Bovine coronaviruses associated with enteric and respiratory diseases in Canadian dairy cattle display different reactivities to anti-HE monoclonal antibodies and distinct amino acid changes in their HE, S and ns4.9 protein.

Bovine coronavirus isolates associated with recent outbreaks of respiratory disease in Ontario and Quebec dairy farms were compared to reference strains known to be responsible for neonatal calf diarrhea (NCD) or winter dysentery (WD) of adult cattle. In respect to their hemagglutinating properties and their higher RDE activities with rat erythrocytes, WDBCoV strains differed from NCDBCoV strains and respiratory bovine coronaviruses RBCoV strains. Serologically, three MAbs directed to the HE glycoprotein of the WDBCoV strain BCQ.2590 recognized two serogroups amongst NCDBCoV strains by hemagglutination inhibition, whereas only one of the MAbs failed to react toward three of the four RBCoV isolates tested. Sequencing analysis of the S (S1 portion), HE, ORF4 and ORF5 genes of BCoV isolates associated with different clinical syndromes indicated that neither insertions or deletions could explain their distinct tropism. For the HE glycoprotein, a total of 15 amino acids (aa) substitutions were identified by comparing field isolates to the prototype Mebus strain. Two specific proline substitutions were identified for virulent strains being located in the signal peptides (aa 5) and aa position 367; one specific aa change was revealed at position 66 for RBCoV field isolates. Analysis of the S1 portion of the S glycoprotein revealed a total of eight aa changes specific to enteropathogenic (EBCoV) strains and eight aa changes specific to RBCoV strains. For all BCoV isolates studied, the region located between the S and M genes (ORF4) apparently encodes for two non-structural (ns) proteins of 4.9 and 4.8 kDa. A specific non-sense mutation was identified for the nucleotide at position 88 of the putative 4.9 kDa protein gene of RBCoV isolates resulting in 29 rather that 43 aa residues. The ORF5, which encodes a 12.7 ns protein and the 9.5 kDa E protein, was highly conserved amongst the BCoV field isolates.

In vitro interaction of the carboxy-terminal domain of lamin A with actin.

The nuclear lamina formed by lamins is localized between the inner nuclear membrane and chromatin. Lamins are thought to be implicated in the higher order chromatin structure. Interactions of lamins with chromatin have been described but the molecular components directly involved in these interactions remain to be identified. Using a GST-C-terminal domain of lamin A fusion protein to probe cellular extracts for interacting proteins, we have found that this domain of lamin A binds to nuclear actin. This result suggests that an actin-based molecular motor linked to the lamina could be involved in the movement of chromatin domains.

Lamin proteins form an internal nucleoskeleton as well as a peripheral lamina in human cells.

The nuclear lamina forms a protein mesh that underlies the nuclear membrane. In most mammalian cells it contains the intermediate filament proteins, lamins A, B and C. As their name indicates, lamins are generally thought to be confined to the nuclear periphery. We now show that they also form part of a diffuse skeleton that ramifies throughout the interior of the nucleus. Unlike their peripheral counterparts, these internal lamins are buried in dense chromatin and so are inaccessible to antibodies, but accessibility can be increased by removing chromatin. Knobs and nodes on an internal skeleton can then be immunolabelled using fluorescein- or gold-conjugated anti-lamin A antibodies. These results suggest that the lamins are misnamed as they are also found internally.

Lamin A precursor is localized to intranuclear foci.

Lamin A is synthesized in the cytoplasm as a precursor bearing a carboxyl-terminal CaaX box or isoprenylation signal. This precursor is post-translationally processed through multiple steps: isoprenylation with a farnesyl residue on the cysteine of the CaaX box, proteolytic removal of the last three amino acids, carboxymethylation of the cysteine residue and, finally, proteolytic removal of 15 amino acids from the carboxyl terminus. This last step gives rise to mature lamin A from which the isoprenylated terminus has been removed. Isoprenylation is a prerequisite for all other steps of processing. The subcellular location of these processing steps for lamin A is still a matter of debate. We have produced an antibody specific to the 18 amino acid carboxyl terminus of the lamin A precursor that does not recognize mature lamin A. This antibody detects intranuclear foci by immunofluorescence. Larger amounts of lamin A precursor were accumulated by treating cells with mevinolin (MVN), an inhibitor of isoprenoid synthesis. In MVN-treated cells, the lamin A precursor accumulated most strikingly in the peripheral nuclear lamina where it was assembled, while intranuclear foci were maintained. The addition of an excess of mevalonate (MVA), which restores isoprenylation activity, to MVN-treated cells led to a progressive disappearance of the lamin A precursor from the peripheral lamina. This process was completed after 4 hours of MVA treatment, after which the lamin A precursor was restricted to intranuclear foci. We conclude from these results that the non-isoprenylated lamin A precursor appears competent for assembly into the peripheral nuclear lamina, and that all the processing steps leading to mature lamin A can occur within the nuclear space.

Modeling the cognitive content of displays.

An approach for measuring the cognitive complexity of visual displays is discussed and applied to a dynamic display of avionic information. A semantic network formalism is used to model two interrelated knowledge systems, world knowledge and display knowledge. The information the operator receives during training about the general display format characteristics and the task requirements, along with other previously stored information, constitutes world knowledge. The semantic content of a particular configuration of information encountered during task performance constitutes display knowledge. Four orthogonal predictor measures of cognitive complexity were derived from the networks. In an experiment three of the orthogonal predictors were significantly correlated with task performance. After averaging across operators, the three significant predictors accounted for 99% of the variation of display effectiveness. Results indicate that a model of cognitive complexity based on a semantic network formalism may provide a useful technique for quantitatively evaluating the quality of competing display format concepts.