Genetic retargeting of adenovirus vectors: functionality of targeting ligands and their influence on virus viability

Background

We studied the ability of adenovirus type 5 (Ad5) to encapsidate new cellular ligands carried by their fibers to yield functional retargeted vectors for gene therapy. Recombinant Ad5 fibers containing shaft repeats 1 to 7 and an extrinsic trimerization motif, and terminated by its native knob or amino acid motifs containing RGD, have been rescued into infectious virions.

Methods

Polypeptide ligands of cell surface molecules, including single‐chain antibodies or epidermal growth factor, were cloned into recombinant fibers. Phenotypic analysis of fiber constructs and rescuing into the Ad5 genome were performed. Recombinant viruses were characterized with reference to fiber content, growth rate and infectivity.

Results

A major limiting factor for recovering viable recombinant Ad5 carrying fiber‐fused polypeptide ligands was apparently the ability of the ligand to fold correctly within the cellular cytoplasm. This constraint has previously not been systematically evaluated in the literature. Phenotypic analysis of the fiber‐ligand fusions showed that their degree of cytoplasmic solubility correlated with their ability to yield viable Ad5 vectors. Our results suggested that the fiber manipulations diminish virus growth rate, probably through different, opposing effects: (i) the reduced shaft length increases fiber solubility in the absence of the knob but (ii) diminishes virus entry, and (iii) the absence of the knob alters the overall protein composition of the virion and decreases its fiber copy number.

Conclusions

Based on our findings, cytoplasmic solubility and cytoplasmic ligand reactivity of fiber‐ligand fusion proteins are the best prediction criterion for viability and recovery of genetically retargeted Ad vectors. Copyright © 2002 John Wiley & Sons, Ltd.

     

Inhibition of Klenow DNA polymerase and poly(A)-specific ribonuclease by aminoglycosides

Aminoglycosides are known to bind and perturb the function of catalytic RNA. Here we show that they also are potent inhibitors of protein-based catalysis using Escherichia coli Klenow polymerase (pol) and mammalian poly(A)-specific ribonuclease (PARN) as model enzymes. The inhibition was pH dependent and released in a competitive manner by Mg2+. Kinetic analysis showed that neomycin B behaved as a mixed noncompetitive inhibitor. Iron-mediated hydroxyl radical cleavage was used to show that neomycin B interfered with metal-ion binding in the active sites of both enzymes. Our analysis suggests a mechanism of inhibition where the aminoglycoside binds in the active site of the enzyme and thereby displaces catalytically important divalent metal ions. The potential causes of aminoglycoside toxicity and the usage of aminoglycosides to probe, characterize, and perturb metalloenzymes are discussed.     

Improved partitioning in aqueous two-phase system of tyrosine-tagged recombinant lactate dehydrogenase

The partitioning of Bacillus stearothermophilus lactate dehydrogenase (LDH) in an aqueous two-phase system was studied. Particularly, the influence of tyrosine tags on the partitioning was evaluated. The hydrophobic effect, caused by the addition of tyrosine residues, was determined in a system based on dextran and the thermoseparating ethylene oxide-propylene oxide random copolymer (EO30PO70). Five different LDH variants were constructed with N-terminal tags containing tyrosines (Y3 and Y6), tyrosines and prolines (Y3P2 and Y6P2), and only prolines (P2). LDH fused with tags containing tyrosines increased the partitioning coefficient, and the more tyrosines added to the protein, the larger improvement in partitioning. When prolines were added between the tyrosine-rich tag and the protein, a further increased partitioning was obtained. The enhanced partitioning was attributed to the rigid structure of the proline, which in turn led to an increase in the exposure of the tag to the surroundings. The best tyrosine tag, Y6P2, increased the partition coefficient four times and additionally, a higher thermostability was observed.     

Survival of Flavobacterium psychrophilum in rainbow trout (Oncorhynchus mykiss) serum in vitro

Virulent and non-virulent strains of Flavobacterium psychrophilum of different serotypes were examined for survival and growth in non-immune and immune rainbow trout serum, in vitro. A majority of the examined strains consumed complement of non-immune serum, but the complement cascade was not able to cause an immediate (after 3 h incubation) notable reduction in viability of the inoculated cells. After 24 h incubation a more pronounced reduction in the number of viable bacteria was observed in untreated serum as well as in serum heated at 45 degrees C. In serum heated at 56 degrees C this reduction in cell number was not observed, but an increase in cell number did not occur either. The serum survival of one of the examined strains was different from the others in showing cell multiplication after 24 h incubation in normal as well as heat-treated (45 and 56 degrees C) serum. In immune serum no immediate reduction in viability of inoculated cells, of all tested strains, was observed. The number of viable cells showed a slow decrease or remained almost unchanged for up to 72 h post-inoculation in untreated serum, at 5 degrees C as well as 15 degrees C. In heat-treated serum (45 degrees C) the number of viable cells decreased slowly at 5 degrees C and 15 degrees C for up to 72 h. The results suggest that the examined strains were unaffected by the alternative complement reaction present in fish serum as well as by antibodies against F. psychrophilum. However, some unknown component(s) in the fish sera, or lack of nutrients or essential growth factors, inhibited the growth of most of the examined strains in the tested fish sera.     

