Saturation of penicillin-binding protein 1 by β-lactam antibiotics in growing cells of Bacillus licheniformis

With the help of a new highly sensitive method allowing the quantification of free penicillin-binding proteins (PBPs) and of an integrated mathematical model, the progressive saturation of PBP1 by various β-lactam antibiotics in growing cells of Bacillus licheniformis was studied. Although the results confirmed PBP1 as a major lethal target for these compounds, they also underlined several weaknesses in our present understanding of this phenomenon. In growing cells, but not in resting cells, the penicillin target(s) appeared to be somewhat protected from the action of the inactivators. In vitro experiments indicated that amino acids, peptides and depsipeptides mimicking the peptide moiety of the nascent peptidoglycan significantly interfered with the acylation of PBP1 by the antibiotics. In addition, the level of PBP1 saturation at antibiotic concentrations corresponding to the minimum inhibitory concentrations was not constant, suggesting that additional, presently undiscovered, factors might be necessary to account for the experimental observations.     

L-Arabinose is not a gratuitous inducer of α-galactosidase from Saccharomyces carlsbergensis

L-Arabinose has been described as a gratuitous inducer of the yeast -galactosidase. This has been found to be an artefact resulting from galactose contamination of commercial samples of L-arabinose. The inactivation produced on UDP-glucose 4-epimerase by the pentose does not amplify the inducer activity of contaminating D-galactose.     

Effect of mexiletine on glucose and oxygen uptake in rat brain, liver and myocardial tissue in vitro (author's transl)

The effect of mexiletine on oxygen and glucose consumption was studied both in homogenate and slices of brain, liver and myocardium of Wistar rats. Oxygen consumption was detected by means of Warburg's manometric techniques, and glucose utilization by the enzymatic method of glucose oxidase. Whilst glucose uptake was not modified in any of the studied preparations, mexiletine promoted a significant increase of oxygen consumption in the homogenized slices, and an inhibition in the intact tissue.     

Processing of an endogenous protein can generate MHC class II-restricted T cell determinants distinct from those derived from exogenous antigen.

Class II MHC molecules on the surface of an APC present immunogenic peptides derived mainly from exogenous proteins to CD4+ T cells. During its transport to the cell surface, class II molecules intersect the endocytic pathway where they acquire peptides derived from endocytosed proteins. However, class II-restricted presentation of endogenously derived peptides can also occur. The current studies were undertaken to examine the ability of different types of APC to generate and present four different T cell determinants derived from an endogenous, nonsecreted, truncated form of hen-egg white lysozyme (HEL[1-80]-Kk). This was compared with the ability of these APC to generate the same determinants from exogenous HEL. All the peptides derived from endogenous HEL[1-80]-Kk tested, were presented by B cells to HEL-specific T cell hybridomas with an efficiency similar to presentation of the same determinants from exogenous HEL. In contrast, an I-Ak-bearing rat fibroblast was unable to generate the HEL peptide 25-43 from exogenous HEL, but could efficiently produce it from endogenous HEL[1-80]-Kk. The results indicate first, that peptides derived from an endogenous Ag can be presented by MHC class II molecules with an efficiency comparable to that of the presentation of the exogenous Ag. Second, that Ag-presenting B cells can generate the same repertoire of antigenic peptides from endogenous Ag as those generated from the exogenous protein. And third, that in contrast to B cells, certain "nonprofessional" APC can generate, from an endogenous protein, T cell determinants distinct from those generated after endocytosis of the exogenous protein. These results suggest that processing of exogenous and endogenous Ag by different APC take place in different intracellular compartments.     

The ICL1 gene from Saccharomyces cerevisiae.

The glyoxylate cycle is essential for the utilization of C2 compounds by the yeast Saccharomyces cerevisiae. Within this cycle, isocitrate lyase catalyzes one of the key reactions. We obtained mutants lacking detectable isocitrate lyase activity, screening for their inability to grow on ethanol. Genetic and biochemical analysis suggested that they carried a defect in the structural gene, ICL1. The mutants were used for the isolation of this gene and it was located on a 3.1-kb BglII-SphI DNA fragment. We then constructed a deletion-substitution mutant in the haploid yeast genome. It did not have any isocitrate lyase activity and lacked the ability to grow on ethanol as the sole carbon source. Both strands of a DNA fragment carrying the gene and its flanking regions were sequenced. An open reading frame of 1671 bp was detected, encoding a protein of 557 amino acids with a calculated molecular mass of 62515 Da. The deduced amino acid sequence shows extensive similarities to genes encoding isocitrate lyases from various organisms. Two putative cAMP-dependent protein-kinase phosphorylation sites may explain the susceptibility of the enzyme to carbon catabolite inactivation.     

