High acyl-CoA cholesterol acyl transferase activity in fetal rabbit aorta: evidence for the presence of stimulating factor(s) in amniotic fluid

The activity of Acyl CoA-cholesterol acyl transferase was markedly high in fetal aortas when compared to maternal and adult male rabbits. This activity dropped by 50% at 1 week of age. This high activity in fetal aorta a) did not appear to be due to changes in plasma cholesterol levels or to the later development of endogenous inhibitor in the aorta, but rather b) due to stimulatory factor(s) present in amniotic fluid.     

Hepatic 3-hydroxy-3-methylglutaryl coenzyme A reductase activity and cholesterol 7 alpha-hydroxylase activity in neonatal guinea pig.

Optimal assay conditions for hepatic HMG-CoA reducatase activity and cholesterol 7 alpha-hydroxylase activity in the guinea pig were determined. These two enzyme activities were studied in the liver of newborn guinea pigs during the first three postnatal weeks. Hepatic HMG-CoA reductase activity of neonatal guinea pigs was similar to that of adult animals. However, cholesterol 7 alpha-hydroxylase activity of newborns was about one-third of that in adult guinea pigs. This finding suggests that the system for bile acid synthesis in the neonatal guinea pigs is underdeveloped.     

Loss of N-linked glycosylation from the hemagglutinin-neuraminidase protein alters virulence of Newcastle disease virus

The hemagglutinin-neuraminidase (HN) protein of Newcastle disease virus (NDV) is an important determinant of its virulence. We investigated the role of each of the four functional N-linked glycosylation sites (G1 to G4) of the HN glycoprotein of NDV on its pathogenicity. The N-linked glycosylation sites G1 to G4 at residues 119, 341, 433, and 481, respectively, of a moderately pathogenic NDV strain Beaudette C (BC) were eliminated individually by site-directed mutagenesis on a full-length cDNA clone of BC. A double mutant (G12) was also created by eliminating the first and second glycosylation sites at residues 119 and 341, respectively. Infectious virus was recovered from each of the cDNA clones of the HN glycoprotein mutants, employing a reverse genetics technique. There was a greater delay in the replication of G4 and G12 mutant viruses than in the parental virus. Loss of glycosylation does not affect the receptor recognition by HN glycoprotein of NDV. The neuraminidase activity of G4 and G12 mutant viruses and the fusogenicity of the G4 mutant virus were significantly lower than those of the parental virus. The fusogenicity of the double mutant virus (G12) was significantly higher than that of the parental virus. Cell surface expression of the G4 virus HN was significantly lower than that of the parental virus. The antigenic reactivities of the mutants to a panel of monoclonal antibodies against the HN protein indicated that removal of glycosylation from the HN protein increased (G1, G3, and G12) or decreased (G2 and G4) the formation of antigenic sites, depending on their location. In standard tests to assess virulence in chickens, all of the glycosylation mutants were less virulent than the parental BC virus, but the G4 and G12 mutants were the least virulent.     

A recombinant Newcastle disease virus (NDV) expressing VP2 protein of infectious bursal disease virus (IBDV) protects against NDV and IBDV

Infectious bursal disease virus (IBDV) causes a highly immunosuppressive disease in chickens. Currently available, live IBDV vaccines can lead to generation of variant viruses. We have developed an alternative vaccine that will not create variant IBDV. By using the reverse genetics approach, we devised a recombinant Newcastle disease virus (NDV) vector from a commonly used vaccine strain LaSota to express the host-protective immunogen VP2 of a variant IBDV strain GLS-5. The gene encoding the VP2 protein of the IBDV was inserted into the most 3'-proximal locus of a full-length NDV cDNA for high-level expression. We successfully recovered the recombinant virus, rLaSota/VP2. The rLaSota/VP2 was genetically stable, at least up to 12 serial passages in chicken embryos, and was shown to express the VP2 protein. The VP2 protein was not incorporated into the virions of recombinant virus. Recombinant rLaSota/VP2 replicated to a titer similar to that of parental NDV strain LaSota in chicken embryos and cell cultures. To assess protective efficacy of the rLaSota/VP2, 2-day-old specific-pathogen-free chickens were vaccinated with the recombinant virus and challenged with a highly virulent NDV strain Texas GB or IBDV variant strain GLS-5 at 3 weeks postvaccination. Vaccination with rLaSota/VP2 generated antibody responses against both NDV and IBDV and provided 90% protection against NDV and IBDV. Booster immunization induced higher levels of antibody responses against both NDV and IBDV and conferred complete protection against both viruses. These results indicate that the recombinant NDV can be used as a vaccine vector for other avian pathogens.     

Different protein sequences can give rise to highly similar folds through different stabilizing interactions.

We report an interesting case of structural similarity between 2 small, nonhomologous proteins, the third domain of ovomucoid (ovomucoid) and the C-terminal fragment of ribosomal L7/L12 protein (CTF). The region of similarity consists of a 3-stranded beta-sheet and an alpha-helix. This region is highly similar; the corresponding elements of secondary structure share a common topology, and the RMS difference for "equivalent" C alpha atoms is 1.6 A. Surprisingly, this common structure arises from completely different sequences. For the common core, the sequence identity is less than 3%, and there is neither significant sequence similarity nor similarity in the position or orientation of conserved hydrophobic residues. This superposition raises the question of how 2 entirely different sequences can produce an identical structure. Analyzing this common region in ovomucoid revealed that it is stabilized by disulfide bonds. In contrast, the corresponding structure in CTF is stabilized in the alpha-helix by a composition of residues with high helix-forming propensities. This result suggests that different sequences and different stabilizing interactions can produce an identical structure.     

The use of side-chain packing methods in modeling bacteriophage repressor and cro proteins.

In recent years, it has been repeatedly demonstrated that the coordinates of the main-chain atoms alone are sufficient to determine the side-chain conformations of buried residues of compact proteins. Given a perfect backbone, the side-chain packing method can predict the side-chain conformations to an accuracy as high as 1.2 Å RMS deviation (RMSD) with greater than 80% of the χ angles correct. However, similarly rigorous studies have not been conducted to determine how well these apply, if at all, to the more important problem of homology modeling per se. Specifically, if the available backbone is imperfect, as expected for practical application of homology modeling, can packing constraints alone achieve sufficiently accurate predictions to be useful? Here, by systematically applying such methods to the pairwise modeling of two repressor and two cro proteins from the closely related bacteriophages 434 and P22, we find that when the backbone RMSD is 0.8 Å, the prediction on buried side chain is accurate with an RMS error of 1.8 Å and approximately 70% of the χ angles correctly predicted. When the backbone RMSD is larger, in the range of 1.6–1.8 Å, the prediction quality is still significantly better than random, with RMS error at 2.2 Å on the buried side chains and 60% accuracy on χ angles. Together these results suggest the following rules-of-thumb for homology modeling of buried side chains. When the sequence identity between the modeled sequence and the template sequence is >50% (or, equivalently, the expected backbone RMSD is <1 Å), side-chain packing methods work well. When sequence identity is between 30–50%, reflecting a backbone RMS error of 1–2 Å, it is still valid to use side-chain packing methods to predict the buried residues, albeit with care. When sequence identity is below 30% (or backbone RMS error greater than 2 Å), the backbone constraint alone is unlikely to produce useful models. Other methods, such as those involving the use of database fragments to reconstruct a template backbone, may be necessary as a complementary guide for modeling.