Catalysis of guanine nucleotide exchange on eIF2 by eIF2B: can it be both a substituted enzyme and a sequential mechanism?

There are conflicting reports over the question of whether the displacement by GTP of GDP bound to eIF2 catalyzed by eIF2B follows a substituted enzyme mechanism, as is believed to be the case for other guanine nucleotide exchange factors, or is a sequential mechanism. Analysis of data recently provided by Williams et al. (J. Biol. Chem. 276, 24697-24703, 2001) showing displacement by eIF2B of GDP bound to eIF2 in the absence of displacing nucleotide appears to offer a way of resolving the dispute and suggests that both mechanisms may be operative.

Antoine Béchamp: pere de la biologie. Oui ou non?

There is an alternative medicine lobby that, in conjunction with antivivisectionists, believes Louis Pasteur to have been a fraud [R. Bottomley's You Don't Have to Feel Unwell! (Newleaf, 1994) is a recent example]. They frame their accusations around a rivalry between Pasteur and a contemporary, Antoine Béchamp, from whom they suggest Pasteur stole his ideas and then distorted them for his own purposes. This article explores some aspects of the controversies between Béchamp and Pasteur.

Biochemistry comes of age: a century of endeavour.

Biochemistry began as an identifiable, separate discipline at the beginning of the twentieth century. This article traces its early development, focusing on three pioneers--Eduard Buchner, Arthur Croft Hill and Arthur Harden.

The use of poly(2-acrylamido-2-methyl-1-propanesulfonic acid) polymers as spacers for isotachophoresis in sieving gel matrices.

The electric field strength gradients generated in isotachophoresis (ITP) may be used for the separation of biomolecules. Poly(2-acrylamido-2-methyl-1-propanesulfonic acid) (polyAMPS) polymers of a uniform distribution of molecular mass were synthesized and used as novel spacers in ITP. Since these polymeric spacers are strongly acidic species, their ionic charges remain constant over a wide pH range, so that their ionic mobilities are governed solely by their molecular masses and not by the pH of the milieu. A modification of ITP known as telescope electrophoresis was used to separate a number of acidic dyes of varying ionic mobility, using polyAMPS polymers as spacers. The resolution obtained was superior to that obtained by polyacrylamide gel electrophoresis (PAGE), due to the focusing effect of the electric field strength gradient. Since these novel polymeric spacers are designed to operate within sieving medium, it was decided to test their suitability for the separation of DNA molecules. DNA molecules up to 1000 bp long were successfully resolved, with a similar resolution to that obtained with conventional PAGE.

Synthesis of zwitterionic acrylic acid buffers for isoelectric focusing in immobilized pH gradients.

A range of zwitterionic acrylic acid derivatives, buffering in the neutral and basic pH ranges, have been synthesized by the Mannich reaction of malonic acid, formaldehyde and a secondary amine. These compounds include 2-(4-morpholinomethyl)propenoic acid pK2 7.59 +/- 0.03 (23 degrees C), 2-[bis(2-hydroxyethyl)aminomethyl]propenoic acid pK2 approximately 8.6 (20 degrees C), 2-[bis(2-hydroxypropyl) aminomethyl]propenoic acid PK2 approximately 8.7 (20 degrees C), 2-[N-(2-hydroxyethyl)-N-methylaminomethyl]propenoic acid pK2 9.22+/-0.08 (22 degrees C), 2-[N-ethyl-N-(2hydroxyethyl)aminomethyl]-propenoic acid pK2 approximately 9.6 (20 degrees C), and 2-[4-(2-carboxyprop-2-enyl)piperazinylmethyl]propenoic acid, which has a sigmoidal buffering profile over the pH range 3-10. These zwitterionic acrylic acid buffers were successfully copolymerized with acrylamide to prepare immobilized pH gradients (IPGs) in the neutral to alkaline portion of the pH range. Bovine erythrocyte carbonic anhydrase isozymes were resolved on a pH 5-8 IPG prepared using 2-[4-(2-carboxyprop-2-enyl)piperazinylmethyl]propenoic acid as the immobilized buffer, and horse heart myoglobin was focused on pH 7.1-8.1 and pH 7.5-7.7 IPGs, using 2-(4-morpholinomethyl)propenoic acid as the immobilized buffer. In both cases the pK 9.3 Immobiline compound was used as the strongly basic titrant. These new compounds, besides possessing more hydrophilic residues than the corresponding commercial basic acrylamido buffers (Immobilines), resist hydrolysis at alkaline pH values.

An alpha subunit-deficient form of eukaryotic protein synthesis initiation factor eIF-2 from rabbit reticulocyte lysate and its activity in ternary complex formation.

