GC-pair distribution in the DNA of simian SA7 and human type 6 adenoviruses

The buoyant density values in cesium chloride of DNA fragments of simian adenovirus SA7 obtained with restricting endonucleases of R. EcoRI, R. BamHI, R. SalI and human adenovirus type 6 DNA fragments obtained with restricting endonucleases of R. EcoRI and R. BamHI were determined. On the basis of the buoyant density values for these fragments the content of GC pairs in them was calculated. Using the known position of the fragments in the physical map of these DNAs, the distribution of GC pairs along the examined DNA molecules was constructed.     

Studies on human adenoviruses as inducers of interferon in chick cells

Summary Purified adenoviruses types 2 and 5 were found to induce a viral inhibitor with the properties of interferon in chick embryo fibroblasts. Soluble antigens alone were ineffective.Trypsin eliminated the interferon-inducing capacity of purified virus. Its effect was not entirely explained. It did not affect the adsorption of the virus to chick or KB cells, but it released acid soluble fragments from labelled virus particles. Adenovirus type 5 produced acid, a late cytopathic effect and occasionally replicated in chick cells in addition to inducing interferon.Dedicated to ProfessorJohn F. Enders on the occasion of his 70th birthday.Senior Fulbright Research Fellow on sabbatical leave from the Graduate School of Public Health, University of Pittsburgh, 1965–1966.Supported in part by the Deutschen Forschungsgemeinschaft.     

密码子优化提高重组腺病毒中人轮状病毒G1VP7和G3VP7基因的表达量

目的 通过密码子优化提高重组腺病毒中人轮状病毒G1VP7和G3VP7基因的表达量.方法 人工合成按照人的优势密码子优化的人轮状病毒G1VP7和G3VP7基因,包装重组腺病毒后,与未优化的相同基因的重组腺病毒用Western Blot方法比较其表达量.结果 密码子优化的人轮状病毒G1VP7和G3VP7基因,与未优化的相同基因的重组腺病毒比较,其蛋白表达量大大提高.结论 密码子优化确实提高了重组腺病毒中人轮状病毒G1VP7和G3VP7基因的表达量,可望满足腺病毒载体疫苗的研发需要.     

Replication of human adenoviruses in guinea pig embryo cells: an ultrastructural study

Summary Guinea pig embryo (GPE) cells showed different degrees of susceptibility to human adenovirus types as determined by virus infectivity assay and electron microscopic examination. Adenovirus 2 and 5 induced extensive cellular changes and produced high titers of infectious virus in GPE cells as in human cells. Mature progeny virus and protein crystals were observed in both cell types. Adenovirus 7 induced some cellular changes in GPE cells but only a small number of cells yielded progeny virus as determined by electron microscopy. Adenovirus 3, 8 and 31 induced some cellular changes but no progeny virus was found under electron microscopic examination. Characteristic fibers were observed in nuclei of adenovirus 31 infected cells. The ability of human adenovirus 2 and 5 to replicate in GPE cells is an example of an unusual cross-species biological property of certain adenovirus types. This property may be useful as a biological marker for these virus types.With 8 Figures     

Properties of the nicotinic-receptor-activated current in adrenal chromaffin cells of the guinea-pig

Properties of acetylcholine(ACh)- and nicotine-induced currents were studied in the guinea-pig chromaffin cell, using the whole-cell and cell-attached versions of the patch-clamp technique. Bath application of ACh or nicotine, but not muscarine, produced an inward current associated with an increase in current noise at a membrane potential of –70 mV. Low concentrations of both agonists produced a sustained inward current whereas high concentrations produced a transient, then a sustained inward current. Nicotine was about twice as potent as ACh in inducing the inward current. Hexamethonium (6 M) inhibited the ACh-induced current but not in a competitive manner. By contrast, atropine (6 M) inhibited the ACh-induced current more strongly with increasing concentrations of ACh. The nicotinic-receptor-activated current (nicotinic current) showed inward rectification and, when Cs⁺ was used instead of K⁺ in the pipette solution, the polarity of the current changed at around –5 mV and a negative slope occurred between +10 mV and +30 mV. The nicotinic channel had a unit conductance of 33 pS. During the initial 20–30 min of whole-cell voltage-clamp recording, the time course of the agonist-induced desensitization was markedly facilitated. Addition of 5 mM MgATP in the pipette solution at least partly prevented this facilitation of the desensitization. The frequency of activation of the nicotinic receptor and the extracellular Ca²⁺ were not primary factors in the acceleration of rate of desensitization.     

