray-based molecular analysis. Major advantages of the new array-based cytogenetic technologies are the high resolution and the high throughput. This technology is the first to offer an autonomous whole-chromosome analysis in one hybridization reaction for the detection of submicroscopic gains/losses. However, as with any new technology, it needs to be validated with regard to its performance in various applications (e.g. clinical genetic testing and cancer applications), comparative cost, and the data interpretation.     

JC virus genomes in progressive multifocal leukoencephalopathy: detection using a sensitive non-radioisotopic in situ hybridization method

JC virus genomes have been localized in formalin-fixed, paraffin-embedded brain tissues of two cases of known progressive multifocal leukoencephalopathy by in situ hybridization utilizing a biotinylated JC virus DNA probe. A three-stage immunoperoxidase system with gold-silver amplification of the diaminobenzidine substrate was used to visualize biotinylated nucleic acid hybrids. Dot-blot quantification of this visualization system indicates that subpicogramme amounts of biotinylated DNA can be detected. Optimal detection of the virus genomes in the brain tissues required a microwave irradiation step prior to hybridization. JC virus genomes were observed in the nuclei of enlarged oligodendrocytes and of some bizarre astrocytes. No other cell types were found to harbour the genomes.     

Persistence of HIV-1 silent infection in seronegative subjects at high risk

Twenty regular sexual partners of HIV-1 infected subjects, without detectable human immunodeficiency virus (HIV-1) antibody and positive for HIV-1 genome by in situ hybridization (ISH), were selected and studied longitudinally for 6-36 months to estimate the duration of silent infection. During the follow-up period, 10 showed atypical Western Blot (WB) patterns. Two seronegative partners seroconverted. Rapid progress to AIDS was observed in 7 seropositive subjects.     

Epidemiology of molluscum contagiosum using genetic analysis of the viral DNA

The molecular epidemiology of molluscum contagiosum virus (MCV) infections was investigated by restriction endonuclease analysis of the genomes of 222 separate isolates collected from 147 patients living in Germany (33 patients), Hong Kong (6 patients), and Scotland (108 patients). MCV type 1 (MCV-1) caused 96.6% of the infections, and MCV type 2 (MCV-2) caused 3.4%. However, isolates from four of the 142 MCV-1-infected patients and two of the five MCV-2-infected patients showed minor differences in their DNA restriction patterns because of the loss of a single or very few recognition sites for the enzymes used. No genome variations were detected amongst isolates collected from different sites or on several occasions from individual patients or from closely related patients. Southern blot hybridization revealed a high level of relatedness between MCV-1 and 2. No differences were seen in the appearance or anatomical localization of lesions caused by either virus type. In particular, there was no preferred genital localization for MCV-2 infections.     

Detection of parvovirus B19 DNA, antigen, and particles in the human fetus

Human parvovirus B19 commonly infects children, causing erythema infectiosum (fifth disease). However, there is a significant adult population which has not been exposed to the virus and, consequently, does not have protective antibody. Recent reports have associated B19 infection during pregnancy with fetal death, although normal outcome of pregnancy is more common. To characterise further the role of B19 infection in fetal deaths, a series of laboratory investigations has been undertaken on tissues obtained at autopsy. These have demonstrated the presence of virion-sized DNA by Southern blotting, viral antigen by radioimmunoassay, and viral particles by electron microscopy, all from tissues of hydrops fetalis. These data confirm that the human parvovirus B19 can cross the placenta and replicate in fetal tissues.     

Characterization of the translocated chromosome using fluorescencein situ hybridization and random amplified polymorphic DNA on twoTriticum aestivum—Thinopyrum intermedium translocation lines resistant to wheat streak mosaic or barley yellow dwarf virus

Fluorescencein situ hybridization (FISH) was used to determine the breakpoint of the translocation chromosome in two bread wheat (Triticum aestivum) germplasm lines withThinopyrum intermedium chromatin carrying resistance to either wheat streak mosaic virus (WSMV) or barley yellow dwarf virus (BYDV). In addition, genome-specific random amplified polymorphic DNA (RAPD) markers were used to ascertain the genomic sources of theTh. intermedium chromosomes carrying the WSMV or BYDV resistance. CI17766, a WSMV-resistant wheat germplasm line derived from induced homoeologous pairing by using theph1b mutant, had a translocation chromosome composed of the complete 4AL and about 45% of proximal 4AS from wheat, and the entire 4ES ofTh. intermedium. The BYDV-resistant translocation line, TC14, derived from tissue culture, had a very short distal segment of 7StL fromTh. intermedium terminally attached to 56% of the proximal 7DL. These observations indicate that translocations in these wheat germplasm lines did not involve centromeric breaks and fusion but were a result of homoeologous chromosome recombination.accepted for publication by J. S. (Pat) Heslop-Harrison     