Selective extraction of lecithin:cholesterol acyltransferase (EC 2.3.1.43) from human plasma

A method for the rapid extraction of lecithin:cholesterol acyltransferase (LCAT) from human plasma or serum has been developed. The method is based on direct treatment of acidified plasma of fully conserved enzyme activity, with the strong ion exchanger Q-Sepharose, which under the experimental conditions bound all LCAT but only about 10% of the total protein content of the plasma, no albumin and essentially no lipoproteins. This corresponds to a 10-fold purification. Only traces of apolipoprotein A-I remained in the quantitatively desorbed LCAT preparation which, however, contained a residual fraction of apolipoprotein D and acidic plasma proteins.The present one-step procedure for extraction of LCAT in high yields from human plasma represents a simple and efficient alternative to the first step in previously described methods for preparation of the enzyme to homogeneity.     

Effects of exposure to 17alpha-ethinylestradiol during early development on sexual differentiation and induction of vitellogenin in zebrafish (Danio rerio)

The effect of acute stress on plasma beta-corticosterone (B), testosterone (T) and estradiol-17beta (E2) concentrations in juvenile alligators collected from sites with varying sediment contaminants was examined in this study. Dramatic increases in plasma B concentrations were observed in alligators from all of the sites after 2 h of capture although females from the intermediate contaminant site exhibited a significantly lower percentage increase in B than females from the other two sites. Males from the site with the highest contaminant levels exhibited elevated initial B concentrations relative to the other sites. This pattern was not observed after 2 h of restraint. Females from the highest contaminant site exhibited depressed initial T when compared to the other sites although this pattern was not observed after 2 h of restraint. Neither E2 nor T decreased after 2 h in females, whereas T concentrations decreased in all males over the same time period. The variance associated with these endpoints was also examined to determine whether it could serve as a more sensitive marker for perturbations of the endocrine system and stress response. Females from the higher and intermediate contaminant sites exhibited the lowest and highest standard errors (respectively) associated with 2 h plasma B concentrations with no differences among mean concentrations suggesting a perturbation of the stress response in these animals that was not detected by examining the means. We concluded that the environmental contaminants could be acting as stressors, leading to the observed differences.     

Long-term history of land-use and vegetation at Ire,an agriculturally marginal area in Blekinge,south Sweden

Pollen records from a small lake and a small peatland at Ire in northern Blekinge, Sweden, reveal that until a.d. 300 there was a mixed deciduous type of woodland dominated by Quercus, Tilia and Corylus, with Betula, Pinus, Populus, Fraxinus, Ulmus and Acer as important constituents. The first, but weak, signs of human influence on the vegetation are detected around 2300 b.c. At this time, the area was probably used for woodland grazing. The regional expansion of Fagus occurred around 100 b.c.–a.d. 200, and later, at about a.d. 500, woods dominated by Fagus were common in the area. Around a.d. 600–700 an isolated farmstead may have been established in the area, as single pollen grains of Secale (rye) were found. This farmstead may represent the first permanent settlement in the area. A more widespread opening of the vegetation occurred around a.d. 1000, probably as an effect of a more pronounced use of the area, and an expansion of settlements in the region. The openness of the area seems to have peaked around a.d. 1400–1800, and during this period the vegetation was highly fragmented with small stands of woodland, intensively grazed pastures, and arable fields where Secale and Triticum were mainly cultivated. Around a.d. 1600 Fagus pollen percentages sharply decrease, most probably caused by a deliberate and selective felling of beech trees for the production of potash, which consumed enormous amounts of wood. Picea seems to have been established around a.d. 1600, but it did not become a regional dominant until the first part of the 20th century, when land use decreased and it became favoured by forestry.     

Flavobacterium psychrophilum, invasion into and shedding by rainbow trout Oncorhynchus mykiss

The infection route of Flavobacterium psychrophilum into rainbow trout Oncorhynchus mykiss was studied using bath and cohabitation challenges as well as oral challenge with live feed as a vector. Additionally, the number of bacterial cells shed by infected fish into the surrounding water was determined in the cohabitation experiment and in challenge experiments at 3 different water temperatures. The experiments showed that skin and skin mucus abrasion dramatically enhanced the invasion of F. psychrophilum into the affected fish in bath and cohabitation challenges. Disruption of the skin is discussed as an important invasion route for F. psychrophilum into the fish. The shedding rate of F. psychrophilum by infected fish was associated with water temperature and the mortality of the infected fish. High numbers of F. psychrophilum cells were released into the water by dead rainbow trout during a long time period compared to the numbers of cells shed by live fish. The results emphasise the importance of removing dead and moribund fish from rearing tanks in order to diminish the infection pressure against uninfected fish in commercial fish farms. In immunohistochemical examinations of organs and tissues of orally infected fish, F. psychrophilum cells were detected in only 1 fish out of 31 studied. Mortality of the orally challenged fish was not observed in the experiment.     

Children with septo-optic dysplasia - how to improve and sharpen the diagnosis

The data in this article are based on investigations performed in 25 children with suspected septo-optic dysplasia (SOD). There are many signs and methods that help in the diagnosis of SOD. In particular, the ocular fundus, abnormalities of the hypothalamo-pituitary axis and other midline brain structures should be described. In order to achieve a more holistic and functional diagnosis, the degree of neurological, neuropsychiatric and psychological involvement should also be stated. It has been suggested that SOD is associated with autosomal recessive inheritance, and it can be speculated that it is the result of genetic and environmental influences early in gestation. An early diagnosis can favourably influence the outcome of the affected child.