Phosphatidylinositol inhibits microtubule assembly by binding to microtubule-associated protein 2 at a single, specific, high-affinity site.

The effects of various anionic phospholipids on the in vitro assembly of MAP2/tubulin microtubules has been examined. We show that the potency to inhibit is related to the polarity of the phospholipids and that this is consistent with a mode of action involving the sequencing of microtubule-associated proteins (MAPs) by nonspecific electrostatic interactions. The inhibitory potency of phosphatidylinositol (PI) is, however, considerably larger than predicted by this model. The effects of PI on MAP2/tubulin microtubule assembly have therefore been examined in greater detail by preparing phosphatidylcholine (PC) liposomes doped with increasing amounts of PI. We show that when the PI is sufficiently dispersed by dilution with PC, it inhibits microtubule assembly by binding to MAP2 with an apparent stoichiometry, after correction for the bilamellar nature of the liposomes, of 1:1 mol.mol-1 PI:MAP2. Furthermore, we show that the Kd of this interaction is in the submicromolar range.     

Mouse monoclonal antibodies can nonspecifically inhibit the proliferation of human T-cell growth factor-dependent T cells

A large series of mouse monoclonal antibodies was found to inhibit the proliferation of T-cell growth factor (TCGF)-dependent human T-cell blasts as measured by the incorporation of tritiated thymidine. The specificity of the antibody appeared to be irrelevant for inhibition and two T-cell-specific antibodies did not prevent the absorption of TCGF by treated T cells. It is suggested that the antibodies function by the indirect release of suppressor factors by Fc receptor-bearing TCGF-dependent cells.     

Influence of neighbouring base sequence on N-methyl-N'-nitro-N-nitrosoguanidine mutagenesis in the lacI gene of Escherichia coli

N-methyl-N'-nitro-N-nitrosoguanidine (MNNG)-induced forward mutations within the first 540 base-pairs of the lacI gene of Escherichia coli were cloned and sequenced. In total, 167 MNNG-induced independent mutations were characterized, with G.C to A.T transitions accounting for all but three of the mutations. This mutagenic specificity is consistent with the mispairing predicted by the methylation of the O6 position of guanine. The characterization of such large numbers of mutations permitted an analysis of the influence of local DNA sequence on mutagenesis. This analysis revealed a strong influence by the 5' flanking base. On average, guanine residues preceded (5') by a guanine or an adenine residue were, respectively, nine times and five times more likely to mutate after treatment with MNNG than those preceded by a pyrimidine residue.     

Stoichiometry of estramustine phosphate binding to MAP2 measured by the disassembly of chick brain MAP2:tubulin microtubules

The concentration of estramustine phosphate required to inhibit the assembly or to induce the disassembly of chick brain MAP2:tubulin microtubules is markedly dependent upon the microtubule protein concentration. Analysis of this relationship shows that estramustine phosphate and tubulin compete for common MAP2 sites, that MAP2 can bind 5-6 moles.mole-1 estramustine phosphate, and that the Kd of these sites is congruent to 20 microM estramustine phosphate. It is proposed that two molecules of estramustine phosphate interact with each of the three tubulin-binding sites of MAP2 and inhibit the MAP2:tubulin interaction by neutralising two highly conserved basic residues.     

A survey of hospital toilet facilities.

OBJECTIVE--To assess the quality of toilet facilities available for disabled people in a large provincial teaching hospital. DESIGN--Survey of toilet facilities for patients on the wards and in the outpatient department. SETTING--Teaching hospital in Leeds. RESULTS--Although the quality of toilet facilities varied, none met the standards recommended by the British Standards Institution. The worst facilities were found on a ward accommodating elderly patients, where the toilets were unsuitable for use by disabled people and bedside commodes had to be used instead. CONCLUSION--Toilet provision within a major hospital failed to meet standards required for disabled people. Admission to hospital may therefore result in loss of independence and dignity. If hospitals are to be centres of excellence, greater consideration must be given to the requirements of disabled people in the design of new wards, and current inadequate facilities should be upgraded.     

Comparison of the amino acid sequences of the transacylase components of branched chain oxoacid dehydrogenase of Pseudomonas putida, and the pyruvate and 2-oxoglutarate dehydrogenases of Escherichia coli

The nucleotide sequence of bkdB, the structural gene for E2b, the transacylase component of branched-chain-oxoacid dehydrogenase of Pseudomonas putida has been determined and translated into its amino acid sequence. The start of bkdB was identified from the N-terminal sequence of E2b isolated from branched-chain-oxoacid dehydrogenase of the closely related species, P. aeruginosa. The reading frame was composed of 65.5% G + C with 82.3% of the codons ending in G or C. There was no intergenic space between bkdA2 and bkdB. No codons requiring minor tRNAs were utilized and the codon bias index indicated a preferential codon usage. The bkdB gene encoded 423 amino acids although the N-terminal methionine was absent from E2b prepared from P. aeruginosa. The relative molecular mass of the encoded protein was 45,134 (45,003 minus methionine) vs 47,000 obtained by SDS/polyacrylamide gel electrophoresis. There was a single lipoyl domain in E2b compared to three lipoyl domains in E2p, and one domain in E2o, the transacylases of pyruvate and 2-oxoglutarate dehydrogenases of Escherichia coli respectively. There was significant similarity between the lipoyl domain of E2b and of E2p and E2o as well as between the E1-E2 binding domains of E2b, E2p and E2o. There was no similarity between the E3 binding domain of E2b to E2p and E2o which may reflect the uniqueness of the E3 component of branched-chain-oxoacid dehydrogenase of P. putida. The conclusions drawn from these comparisons are that the transacylases of prokaryotic pyruvate, 2-oxoglutarate and branched-chain-oxoacid dehydrogenases descended from a common ancestral protein probably at about the same time.