While K-ras is essential for mouse development, expression of the K-ras 4A splice variant is dispensable

In mammals, the three classical ras genes encode four highly homologous proteins, N-Ras, H-Ras, and the isoforms K-Ras 4A and 4B. Previous studies have shown that K-ras is essential for mouse development and that while K-ras 4A and 4B are expressed during development, K-ras 4A expression is regulated temporally and spatially and occurs in adult kidney, intestine, stomach, and liver. In the present study, the pattern of K-ras 4A expression was examined in a wide range of wild-type adult mouse tissues, and gene targeting was used to generate K-ras 4A-deficient mice to examine its role in development. It was found that K-ras 4A is also expressed in uterus, lung, pancreas, salivary glands, seminal vesicles, bone marrow cells, and cecum, where it was the major K-Ras isoform expressed. Mating between K-ras(tmDelta4A/+) mice produced viable K-ras(tmDelta4A/tmDelta4A) offspring with the expected Mendelian ratios of inheritance, and these mice expressed the K-ras 4B splice variant only. K-ras(tmDelta4A/tmDelta4A) mice were fertile and showed no histopathological abnormalities on inbred (129/Ola) or crossbred (129/Ola x C57BL/6) genetic backgrounds. The results demonstrate that K-Ras 4A, like H- and N-Ras, is dispensable for normal mouse development, at least in the presence of functional K-Ras 4B.     

Identification of critical residues in the active site of porcine membrane-bound aminopeptidase P

The membrane-bound form of mammalian aminopeptidase P (AP-P; EC 3.4. 11.9) is a mono-zinc-containing enzyme that lacks any of the typical metal binding motifs found in other zinc metalloproteases. To identify residues involved in metal binding and catalysis, sequence and structural information was used to align the sequence of porcine membrane-bound AP-P with other members of the peptidase clan MG, including Escherichia coli AP-P and methionyl aminopeptidases. Residues predicted to be critical for activity were mutated and the resultant proteins were expressed in COS-1 cells. Immunoelectrophoretic blot analysis was used to compare the levels of expression of the mutant proteins, and their ability to hydrolyze bradykinin and Gly-Pro-hydroxyPro was assessed. Asp449, Asp460, His523, Glu554, and Glu568 are predicted to serve as metal ion ligands in the active site, and mutagenesis of these residues resulted in fully glycosylated proteins that were catalytically inactive. Mutation of His429 and His532 also resulted in catalytically inactive proteins, and these residues, by analogy with E. coli AP-P, are likely to play a role in shuttling protons during catalysis. These studies indicate that mammalian membrane-bound AP-P has an active-site configuration similar to that of other members of the peptidase clan MG, which is compatible with either a dual metal ion model or a single metal ion in the active site. The latter model is consistent, however, with the known metal stoichiometry of both the membrane-bound and cytosolic forms of AP-P and with a recently proposed model for methionyl aminopeptidase.     

Prediction of genetic structure in eukaryotic DNA using reference point logistic regression and sequence alignment

MOTIVATION: Current software tools are moderately effective in predicting genetic structure (exons, introns, intergenic regions, and complete genes) from raw DNA sequence data. Improvements in accuracy and speed are needed to deal with the increasing volume of data from large scale sequencing projects. RESULTS: We present a two-stage computer program to predict genetic structure in eukaryotic DNA. The first stage makes use of a novel statistical technique, called reference point logistic (RPL) regression, to calculate scores for potential functional sites. These site scores are combined with interval content, length, and state scores, via a Generalized Hidden Markov Model, to determine a combined score for each possible parse of a given DNA sequence into exons, introns, and intergenic regions. An optimal parse is found using a dynamic programming algorithm. In the second stage, protein sequence alignment methods are applied to improve the accuracy of the initial parse. Computation in the first stage of the program is very fast (1 s on a 360 MHz CPU for a 16 kb sequence) and its predictive accuracy typically matches or exceeds the best results reported for other methods (Sensitivity = 0.93 and Specificity = 0.93 for the Burset/Guigótest set). Computation in the second stage is slower, but the final predictions are more accurate (Sn = 0.97, Sp = 0.97). The program (called GRPL) can handle partial, single, and multi-gene sequences. The program is also capable of predicting the genetic structure of vertebrate, invertebrate, and plant DNA with nearly equal accuracy. Statistical techniques have also been introduced to model the effects of varying C+G content in a continuous manner and to control overfitting of parameters for smaller training sets. AVAILABILITY: An academic implementation of GRPL, compiled for SUN workstations, is available by anonymous ftp from snipe.pharmacy. ualberta.ca/pub. The training and test sets used in this work, together with supplementary material, can be found at the same location. A commercial implementation is available as a component of GeneTool (BioTools Inc., http://biotools.com).     