Eukaryotic protein synthesis initiation factor eIF-2 is usually isolated as a heterotrimer (alphabeta gamma). By use of Sephacryl S-300 fractionation an alpha subunit-deficient form of eIF-2 was identified in impure preparations from rabbit reticulocyte lysate and it appeared in these preparations to be still active in formation of the ternary complex (eIF-2.GTP.Met-tRNAi). Subsequently alpha subunit-deficient eIF-2 was further purified and this appeared to have retained ternary complex forming activity. Together with a suggested lack of involvement of the beta subunit this implies that the alpha subunit was not required for activity and the gamma subunit bound both GTP and Met-tRNAi in formation of the ternary complex. The identification and study of alpha subunit-deficient eIF-2 thus elucidated the involvement of the subunits in binding of GTP and Met-tRNAi to produce the ternary complex in polypeptide chain initiation.

Catalysis of guanine nucleotide exchange on eIF-2 by eIF-2B: is it a sequential or substituted enzyme mechanism?

The mechanism of action of the eukaryotic initiation factor eIF-2B in catalyzing the exchange of guanine nucleotides bound to eIF-2 is uncertain--evidence having been adduced for a sequential mechanism and for a substituted enzyme mechanism. Data purporting to support a substituted enzyme mechanism have been analysed and shown to be ambiguous and equally consistent with a sequential mechanism. Suitable rate constants for a sequential mechanism involving the transient formation of the quaternary complex eIF-2.eIF-2B.GDP.GTP are suggested.

Binding constants in the formation of mammalian protein synthesis initiation complexes and the role of mRNA.

The findings of Parkhurst et al. (Biochemistry 33, 15168-15177:1994) that a 10-mer oligoribonucleotide containing the AUG triplet enhances the binding of the eIF-2 x Met-tRNAi complex to the 40S ribosomal subunit are questioned on the basis of a re-evaluation of their calculations. It is not possible to conclude, as they did, that addition of the AUG-containing oligonucleotide produces an exceptionally large increase (as judged by the magnitude of the coupling free energy) in the binding of the eIF-2 x Met-tRNAi complex to the 40S subunit, or that their results are more consistent with internal initiation than with the scanning initiation model.

The quest by three giants of science for an understanding of cancer.

Understanding the causes of cancer remains a major area of uncertainty, and many serious proposals have been made from a variety of standpoints over the years. This article discusses the first genetic hypothesis from the turn of the century and two other totally different early approaches to the problem.

Exploring the gene with X-rays.

X-rays were discovered 100 years ago. Their effects on living cells and on mutation were quickly recognized. This article discusses a classic paper that sought to use X-rays to estimate the size of the gene and question mechanics to explain its impressive stability.

A comparative analysis of neuroblastic and substrate-adherent human neuroblastoma cell lines.

In human neuroblastoma cell cultures, two phenotypes with differing biological properties have been described: neuroblastic (N) and substrate-adherent (Schwannian or S). In nude mice, N cells are rapidly growing, clonigenic in soft agar, and tumorigenic, whereas S cells are not. The difference in malignant properties between these two cell types and their ability to interconvert in vitro may have clinical relevance. In an attempt to identify genes that may be important in the phenotypic differences and the interconversion, the authors analyzed five representative N and three S cell lines for differential expression of six genes relevant to uncontrolled growth. Beta2-microglobulin was the only gene measured that showed differential expression between the N and S cell lines. This finding supports the theory that beta2-microglobulin and class I MHC expression are markers for differentiation as well as the use of beta2-microglobulin as a differentiation marker in neuroblastoma. The data also suggest that a disregulation of the beta2-microglobulin gene may be partly responsible for the tumorigenicity of the N phenotype.

Louis Pasteur (1822-1895)--chance and the prepared mind.

Louis Pasteur is often regarded as one of the founders of microbiology, and is chiefly famous for his discovery of the role that microorganisms play in health and disease. However, he was trained as a physical scientist, and his research began with the discovery of the stereoisomerism of the different forms of tartaric acid. As a biotechnologist, he is remembered for his studies on the nature of fermentation, and for his rebuttal of the theory of spontaneous generation.

Maxillary sinusitis in Medieval Chichester, England.

Maxillary sinusitis is a common medical complaint, affecting more than 30 million people per year in the United States alone. Very little palaeopathological work on this disease has been carried out, probably because of the enclosed nature of the sinuses in intact skulls and the lack of a suitable method for examination. This study tested the hypothesis that maxillary sinusitis was more common in people with leprosy than in people without it in Medieval England. The prevalence of maxillary sinusitis by age and sex was recorded in 133 individuals, some diagnosed as being leprous, derived from a later Medieval (12th to 17th centuries AD) urban hospital population at Chichester, Sussex, England using both macroscopic and endoscopic methods of examination. Of the 133 individuals with one or both sinuses available for examination, 54.9% (73) had evidence of bone change within the sinuses. There was no difference in prevalence between those with leprosy and those without, although clinical studies suggest that over 50% of lepromatous leprous individuals may develop sinusitis. Comparison with another study on Medieval British sites with a 3.6% prevalence (3 of 83) indicates that the prevalence at Chichester is much greater. The problems with diagnosing sinusitis are addressed and reasons behind the high frequency in this study are discussed. Aetiological factors predisposing to maxillary sinusitis are considered with reference to possible environmental conditions prevailing in the later Medieval period in Britain.