The gene for ricin toxin A chain was modified by site-specific mutagenesis to change arginine 180 to alanine, glutamine, methionine, lysine, or histidine. Separately, glutamic acid 177 was changed to alanine and glutamic acid 208 was changed to aspartic acid. Both the wild-type and mutant proteins were expressed in Escherichia coli and, when soluble, purified and tested quantitatively for enzyme activity. A positive charge at position 180 was found necessary for solubility of the protein and for enzyme activity. Similarly, a negative charge with a proper geometry in the vicinity of position 177 was critical for ricin toxin A chain catalysis. When glutamic acid 177 was converted to alanine, nearby glutamic acid 208 could largely substitute for it. This observation provided valuable structural information concerning the nature of second-site mutations.

Oncostatin M (OM) is a cytokine that shares a structural and functional relationship with interleukin-6, leukemia inhibitory factor, and granulocyte-colony stimulating factor, which regulate the proliferation and differentiation of a variety of cell types. A mutant version of human OM in which two N-linked glycosylation sites and an unpaired cysteine have been mutated to alanine (N76A/C81A/N193A) has been expressed and shown to be active. The triple mutant has been doubly isotope-labeled with 13C and 15N in order to utilize heteronuclear multidimensional NMR techniques for structure determination. Approximately 90% of the backbone resonances were assigned from a combination of triple-resonance data (HNCA, HNCO, CBCACONH, HBHACONH, HNHA and HCACO), intraresidue and sequential NOEs (3D 15N-NOESY-HMQC and 13C-HSQC-NOESY) and side-chain information obtained from the CCONH and HCCONH experiments. Preliminary analysis of the NOE pattern in the 15N-NOESY-HMQC spectrum and the 13C alpha secondary chemical shifts predicts a secondary structure for OM consisting of four alpha-helices with three intervening helical regions, consistent with the four-helix-bundle motif found for this cytokine family. As a 203-residue protein with a molecular weight of 24 kDa, Oncostatin M is the largest alpha-helical protein yet assigned.

Purified nuclei retaining a high degree of ultrastructural integrity were isolated by conventional centrifugation techniques. The cytoplasmic surface of these nuclei was iodinated using lactoperoxidase immobilized onto giant Sepharose beads; thus the outer nuclear membrane and the cytoplasmic surface of nuclear pore complexes were selectively labelled. Pore complexes in association with a fibrous lamina were isolated from these nuclei by removal of the nucleoplasm and extraction with Triton X-100. The chemical composition of the pore-lamina fraction was 93.6% protein, 6% RNA, 0.4% phospholipid. The labelling suggests that major polypeptides N1 (70 000) and N2 (67 000) and more than 10 other more minor polypeptides, ranging from 33 000 to 200 000 mol. wt, as being components of the nuclear pore complex. Polypeptide N3 (58 000) is shown to be present only on the nucleoplasmic face of nuclear envelopes, probably in the fibrous lamina.

The gene for ricin toxin A chain was modified by site-specific mutagenesis to change arginine 180 to alanine, glutamine, methionine, lysine, or histidine. Separately, glutamic acid 177 was changed to alanine and glutamic acid 208 was changed to aspartic acid. Both the wild-type and mutant proteins were expressed in Escherichia coli and, when soluble, purified and tested quantitatively for enzyme activity. A positive charge at position 180 was found necessary for solubility of the protein and for enzyme activity. Similarly, a negative charge with a proper geometry in the vicinity of position 177 was critical for ricin toxin A chain catalysis. When glutamic acid 177 was converted to alanine, nearby glutamic acid 208 could largely substitute for it. This observation provided valuable structural information concerning the nature of second-site mutations.

Harvey Brooks and I well know, involved more unfounded models). The real critiques that I know of come from outside IIASA, for example, from an independent group in Britain studying "Models of Doom" (1).One essential difficulty with IIASA is that it does not appear to have an adequate critical mechanism, by discipline or by report review. IIASA suffers from its heritage of systems analysis, a field with no disciplinary tradition, from the burden of helping intemational cooperation (note the enthusiasm for cybernetics in the U.S.S.R.), and from its location in an discarded imperial palace on the outskirts of a city whose intellectual distinction lies well in the past.