Inhibition of nitric oxide synthase increases synaptophysin mRNA expression in the hippocampal formation of rats

Synaptophysin is a protein involved in the biogenesis of synaptic vesicles and budding. It has been used as an important tool to investigate plastic effects on synaptic transmission. Nitric oxide (NO) can influence plastic changes in specific brain regions related to cognition and emotion. Experimental evidence suggests that NO and synaptophysin are co-localized in several brain regions and that NO may change synaptophysin expression. Therefore, the aim of the present work was to investigate if inhibition of NO formation would change synaptophysin mRNA expression in the hippocampal formation. Male Wistar rats received single or repeated (once a day for 4 days) i.p. injections of saline or l-nitro-arginine (l-NOARG, 40 mg/kg), a non-selective inhibitor of nitric oxide synthase (NOS). Twenty-four hours after the last injection the animals were sacrificed and their brains removed for ‘in situ’ hybridization study using ³⁵S-labeled oligonucleotide probe complementary to synaptophysin mRNA. The results were analyzed by computerized densitometry. Acute administration of l-NOARG induced a significant (p < 0.05, ANOVA) increase in synaptophysin mRNA expression in the dentate gyrus, CA1 and CA3. The effect disappeared after repeated drug administration. No change was found in the striatum, cingulated cortex, substantia nigra or nucleus accumbens. These results reinforce the proposal that nitric oxide is involved in plastic events in the hippocampus.     

Effect of H+ and elevated PCO 2on membrane electrical properties of rat cerebral arteries

Some membrane electrical properties of muscle cells from the middle cerebral artery of the rat were recorded with intracellular microelectrodes. The resting membrane potential (E _m) of this preparation was –63 mV. Reduction of extracellular pH to 7.0 in the face of a constantP _{CO 2}of 40 mm Hg had no significant effect onE _m. Similarly the slope of the steady-state voltage/current curves was not different at pH 7.0 compared to control at pH 7.4. In marked contrast, whenP _{CO 2}was elevated to around 60 to 70 mm Hg there was a rapid hyperpolarization and reduction in the slope of the voltage current curve suggesting an increased conductance for one or more ionic species. In addition elevation ofP _{CO 2}increased the slope of theE _m vs. log[K]₀ curve from 46 mV/decade to 59 m V/decade which is in good agreement with a Nernstian potential for a K⁺ selective membrane. These data suggest that while the smooth muscle cells of rat cerebral arteries are relatively insensitive to a small reduction in extracellular pH; reduction of intracellular pH by elevatingP _{CO 2}induces hyperpolarization by increasing K⁺ conductance (g _k). However, it is not clear from these experiments if theP _{CO 2}effects are mediated entirely by changes in pH or if there is a direct membrane action of CO₂.This work is supported by Grant no. HL27862     

Voltage clamp of a small muscle membrane area by means of a circular sucrose gap arrangement

Summary A new method is described which permits the measurement of membrane currents of thick muscle fibres (diameter 300 m or more) ofAstacus fluviatilis orBalanus balanus under voltage clamp conditions.The potential difference across a small patch of membrane (60–100 m in diameter) is controlled by connecting a voltage source across it with two external electrodes. One of them is connected to the fluid bathing the muscle fibre. The other, tubular one is in touch with the test area. The current flowing through the electrodes represents the sum of the membrane current flowing across the test area and the leak current flowing in the external fluid between the electrodes. In the first version of the method the leak current is limited by a circular sucrose gap around the test area. In the second, more elaborate method, the leak current is eliminated by a system of two concentric sucrose rings with a guard ring electrode between them. This method permits in addition the measurement of full sized action potentials in the test area.This work has been briefly reported in Cs. fysiol.17, 48 (1968).     

用非同位素原位杂交组化在光镜和电镜水平观察前阿黑皮素mRNA在垂体的分布

用地高辛标记反意前阿黑皮素(pro-opiomelanocortin,POMC)cRNA探针原位杂交组化在光镜和电镜水平观察了POMC mRNA在大鼠垂体的分布,并比较了碱性磷酸酶(AKP)显色系统和辣根过氧化酶(HRP)显色系统在光镜水平上的敏感性.结果:POMC mRNA广泛地分布于垂体的中间叶和前叶.中间叶全部细胞均为POMC mRNA阳性.前叶中除前叶的腹侧缘和前叶与中间叶交界处阳性细胞较少外,都有较多的POMC mRNA阳性细胞分布.AKP显色系统比HRP显色系统敏感.在电镜水平,POMC mRNA主要分布于粗面内质网,少数分泌颗粒可能呈阳性反应,胞核未见阳性反应沉淀.文内还就阳性分泌颗粒在调节细胞合成功能方面可能起的作用进行了讨论.