Genetic probes of ribosomal RNA function

We have used a genetic approach to uncover the functional roles of rRNA in protein synthesis. Mutations were constructed in a cloned rrn operon by site-directed mutagenesis or isolated by genetic selections following random mutagenesis. We have identified mutations that affect each step in the process of translation. The data are consistent with the results of biochemical and phylogenetic analyses but, in addition, have provided novel information on regions of rRNA not previously investigated.     

Reduction cranioplasty

Reduction cranioplasty can greatly improve the quality of life for selected patients with severe macrocephaly and can significantly diminish some of the difficulties in the long-term chronic care of others. Because of differences in age, cranial morphology and surgical goals, the surgical plan must be tailored to the individual patient. Three techniques for reduction cranioplasty, with the advantages and disadvantages of each, are described. Four patients, representing the spectrum of severe macrocephaly and also the problems associated with reduction cranioplasty, are presented. Surgical indications, tactical considerations and risks are discussed.     

Economic control of fertilizer in highly productive pastoral systems. III. A hypothetical model to quantify upgrading from a primitive into a highly productive state

A model suitable for quantifying the upgrading of primitive pasture by phosphatic fertilizer is described; the model, based on the Mitscherlich-Spillman equation, provides a criterion for judging when upgrading is complete. Such information is essential to permit a successful experimental attack on the problem of providing fertilizer-advice for highly productive systems where the nutritional requirements both of pasture plants and grazing animals have to be satisfied. The advice must therefore have a sound basis in physiology as well as in economics; but, the generally accepted modern approach cannot be integrated into a logically consistent and practically possible solution to this complex problem. Consequently, the present proposals involve a number of innovations to replace inadequate concepts in the current approach; the most important of these are as follows.

1. Replacement of the economic theory of isoquantal substitution by production function analysis of long term average response by pasture to a complete (physiologically balanced) mixture of fertilizers.
2. Use of a hypothetical model justified by biological theory rather than an empirical model justified by a least squares-best fit criterion.
3. Replacement of multilevel factorial field experiments by simple 3-level trials representatively dispersed over the whole of the commercially productive area and carried on for a decade.
4. Exclusion of grazing animals from the main phase of a simple trial, but with their heterotrophic cycling of nutrients effectively simulated by a modified experimental technique.
5. Systematic use of foliar chemical analysis, which in one way or another is essential to supplement each of the first 4 innovations.

     

Epitope-labeled soluble human interleukin-5 (IL-5) receptors. Affinity cross-link labeling, IL-5 binding, and biological activity

The human receptor for the potent eosinophilopoietic cytokine interleukin-5 (IL-5) consists of two components: a 60-kDa ligand-binding alpha chain (IL-5 alpha R) and a 130-kDa beta chain (IL-5 beta R). Three ectodomain constructs of the alpha chain (alpha RED) bearing C-terminal epitope tags were engineered and expressed in baculovirus-infected Sf9 cells. Each recombinant alpha chain was secreted into the medium, maximum expression occurring 72 h post-infection. The various soluble alpha chains were shown by affinity cross-link labeling and competition with unlabeled IL-5 to bind recombinant human (rh) 125I-IL-5 specifically with an ED50 of 2-5 nM. The epitope tag provided a simple purification of the receptor from conditioned medium using immunoaffinity chromatography. The purified material had an apparent molecular mass of 43 kDa and was heterogeneously glycosylated. Sedimentation analysis revealed a 1:1 association of the purified epitope-tagged soluble receptor with its ligand, resulting in the formation of a 70-74-kDa complex. Circular dichroism analysis revealed that the soluble alpha chain existed with a significantly ordered structure consisting of 42% beta-sheet and 6% alpha-helix. Such analyses combined with fluorescence spectrometry suggested that ligand-receptor complex formation in solution resulted in minimal conformational changes, consistent with the suggestion that the membrane-associated form of the alpha chain itself has minimal signal transduction capability. Surface plasmon resonance studies of the interaction of the purified alpha RED with immobilized rhIL-5 revealed a specific, competable interaction with a dissociation constant of 9 nM. Preincubation of an IL-5-dependent cell line with the epitope-tagged alpha RED also dose-dependently neutralized rhIL-5-induced proliferation. These data demonstrate that biologically active epitope-tagged recombinant soluble IL-5 receptors are facile to produce in large quantities and may have therapeutic utility in the modulation of IL-5-dependent eosinophilia in man.