Uncoupler-induced relocation of elongation factor Tu to the outer membrane in an uncoupler-resistant mutant of Escherichia coli

Escherichia coli UV6, a mutant which is resistant to the uncoupler carbonyl cyanide m-chlorophenylhydrazone (CCCP), when grown in the presence of CCCP, but not in its absence, incorporated a new protein (Mr, 42 000) into the cell envelope. This protein was found in both cytoplasmic and outer-membrane fractions. In the outer membrane it was one of three or four most abundant proteins. The protein was tightly bound to the membranes and was not solubilized by several detergents. Solubilization was achieved with sodium lauroylsarcosinate (sarkosyl). The protein was purified close to homogeneity by affinity chromatography on a column of GDP-Sepharose. It was identified as elongation factor Tu (EF-Tu) on the basis of electrophoretic mobility, profiles of peptide fragments produced by proteolysis, and by its ability to bind to GDP-Sepharose. Disruption of cells in the presence of CCCP or incubation of envelopes with EF-Tu did not result in incorporation of EF-Tu into the membranes. It is suggested that this protein is incorporated into the outer membrane as a consequence of an alteration in the normal protein biosynthetic mechanisms of the mutant induced by the presence of CCCP.     

Diversity of free-living ‘naked’ amoeboid organisms

Amoeboid organisms are phylogenetically diverse, some being more closely related to plants or metazoans than to each other. Amoeboid organisms are ecologically successful, having been isolated on all continents, including Antarctica, as well as being the main predators controlling bacterial populations in soil. The classification of these organisms has historically relied upon morphological characteristics. The application of electron microscopy, comparison of enzymic profiles after electrophoretic separation, and analysis of nucleic acid fractions have provided reliable bases for classifying amoeboid organisms. The extent of diversity of these organisms has been recognized, as methods to detect, culture, characterize and identify them has increased. It is reasonable to anticipate that the current 40 000 species of protists will increase substantially as amoeboid organisms are cultivated from poorly accessible niches and from extreme environs.     

Potent selective inhibitors of protein kinase C

A series of potent, selective inhibitors of protein kinase C has been derived from the structural lead provided by the microbial broth products, staurosporine and K252a. Our inhibitors block PCK in intact cells (platelets and T cells), and prevent the proliferation of mononuclear cells in response to interleukin 2 (IL2).     

Fast-atom-bombardment mass spectra of enkephalins.

The positive- and negative-ion mass spectra of [methionine]enkephalin and [leucine]enkephalin have been obtained by using a fast-atom-bombardment source described previously by Barber, Bordoli, Sedgwick & Tyler [(1981) J. Chem. Soc. Chem. Commun., in the press]. This technique has allowed the spectra to be obtained without conversion of the enkephalins into volatile derivatives. The fast-atom-bombardment spectra show good pseudo-molecular-ion sensitivity and fragmentation that can be interpreted on the basis of the known molecular structure.     

Induction of IgE-secreting cells and IgE isotype-specific suppressor T cells in the respiratory lymph nodes of rats in response to antigen inhalation

Repeated exposure of high-IgE-responder Brown Norway (BN) rats to an aerosol of ovalbumin (OVA) once weekly triggered progressively increasing levels of OVA-specific IgG in serum. In contrast, responses in the IgE class were transient, declining from peak titers during the third week to background levels by Week 5, despite continuing aerosol exposure. Subsequent parenteral challenge of these animals revealed a state of antigen- and IgE isotype-specific tolerance. Adoptive transfer of splenocytes or pooled respiratory tract lymph node (RTLN) cells from aerosol-exposed animals to naive rats abrogated subsequent OVA-specific primary IgE responses in the recipients, but did not affect specific IgG responses, and kinetic studies indicated that these suppressor cells arose first in the RTLN. Transfer studies employing individual lymph node groups which constituted the RTLN pool pinpointed the superficial cervical nodes, which drain the uppermost portion of the respiratory tract, as the major source of suppressor cells. Fractionation of cell populations before adoptive transfer employing monoclonal antibodies directed against T-cell markers, defined a population of suppressor cells generated by aerosol exposure which expressed both the W3/13 (pan T-cell) and OX8 (cytotoxic/suppressor T-cell) antigens, but which was negative for the W3/25 (helper T-cell) marker. Analysis of the IgE and IgG responses induced by OVA inhalation was performed employing the ELISA plaque technique, recently developed in this laboratory. These studies revealed the parathymic and posterior mediastinal nodes draining the lower lung, as the major sites of specific IgE and IgG production; smaller numbers of OVA-specific IgG-secreting cells (but none secreting specific IgE) were detected in the nodes draining the upper respiratory tract, while antibody secretion outside the respiratory tract was restricted to comparatively few cells in the spleen. The ELISA plaque assay was also employed to enumerate total numbers of cells secreting the IgE isotype in aerosol-exposed and control rats, employing samples from 10 different lymphoid organs. Approximately 50% of the IgE-secreting cells in these animals were localized in RTLN, as opposed to 25% in gut-associated lymphoid tissues. These data are discussed in relation to the pivotal role of respiratory-tract associated lymphoid tissues in regulation of IgE responses to aeroallergens.