Regulation of heme-controlled eukaryotic polypeptide chain initiation factor 2 alpha-subunit kinase of reticulocyte lysates.

We have obtained highly purified preparations of the heme-controlled eukaryotic initiation factor 2 alpha-subunit (eIF-2 alpha) kinase (HCI) from rabbit reticulocyte lysates containing five different polypeptides. One of these is a 87-kDa (p87) phosphopeptide which appears to show an autokinase activity. The controlled digestion with trypsin of HCI preparations leads to the suggestion that phosphorylation of p87 is not needed for kinase activity and, furthermore, that another 89-kDa polypeptide could be the kinase catalytic subunit. In agreement with this, monoclonal antibodies directed against p87 do not interfere with eIF-2 alpha kinase activity. Moreover, the anti-p87 antibodies and those directed against the mammalian 90-kDa heat shock protein recognize the same p87 polypeptide from rabbit reticulocyte lysates. Upon incubation of the HCI preparation with hemin (5-10 microM), the eIF-2 alpha kinase is converted into an inactive form and appears to become associated with related peptides forming high molecular weight complexes which can be reversibly activated by 2-mercaptoethanol. The maintenance of the integrity of the porphyrin ring is absolutely required for kinase inactivation and although the presence of metal ion is not essential, the iron and cobalt metalloporphyrins are more effective than protoporphyrin IX. The formation of the inactive form of HCI by hemin is prevented by either N-ethylmaleimide, monoclonal antibodies directed against p87, or phosphorylation of p87. The data strongly suggest that hemin regulates eIF-2 alpha kinase activity by promoting formation of the inactive dimer HCI.p87 via disulfide bonds and direct binding of hemin. A model of HCI regulation is discussed.     

Mating behavior and male mating success in wildAnastrepha Ludens (Diptera: Tephritidae) on a field-caged host tree

Mating behavior and factors affecting mating success of males were studied using wild Anastrepha ludens on a fieldcaged host tree. The most common courtship sequence had five components: (1) male calls from the underside of a leaf, (2) female arrives to the maleoccupied leaf, (3) male orients to female and stops calling, (4) one or both approach to a face-to-face position 1–3 cm apart, and (5) male mounts female after 1–2 s. Courtship behavior was almost identical to that of laboratoryculture flies observed previously under laboratory conditions. Most malefemale encounters occurred at a height of 1–2m, well inside the outer canopy of the tree. Differential mating success by males occurred. No male mated more than once per day, owing possibly to a very short sexual activity period. Factors favoring mating success of males were survival ability and tendency to join male aggregations and to fight other males. Thorax length and age (9–11 days difference) had no effects on male copulatory success. Overall win/loss percentage was not related to mating success because the males that were most successful at mating fought mostly among themselves, driving their win/loss percentage down. However, these successful males (at mating) won most of their fights against less successful males. Results confirmed a lek mating system: males aggregated, called, and defended territories; territories did not contain femalerequired resources; and females exercised mate choice, apparently through selection of sites within leks.     

Differentiation between thermophilic Campylobacter species by species-specific antibodies

P.L. GRIFFITHS. G.S. MORENO AND R.W. A. PARK. 1992. The four species of thermophilic camplyobacters, Campylobacter jejuni, C. coli, C. upsaliensis and C. lari , are difficult to distinguish from each other because of their lack of reactivity in many conventional biochemical and physiological tests. Those tests which do discriminate sometimes give discordant results. Species-specific antibody preparations (APs), capable of discriminating between the thermophilic campylobacter species by dot-ELISA, were raised by inoculation of mice with partially purified membrane protein. The APs produced were absorbed with cells of cross-reactive species and tested by dot-ELISA against reference and natural strains, the identities of which were confirmed by DNA/DNA hybridization. The results showed that such APs could be useful as an alternative to DNA/DNA hybridization for rapid species identification, for example in epidemiological surveys. Western blotting experiments with the APs showed that the specificity of the antibodies was not due to a single antigen.