Candidate adenoviruses 40 and 41: fastidious adenoviruses from human infant stool

About 200 antigenically related adenoviruses were isolated from cases of infantile diarrhoea in the Netherlands and North-West Germany. The viruses were fastidious and failed to replicate serially in human diploid fibroblasts and in primary human embryonic kidney cells. A number of strains were established in HeLa, HEp-2, Graham (293), cynomolgus monkey kidney, and Chang conjunctival cells. The viruses were mammalian adenoviruses by the usual criteria. No relationship to the 39 known human adenovirus species was found, either by neutralization tests or by haemagglutination inhibition tests. Neutralization tests showed two distinct variants, represented by strains Tak and Dugan. The variants were identical in haemagglutination inhibition tests. DNA restriction enzyme analysis showed Tak and Dugan to have considerably different genomes, indicating that these variants should be classified as different species (Wadell et al, 1983). It is proposed that the variants should be called Mastadenovirus h 40 (with reference strains Dugan and Hovi X) and Mastadenovirus h 41 (with reference strain Tak). Neutralization and haemagglutination inhibition tests demonstrated that the viruses from Glasgow and Helsinki (Hovi X) described by Johansson et al [1980] and by Kidd and Madeley [1981] belong to these two adenovirus species.     

Properties of membrane currents in isolated smooth muscle cells from guinea-pig trachea

Tracheal smooth muscle cells were enzymatically isolated from guinea-pig trachea. These cells contracted in response to acetylcholine (0.01–10 M) in a concentration-dependent fashion. Under current-clamp conditions with 140 mM K⁺ in the pipette solution, the membrane potential oscillated spontaneously at around –30 mV. Under voltage-clamp conditions, there appeared spontaneous but steady oscillations of outward current (I _o). On depolarization from a holding potential at –40 mV, three components of outward current were elicited: transient outward current (I _T), steady-state outward current (I _s) and I _o. These three components of outward current reversed around the K⁺ equilibrium potential and were abolished by Cs⁺ in the pipette, indicating that K⁺ was the major charge carrier of these outward currents. All these three components were completely suppressed by extracellular tetraethylammonium (10 mM). Both I _T and I _o were depressed by quinidine (1 mM), 4-aminopyridine (10 mM) and nifedipine (100 nM), but I _s was not affected. I _T and I _o were suppressed by a Ca²⁺-free perfusate with less than 1 nM Ca²⁺ in the pipette, while with 10 nM Ca²⁺ in the pipette, only I _o was suppressed. In both conditions, I _s was not affected by the Ca²⁺-free perfusate. Therefore, it is suggested that I _o, I _T and I _s are separate types of K⁺ current. With Cs⁺ in the pipette, K⁺ currents were almost completely suppressed and a transient inward current was observed during depolarizing pulses. The inward current was not affected by tetrodotoxin and increased when the concentration of extracellular Ca²⁺ was raised, indicating that the current is a Ca²⁺ channel current. Even with a holding potential of –80 mV, the low-threshold inward current could not be observed. The high-threshold Ca²⁺ current was abolished by nifedipine (100 nM) and was enhanced by Bay K 8644 (100 nM). The order of permeation of divalent cations through the Ca²⁺ channel was Ba²⁺ >Sr²⁺ Ca²⁺. Cd²⁺ blocked the Ca²⁺ current more effectively than Ni²⁺. These results may indicate that the Ca²⁺ current of tracheal smooth muscle cells is mainly composed of the current through an L-type Ca²⁺ channel.     

Characterization of human thymic epithelial cells grown in serum-free medium

Thymic epithelial cells have a critical influence on T-cell differentiation. In order to characterize these cells in humans, a serum-free growth medium was developed for their long-term culture. Important components of this medium included transferrin, epidermal growth factor, prostaglandin E1, and selenious acid. The presence of a keratin cytoskeleton, tonofilaments, and desmosomes confirmed the epithelial nature of these cells. Indirect immunofluorescence study of these epithelial cells demonstrated the presence of Ia and B-2 microglobulin antigens. The availability of highly enriched thymic epithelial cultures should simplify the functional characterization of this cell.     

Cross-reactivity between human adenoviruses in delayed-type hypersensitivity

The aim of this study was to determine the antigen responsible for the induction of delayed-type hypersensitivity (DTH) by human adenoviruses (Ads). The estimation of DTH was based on measurement of the extent of swelling of the hind footpads of mice. CsCl density gradient-purified human Ad serotype 6 (Ad6) induced DTH in a dose-dependent manner. In Ad6-sensitized mice, DTH could be elicited by serotypes belonging to the same species of human Ads (types 1 and 5) and by a serotype (type 3) belonging to another species. Latex particles coated with purified hexon antigen prepared from Ad5 had the capacity to sensitize mice and elicit a DTH reaction. We suggest that, for serotypes belonging to species C, the cross-reactive highly conserved T cell epitope of the hexon protein might be responsible for the DTH induction, and furthermore the same epitope might result in the cross-reactivity between serotypes 3 and 6. The possible importance of these data is discussed in relation to human gene therapy through the application of Ad vectors.     