The spasmodic peptide defines a new conotoxin superfamily

We purified and characterized a peptide from the venom of Conus textile that makes normal mice assume the phenotype of a well-known mutant, the spasmodic mouse. This "spasmodic" peptide has 27 amino acids, including two gamma-carboxyglutamate (Gla) residues. A cDNA clone encoding the precursor for the peptide was identified; a gamma-carboxylation recognition signal sequence (gamma-CRS) is present in the -1 --> -20 region of the peptide precursor. Both the gamma-CRS and the position of the Gla residues in the mature toxin are notably different from other Gla-containing conopeptides. The spasmodic peptide has a novel disulfide framework and distinct signal sequence which together define a new P-superfamily of conopeptides. A cDNA encoding another member of the P-superfamily was identified from a different species, Conus gloriamaris.     

Primary sequence and solution conformation of ferrocytochrome c-552 from Nitrosomonas europaea.

Cytochrome c-552 from Nitrosomonas europaea is a 9.1-kDa monoheme protein that is a member of the bacterial cytochrome c-551 family. The gene encoding for c-552 has been cloned and sequenced and the primary sequence of the product deduced. Proton resonance assignments were made for all main-chain and most side-chain protons in the diamagnetic, reduced form by two-dimensional NMR techniques. Distance constraints (1056) were determined from nuclear Overhauser enhancements, and torsion angle constraints (88) were determined from scalar coupling estimates. Solution conformations for the protein were computed by the hybrid distance geometry-simulated annealing approach. For 20 computed structures, the root mean squared deviation from the average position of equivalent atoms was 0.84 A (sigma = 0.12) for backbone atoms over all residues. Analysis by residue revealed there were three regions clearly less well defined than the rest of the protein: the first two residues at the N-terminus, the last two at the C-terminus, and a loop region from residues 34 to 40. Omitting these regions from the comparison, the root mean squared deviation was 0.61 A (sigma = 0.13) for backbone atoms, 0.86 A (sigma = 0.12) for all associated heavy atoms, and 0. 43 A (sigma = 0.17) for the heme group. The global folding of the protein is consistent with others in the c-551 family. A deletion at the N-terminus relative to other family members had no impact on the global folding, whereas an insertion at residue 65 did affect the way the polypeptide packs against the methionine-ligated side of the heme. The effects of specific substitutions will be discussed. The structure of c-552 serves to delineate essential features of the c-551 family.     

The correlation of protein structure and evolution of a protein-coding gene: phylogenetic inference using cytochrome oxidase III

Discriminating phylogenetic signal from noise in DNA sequence data is a difficult problem in phylogenetic inference at higher systematic levels. For protein-coding genes, noise at synonymous (silent) positions can be filtered by deleting entire codon positions or types of change at a codon position. This method is not appropriate for replacement sites, because changes at each site within a codon may not be independent. This research presents a method using information from protein structure to evaluate variation in replacement sites. Analysis of the correlation of amino acid variation with protein structure identified rapidly evolving codons in the COIII gene. In a series of phylogenetic analyses attempting to recover a known set of vertebrate relationships, downweighting these labile codons produced the most accurate results. Structural correlates of variable and invariant residues identified in this study can be used to increase the accuracy of models used for phylogenetic inference. Viewing amino acid variation within a phylogenetic framework provided insight into residue changes important in the secondary and tertiary structures of the molecule, changes that were correlated between pairs of neighboring residues or between residues in neighboring helices.      

Tumour suppressor gene mutations in humans and mice: parallels and contrasts.

