Use of immunoperoxidase for the rapid identification of human myxoviruses and paramyxoviruses in tissue culture

The immunoperoxidase method, using commercially available antisera, was compared with standard virological methods for the identification and typing of 77 isolates of human myxoviruses and paramyxoviruses. Results of typing using neutralization tests and the immunoperoxidase technique were identical for 76 of the 77 isolates. With the immunoperoxidase method there was one false negative reaction, but no false positive reactions. Cross-reactivity between influenza A (soluble) and A₂/HK antisera with influenza A isolates was noted, but did not interfere with the interpretation of results. It is concluded that the immunoperoxidase method is ideally suited for the rapid identification and typing of common human respiratory viruses on a routine basis. It offers a number of decided advantages over both immunofluorescence and standard virological methods.     

Recovery and identification of DNA sequences harboured in preserved ancient human bones.

A new method is presented to extract and identify specific DNA fragments from well preserved human bones, dating from three different time periods. Bone samples were thoroughly freed from surfacial contaminating DNA. Access to the inner bone spongiosum was achieved by removing the covering bone layers of the vertebra or sternum, whereas the patella, tibia and caput of the femur or humerus were cleaved with an iron saw. After the spongiosum was taken out, extraction of nucleic acids from this "sand" like material was performed by heating at 94 degrees C during 20 min in a buffer containing essentially minor concentrations of detergent, chelating and reducing agents. The extracts were used in various Polymerase Chain Reaction (PCR) protocols to amplify different human specific DNA fragments (originating from chromosomes X and 12). From 15 out of 20 bone samples human-specific gene fragments could thus be identified.     

Recent contributions of molecular biology to the clinical virology of myxoviruses.

Recent advances in the clinical virology of influenza are based on non-pragmatically oriented research on the genetics and biochemistry of the influenza virus. Antigenically hybrid recombinant viruses can be tailored to provide monospecific reagents for serological studies. Basic research on viral structure and the mechanism of viral replication has directly influenced the establishment of a cell culture system suitable for the isolation of most influenza viruses. Identification of viral genotype by RNA gel electrophoresis and mapping of oligonucleotides of viral RNA has already facilitated epidemiologic investigations. The clinical virologist of the future must have an understanding of the potential limitations of these techniques for specific strain identification.     

抗人血栓调节蛋白单克隆抗体的制备与鉴定

为了制备特异性抗人血栓调节蛋白(human thrombomodulin,hTM)的单克隆抗体(monoclonal antibody,McAb),利用脂质体Lipofectamine 2000将包含hTM全长cDNA序列的重组表达质粒pThr402转染CHO细胞,经G418筛选及相关鉴定后获得高效稳定表达hTM的CHO-TM5细胞株。将CHO-TM5细胞直接免疫Balb/c小鼠,应用杂交瘤技术,通过细胞ELISA (cellular enzyme-linked immunoabsorbent assay,CELISA)筛选出阳性克隆后,将杂交瘤细胞株腹腔注射Balb/c小鼠诱生腹水。用CELISA、流式细胞术、免疫组织化学染色法及免疫印迹法对所获McAb的特异性进行鉴定。我们获得了1株可稳定分泌抗hTM的McAb的杂交瘤细胞株NH-1,其亚型为IgGl,McAb腹水效价为1×10~(-6),腹水抗体含量为20 mg/ml。NH-1对相应抗原具有较高的组织特异性,在体内与正常组织的交叉反应少,对人脐静脉内皮细胞、CHO-TM5有特异性结合反应,说明NH-1可特异性识别天然的hTM分子,为进一步应用此McAb进行hTM生物学功能及临床意义研究提供了基础。     

