Binding of Cellular Proteins to the Leader RNA of Equine Arteritis Virus

The genome of equine arteritis virus (EAV) produces a 3 coterminal-nested set of six subgenomic (sg) viral RNAs during virus replication cycle, and each set possesses a common leader sequence of 206 nucleotides (nt) in length derived from the 5 end of the viral genome. Given the presence of the leader region within both genomic and sg mRNAs, it is likely to contain cis-acting signals that may interact with cellular or viral proteins for RNA synthesis. Gel mobility shift assays indicated that proteins in Vero cell cytoplasmic extracts formed complexes with the positive (+) and negative (-) strands of the EAV leader RNA. Several cell proteins with molecular masses ranging from 74 to 31 kDa and 58 to 32 kDa were detected in UV-induced cross-linking assays with the EAV leader RNA (+) and (-) strands, respectively. In both cases, intense bands were observed at the 58–52 kDa molecular weight markers. Results from competition gel mobility shift assays using overlapping cold RNA probes spanning the leader RNA (+) strand indicated that nt 140–206 are not necessary for binding to cell proteins.     

Detection of numerical chromosomal abnormalities in malignant cells on body fluids by fluorescence in situ hybridization of interphase cell nuclei with chromosome-specific probes.

To detect numerical chromosomal abnormalities (NCA) in malignant cells on body fluids, Fluorescence in situ hybridization (FISH) technique was tested in clinical specimens from patients with metastatic disease. Directly labeled DNA probes specific for chromosomes 8, 12, X, and Y (Imagenetics, Naperville, IL) were used for in situ hybridization to interphase cell nuclei. Fifteen body fluids (BF) from various sites were studied. Based initially on the Papanicolaou-stained slides, there were seven malignant and eight benign samples. Blind analysis (200 cells/sample) showed that all benign samples had a normal number of chromosomes, whereas six of seven malignant samples showed different NCA comprising 5-60% of the cell population ranging from three to 10 chromosome signals per cell. We conclude that interphase cytogenetic cell analysis of BF by FISH is: (1) feasible and gives superior signals for detection of NCA, (2) helpful in detecting malignant cells, (3) relatively simple with a turnaround time of less than 24 hr. This method may have diagnostic and prognostic application in the study of the biologic behavior of malignant neoplasms.     

Minimising stimulator artefacts in electromagnetic flow signals

Simultaneous electrical stimulation of tissue with the measurement of blood flow using an electromagnetic flowmeter system almost invariably results in large flow measurement inaccuracies. These inaccuracies are because the electrical energy from stimulating artefacts is amplified along with the flow signals. The paper describes the building and use of an inexpensive circuit to remove stimulation artefacts from electromagnetic flow measurements.     

Quantitation of hepatitis B viral DNA by solution hybridization: Comparison with DNA polymerase and hepatitis B e antigen during antiviral therapy

Serological markers of hepatitis B virus (HBV) replication were assessed in a randomized, controlled trial of prednisone withdrawal followed by α -interferon in the treatment of chronic hepatitis B. HBV DNA levels in more than 700 serial serum samples from 41 patients were determined by a sensitive and quantitative solution hybridization assay. Results were compared with HBV DNA polymerase (DNAp) activity and hepatitis B e antigen (HBeAg) in 21 untreated controls and 20 treated patients. Among treated patients, the mean pretherapy HBV DNA values were higher in nonresponders than in responders. During prednisone treatment, DNA levels increased an average of 2.1-fold in responders and 1.4-fold in nonresponders. During the 2-week rest interval between prednisone and interferon, DNA values fell an average of 57% in responders. In contrast, the mean DNA values in nonresponders did not change during the same interval. This early distinction between responders and nonresponders was not apparent from DNAp or HBeAg results. During interferon treatment, HBV DNA became undetectable in responders and remained negative during a 1-year follow-up. DNA in nonresponders declined to 14% of baseline during interferon treatment but increased to pretherapy levels after treatment. DNAp values generally paralleled HBV DNA values, but DNAp activity showed more variability and lower sensitivity than did the hybridization assay results. HBeAg values varied independently of HBV DNA and DNAp with a much delayed decline in responders. These results indicate that HBV DNA, when measured quantitatively by a sensitive solution hybridization assay, is an early predictor of the effects of antiviral agents on replication.     

Action of remantadine on fusion of the lipid envelope of influenza a virus with plasma and internal membranes in lymphoblastoid cells

Department of Biomembranes, Research Center for Development and Introduction of Modern Methods of Molecular Diagnosis, Ministry of Health of the USSR, Moscow. (Presented by Academician of the Academy of Medical Sciences of the USSR I. P. Ashmarin.) Translated from Byulleten' Éksperimental'noi Biologii i Meditsiny, Vol. 109, No. 5, pp. 483–485, May, 1990.     