Tumour suppressor genes prevent cancer development. They can be identified by studying humans, but a full understanding of the mechanisms of their action requires the production of animal models. Mice with mutations in tumour suppressor genes can be produced by gene targeting. The phenotypic consequences of tumour suppressor gene mutations in mice and humans show parallels and contrasts, and both can contribute to the elucidation of disease processes.     

Very Preterm Infants Failing CPAP Show Signs of Fatigue Immediately after Birth

Objective

To investigate the differences in breathing pattern and effort in infants at birth who failed or succeeded on continuous positive airway pressure (CPAP) during the first 48 hours after birth.

Methods

Respiratory function recordings of 32 preterm infants were reviewed of which 15 infants with a gestational age of 28.6 (0.7) weeks failed CPAP and 17 infants with a GA of 30.1 (0.4) weeks did not fail CPAP. Frequency, duration and tidal volumes (VT) of expiratory holds (EHs), peak inspiratory flows, CPAP-level and FiO₂-levels were analysed.

Results

EH incidence increased <6 minutes after birth and remained stable thereafter. EH peak inspiratory flows and VT were similar between CPAP-fail and CPAP-success infants. At 9-12 minutes, CPAP-fail infants more frequently used smaller VTs, 0-9 ml/kg and required higher peak inspiratory flows. However, CPAP-success infants often used large VTs (>9 ml/kg) with higher peak inspiratory flows than CPAP-fail infants (71.8 ± 15.8 vs. 15.5 ± 5.2 ml/kg.s, p <0.05). CPAP-fail infants required higher FiO₂ (0.31 ± 0.03 vs. 0.21 ± 0.01), higher CPAP pressures (6.62 ± 0.3 vs. 5.67 ± 0.26 cmH₂O) and more positive pressure-delivered breaths (45 ± 12 vs. 19 ± 9%) (p <0.05)

Conclusion

At 9-12 minutes after birth, CPAP-fail infants more commonly used lower VTs and required higher peak inspiratory flow rates while receiving greater respiratory support. VT was less variable and larger VT was infrequently used reflecting early signs of fatigue.     

Antiviral Biologic Produced in DNA Vaccine/Goose Platform Protects Hamsters Against Hantavirus Pulmonary Syndrome When Administered Post-exposure

Andes virus (ANDV) and ANDV-like viruses are responsible for most hantavirus pulmonary syndrome (HPS) cases in South America. Recent studies in Chile indicate that passive transfer of convalescent human plasma shows promise as a possible treatment for HPS. Unfortunately, availability of convalescent plasma from survivors of this lethal disease is very limited. We are interested in exploring the concept of using DNA vaccine technology to produce antiviral biologics, including polyclonal neutralizing antibodies for use in humans. Geese produce IgY and an alternatively spliced form, IgYΔFc, that can be purified at high concentrations from egg yolks. IgY lacks the properties of mammalian Fc that make antibodies produced in horses, sheep, and rabbits reactogenic in humans. Geese were vaccinated with an ANDV DNA vaccine encoding the virus envelope glycoproteins. All geese developed high-titer neutralizing antibodies after the second vaccination, and maintained high-levels of neutralizing antibodies as measured by a pseudovirion neutralization assay (PsVNA) for over 1 year. A booster vaccination resulted in extraordinarily high levels of neutralizing antibodies (i.e., PsVNA₈₀ titers >100,000). Analysis of IgY and IgYΔFc by epitope mapping show these antibodies to be highly reactive to specific amino acid sequences of ANDV envelope glycoproteins. We examined the protective efficacy of the goose-derived antibody in the hamster model of lethal HPS. α-ANDV immune sera, or IgY/IgYΔFc purified from eggs, were passively transferred to hamsters subcutaneously starting 5 days after an IM challenge with ANDV (25 LD₅₀). Both immune sera, and egg-derived purified IgY/IgYΔFc, protected 8 of 8 and 7 of 8 hamsters, respectively. In contrast, all hamsters receiving IgY/IgYΔFc purified from normal geese (n=8), or no-treatment (n=8), developed lethal HPS. These findings demonstrate that the DNA vaccine/goose platform can be used to produce a candidate antiviral biological product capable of preventing a lethal disease when administered post-exposure.