A possible mechanism for the action of some myxoviruses

Summary The redox properties of some myxoviruses [Fowl plaque virus strain Rostock (FPV), New Castle Disease virus strain Italy (NDV), B/Hong Kong, A/Port Chalmers, A/Victoria, A/Scotland, and A/Fort Dix (FD)] have been investigated by means of electron spin resonance (ESR) and electron microscopic studies as well as by the determination of the hemagglutination (HA) titer (antigen efficiency). The results have shown that viruses decrease the spin concentration of Cu²⁺ by acting as a reducing species (electron donor) which will result in the inactivation (oxidation) of the virus. Addition of an oxidizing substance, such as H₂O₂, to a virus suspension also leads to an oxidation of the viruses and, thus, to their inability to reduce Cu²⁺. This result is confirmed by the decrease of the HA titer of viruses with increasing Cu²⁺ concentrations. H₂O₂ could not be applied for the HA titer test since it interacts with the erythrocytes of the chicken blood used for this determination. Therefore, another oxidizing substance (oxidized glutathione, GSS) was selected which exhibited a slightly less pronounced effect than Cu²⁺. Since reduced glutathione (GSH) exerts a similar but less pronounced effect than GSS, it might be concluded that viruses have a redox system of their own and act as reducing or oxidizing substance depending on the biological receptor system. Electron microscopic studies confirm this hypothesis. As can be seen by the electron micrographs, increasing concentrations of either Cu²⁺, GSS, H₂O₂, KMnO₄, or GSH will, finally, result in a complete destruction of the virus. Because of structural similarities it might be assumed that other types of viruses behave very similarly.     

Individual DNA identification from ancient human remains.

Individual identification of ancient human remains is one of the most fundamental requisites for studies of paleo-population genetics, including kinship among ancient people, intra- and interpopulation structures in ancient times, and the origin of human populations. However, knowledge of these subjects has been based mainly on circumstantial archaeological evidence for kinship and intrapopulation structure and on genetic studies of modern human populations. Here we describe individual identification of ancient humans by using short-nucleotide tandem repeats and mtDNAs as genetic markers. The application of this approach to kinship analysis shows clearly the presence or absence of kinship among the ancient remains examined.     

Preliminary identification and role of phosphodiesterase isozymes in human basophils.

We attempted to identify and establish the role of cyclic nucleotide phosphodiesterase (PDE) isozymes in human basophils by using standard biochemical techniques as well as describing the effects of isozyme-selective and nonselective inhibitors of PDE. The nonselective PDE inhibitors, theophylline and 3-isobutyl-1-methylxanthine, inhibited anti-IgE-induced release of histamine and leukotriene C4 (LTC4) from basophils. This inhibition was accompanied by elevations in cAMP levels. Rolipram, an inhibitor of the low Km cAMP-specific PDE (PDE IV), inhibited the release of both histamine and LTC4 from activated basophils and increased cAMP levels in these cells. In contrast, mediator release from basophils was not inhibited by either siguazodan or SK&F 95654, inhibitors of the cGMP-inhibited PDE (PDE III) or zaprinast, an inhibitor of the cGMP-specific PDE (PDE V). SK&F 95654 failed to elevate basophil cAMP in these experiments whereas zaprinast induced significant increases in cAMP content. The inhibitory effect of rolipram on mediator release was potentiated by siguazodan or SK&F 95654, but not by zaprinast. SK&F 95654 also enhanced the ability of rolipram to increase cAMP content. Forskolin, a direct activator of adenylate cyclase, inhibited IgE-dependent release of mediators from basophils and increased cAMP levels in these cells. These effects were enhanced by rolipram, but not by SK&F 95654 or zaprinast. The cell permeant analog of cAMP, dibutyryl cAMP, inhibited mediator release from these cells, a property not shared by either dibutyryl-cGMP or sodium nitroprusside, an activator of soluble guanylate cyclase. The presence of both PDE III and PDE IV was confirmed by partially purifying and characterizing PDE activity in broken cell preparations. Overall, these data lend support to the hypothesis that cAMP inhibits mediator release from basophils and suggest that the major PDE isozyme responsible for regulating cyclic AMP content in these cells is PDE IV, with a minor contribution from PDE III. However, the finding that zaprinast caused increases in cAMP without inhibiting mediator release indicates that cAMP accumulation is not invariably linked to an inhibition of basophil activation.