Interaction of Zn2+ with guinea-pig C5 convertase and guinea-pig C5

A comparison of two methods of C5 activation, the standard method (EAC14 + C2, C3, C5, C6, and C7) and the washed-cell intermediate method (EAC1423 + C5 and washed), demonstrated that formation of hemolytically competent SAC14235 was reduced in the washed-cell method. Addition of Zn2+ in this method increased the formation of competent SAC14235 to the approximate level of the standard method. The optimum concn range of Zn2+ was 0.006-0.025 mM. In the standard method Zn2+ had no significant enhancing effect on the formation of competent SAC14235. Zn2+ had a greater affinity for EAC1423 than for fluid-phase C5 when reacted with each separately. Maximum enhancement, however, was obtained when Zn2+ was present during the reaction of C5 with EAC1423. The effect of Zn2+ on C5 activity was not related to the stabilizing property of C6 on cell-bound C5. In the washed-cell method there was an inverse relationship between the concn of C2 or C3 and the concn of C5 required to generate one competent SAC14235/cell. Over a 10-fold range of C2 or C3 concn there was an approximate four-fold increase in the number of competent SAC14235/cell formed in the presence of Zn2+. Zn2+ enhances formation of competent SAC14235 on cells that have a limited ability to activate C5 even though the C2 and C3 on the convertase are present in excess.     

Quantitative detection and rapid identification of human adenoviruses

We have established a method of quantitative detection and rapid identification of human adenoviruses (hAdVs). Using LightCycler PCR with a primer set, we were able to amplify 554 bp of the hexon gene from each of 51 prototype strains of hAdVs. The sensitivity of LightCycler PCR was 10 copies of hAdV DNA/reaction. When LightCycler PCR was performed using a set of primers, hAdV was positive for 74.4% (99 of 133) of conjunctivitis patients and for 27.3% (81 of 297) of respiratory infection patients. We also attempted to measure hAdV in the potentially contaminated eye drops used by patients, detecting 5.4 x 10(2) to 1.6 x 10(6) copies/ml of hAdV. We determined the 350-bp nucleotide sequence of the amplified hexon gene and compared it with the sequences of the 51 prototype strains. Phylogenetic analysis based on 350 bp of the hexon gene identified 99 positive conjunctival swabs as 24 cases of AdV type 3 (AdV-3), 14 cases of AdV-4, 1 case of AdV-8, 19 cases of AdV-19a, and 41 cases of AdV-37. The 81 sequences from pharyngeal or nasal mucus swabs were identified as 29 cases of AdV-2, 18 cases of AdV-1, 18 cases of AdV-5, 12 cases of AdV-4, 2 cases of AdV-37, 1 case of AdV-3, and 1 case of AdV-6. LightCycler PCR followed by phylogenetic analysis provides an effective tool for the rapid identification of hAdVs and for studying molecular epidemiology.     

Properties and expression of human tartrate-resistant acid phosphatase isoform 5a by monocyte-derived cells

Human serum tartrate-resistant acid phosphatase exists as two enzyme isoforms (TRACP 5a and 5b), derived by differential, post-translational processing of a common gene product. Serum TRACP 5b is from bone-resorbing osteoclasts (OC) and becomes elevated in diseases of increased bone resorption. TRACP 5a is secreted by macrophages (MPhi) and dendritic cells (DC) and is increased in many patients with rheumatoid arthritis. Our purpose was to fully characterize the properties of human TRACP isoforms and to produce an antibody specific to TRACP 5a for use as a biomarker in chronic inflammatory diseases. Partially purified, natural serum TRACP isoforms and recombinant TRACP 5a (rTRACP 5a) were compared with respect to specific activity and subunit structure and presence of sialic acid. Mice were immunized with rTRACP 5a, and resulting hybridomas were screened for monoclonal antibody to serum TRACP 5a. One antibody, 220, was tested for its epitope specificity and use in various immunological techniques. rTRACP 5a had properties identical to serum TRACP 5a. Antibody 220 was specific for the trypsin-sensitive epitope in the loop peptide, present only in TRACP 5a. Antibody 220 was effective for specific immunoprecipitation, immunoassay, and immunoblot of TRACP 5a. Intact TRACP was present in MPhi, DC, and OC. TRACP 5a was the predominant isoform secreted by MPhi and DC, whereas TRACP 5b was the predominant isoform secreted by OC. TRACP isoforms 5a and 5b may have different functions inside and outside of monocyte-derived cells. Antibody 220 is an important resource for studies of the biosynthetic relationship among TRACP isoforms and of the significance of serum TRACP 5a as a marker in diseases of bone metabolism and inflammation.     

Sensitivity of FL cells to polio- and adenoviruses

Summary Poliovirus plaque counts on the FL strain of human amnion cells were almost double the counts on rhesus monkey kidney for the same virus preparation, repeatedly assayed over more than six months. FL cells gave more consistent results in virus assay than monkey kidney cells. Plaque counts obtained with FL cells were 2 1/2 to 5 times higher than those obtained with HeLa, Chang's conjunctiva and Detroit-6 cells.Using the chick embryo-adapted MEF-1 strain of poliovirus, FL cells and primary human amnion cells reacted similarly in respect to plaque count and morphology, while no distinct plaques were seen on monkey kidney or HeLa monolayers under comparable conditions. For infectivity assays of adenovirus suspensions based on cytopathogenic effect, FL cells were found suitable, although no plaque formation was obtained.Aided by American Cancer Society Grant E-82 and a grant from the National Foundation.