Exclusion mapping of 12 X-linked disease loci and 10 DNA probes from the long arm of the X-chromosome

Specific chromosome rearrangements associated with disease entities are invaluable resources for physical mapping. A deletion on the X chromosome of a male leads to the nullisomy for X-linked genes, resulting in the onset of genetic diseases and/or the absence of the DNA probe detectable sequences. This permits the localization of these loci within the deleted area. On the other hand, the region for some other X-linked loci can be excluded from the deleted area according to the absence of the characteristic symptoms of the disease and/or the presence of the hybridization signals. An interstitial deletion on the long arm of the X chromosome of a male has been characterized by high resolution banding. The karyotype of the proband is 46,Y,del(X)(pter----q21.1::q21.33----qter). The regions for 12 X-linked disease loci as well as 10 DNA probes are excluded from the deleted area, and localized either proximally or distally to the deletion. The results also reveal a controversy in the present linkage data concerning the assignment of these loci.     

Investigation and improved performance of optical fibre probes in laser Doppler blood flow measurement

Laser Doppler flowmetry with optical-fibre beam transmission is a sensitive fast and convenient method of measuring tissue blood flow. However, its sensitivity can also be a problem because of movement artefacts. This study applies some basic considerations of fibre optics and Rayleigh light scattering to the field of laser Doppler blood flow meters. Practical suggestions are given by which movement arterfacts can be reduced by choice of optical fibres, attention to probe geometry, cladding the fibres to reduce their movements and in the method of application. Experiments which test the normalisation circuitry of laser Doppler instruments are described and the effects of movement artefacts on the interpretation of the pulsatile component of laser Doppler records are also discussed. Probe and fibre line movements cause high-frequency intensity fluctuations due to speckle movement. The intensity fluctuations produce an apparent Doppler shift much greater than the Doppler shift produced by the relative movements of probe and tissue. It has been found that it is important to ensure that the fields of view of the illuminating and detecting fibres do not overlap at the skin surface and that probe contact with the skin surface should be maintained.     

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.     

Error rate for HLA-B antigen assignment by serology: implications for proficiency testing and utilization of DNA-based typing methods

Until recently, the majority of HLA class I typing has been performed by serology. Expensive commercial typing trays are frequently used for testing non-Caucasian subjects and new strategies using DNA-based methods have been adopted for improving clinical histocompatibility testing results and adapted as supplements in proficiency testing. A double-blind comparison of the typing of HLA-B specificities in 40 samples was carried out between serology and two polymerase chain reaction (PCR) methods, PCR amplification with sequence-specific primers (PCR-SSP) and PCR amplification and subsequent hybridization with sequence-specific oligonucleotide probes (PCR-SSOP). The results demonstrated 22.5% misassignments of HLA-B antigens by serology. There was complete concordance between the results obtained with the two PCR based typing methods. A second panel of 20 donor samples with incomplete or ambiguous serologic results was analyzed by PCR-SSP and SSOP. Both PCR methods identified correctly the HLA-B antigens. Our results suggest that more accurate typing results can be achieved by complementing serologic testing with DNA-based typing techniques. The level of resolution for HLA-B antigen assignment can be obtained by this combination of serology and limited DNA-based typing is equivalent to the HLA-B specificities defined by the WHO-HLA Committee. This level of resolution cannot routinely be achieved in clinical histocompatibility testing or in proficiency testing using serologic reagents only.     

人乳头状瘤病毒及其相关基因在中耳胆脂瘤中的表达

目的　探讨人乳头状瘤病毒 (humanpapillomavirus ,HPV)感染在中耳胆脂瘤发生发展中的作用。方法　运用共同引物聚合酶链反应 (polymerasechainreaction ,PCR)和核酸分子斑点杂交法对44例 ( 44耳 )中耳胆脂瘤标本组织中的HPVDNA进行检测 ,并结合其中 35例 ( 35耳 )的病理学检查结果进行对比分析。结果　 12耳 ( 34.3 % )中耳胆脂瘤组织中观察到了HPV感染的损害特征 ;用共同引物PCR法及核酸分子斑点杂交法对 44耳中耳胆脂瘤组织标本进行HPVDNA扩增的阳性率分别为2 9 .5 % ( 13 44 )及 2 5 .0 % ( 11 44 ) ;表现有人乳头瘤病毒损害特征的 12耳中耳胆脂瘤组织HPVDNA检测阳性率为 5 8.3% ( 7 12 ) ,而无此损害特征的 2 3耳中耳胆脂瘤组织HPVDNA检测阳性率为 13.0 %( 3 2 3) ,统计学检验差异有显著性 ( χ2 =7.92 6 ,P <0 .0 0 5 )。结论　HPV感染可能激发中耳胆脂瘤上皮的分裂增殖 ,在中耳胆脂瘤发生发展中起一定的作用 ;侵蚀性乳头瘤样生长和空晕细胞改变可以作为中耳胆脂瘤组织中HPV感染的病理学证据