Epitope mapping by cysteine mutagenesis: Identification of residues involved in recognition by three monoclonal antibodies directed against LamB glycoporin in the outer membrane of Escherichia coli

Abstract Site-directed mutagenesis of the lamB gene was used to introduce individual cysteine substitutions at 20 sites in two regions (surface loops L7 and L8) of LamB protein significant in antibody recognition. Characterisation of cysteine mutants involved immunoblotting with three surface-specific monoclonal antibodies (mAb72, mAb302, mAb347) before and after incubation with thiol-specific reagents. In contrast to an earlier study that showed no amino acid changes affecting recognition by all three antibodies, changes at six amino acids were found to influence a common core epitope. These core sites included one residue (T336) in the predicted loop L7 containing amino acids 329–342 and four (Y379, N387, N389, K392, F398) in the large surface loop involving residues 370–412. Individual antibodies made additional but distinct contacts within the two studied regions, with mAb347 binding the most different and affected by seven substitutions in the 328–338 regions. The lamB mutants were also tested for phage λ receptor activity and starch binding before and after thiol modification and were useful in extending previous maps of these ligand binding sites.     

Factors affecting maintenance, proliferation, and germination of secondary somatic embryos of Eucalyptus globulus Labill

The described protocol for repetitive somatic embryogenesis (SE) in Eucalyptus globulus produced more somatic embryos than the primary SE protocol. Primary somatic embryos (induced on MS_3NAA) were transferred to the same medium, leading to new cycles of somatic embryos, for at least 2 years. The influence of medium (MS and B5), plant growth regulators (auxins and cytokinins), and light on secondary SE was tested. The MS medium without growth regulators (MS_WH) was more efficient for cotyledonary embryo formation and germination than the B5 medium. Reducing auxin (NAA) levels increased the proliferation of globular somatic embryos and allowed SE competence to be maintained on medium free of plant growth regulators. The addition of two cytokinins (BAP and KIN) to the MS medium did not improve proliferation of globular secondary embryos, but was crucial during later stages of the SE process (germination and conversion). Data also show that light may influence the quality of the process, depending on its stage. Darkness should be maintained until the cotyledonary stage is reached, after which exposure to light is recommended.     

Secondary Adrenal Insufficiency: Recent Updates and New Directions for Diagnosis and Management

Secondary adrenal insufficiency is the most common subtype of adrenal insufficiency; it is caused by certain medications and pituitary destruction (pituitary masses, inflammation, or infiltration) and is rarely associated with certain germline variants. In this review, we discuss the etiology, epidemiology, and clinical presentation of secondary adrenal insufficiency and focus on the diagnostic and management challenges. We also review the management of selected special populations of patients and discuss patient-important outcomes associated with secondary adrenal insufficiency.     

ADP phosphorylation and glutamate oxidation in mitochondria isolated from Dictyostelium discoideum amoebae

Mitochondria have been isolated from $\text{D.}\text{discoideum}$ amoebae in which respiration is coupled to ADP phosphorylation. P:O ratios and respiratory control ratios have been obtained for a number of metabolites. In rat liver mitochondria, glutamate is oxidized almost exclusively by a respiration-dependent cyclic transamination pathway, in which glutamate is converted to aspartate. When $\text{D.}\text{discoideum}$ amoebae are incubated with glutamate alone, aspartate does not accumulate appreciably. Furthermore, when the mitochondria are incubated with glutamate plus malonate at a concentration sufficient to inhibit respiration, their utilization of glutamate is depressed only slightly. Thus, it appears that glutamate oxidation within the mitochondria of $\text{D.}\text{discoideum}$ amoebae does not, for the most part, proceed by the cyclic transamination pathway.     

Inhibition of anti-CD3 monoclonal antibody-induced T-cell proliferation by dexamethosone,isoproterenol, or prostaglandin E2 either alone or in combination

1. The purpose of these studies was to investigate the modulation of the proliferation of human T cells obtained from peripheral blood by dexamethasone (DEX), isoproterenol (ISO), and prostaglandin E2 (PGE2). The former two substances interact with T cells via the glucocorticoid and beta-adrenergic receptors respectively. When occupied by their natural ligands, glucocorticosteroids and catecholamines, these receptors have a role in modulating T-cell function during stress. During the inflammatory response increased levels of PGE2 bind to their receptors on T cells and thus alter responsiveness. Proliferation of T cells was induced by immobilized anti-CD3 monoclonal antibody (mAb) in the presence or absence of an additional costimulatory signal delivered by anti-CD28 mAb. 2. Various physiologic concentrations of DEX, ISO, or PGE2 were added at the time of initiation of the cultures and subsequent proliferation of the unstimulated T cells was determined. The results demonstrate that physiologic concentrations of all three of these agents inhibit the anti-CD3 mAb-induced proliferation of T cells. 3. Although DEX and PGE2 were equipotent in suppressing T-cell proliferation, ISO was much less effective. 4. Because concomitant elevations in the peripheral levels of these substances may occur, experiments were performed to determine the T-cell inhibitory effects of DEX together with either PGE2 or ISO. Synergistic suppression of T-cell proliferation was observed when various concentrations of DEX and PGE2, but not DEX and ISO, were added to cultures. This synergistic suppression could not be explained by an increase in cAMP accumulation in T cells stimulated with DEX and PGE2. 5. Finally, the addition of anti-CD28 mAb to anti-CD3 mAb-stimulated T cells overcame much of the suppression of proliferation induced by PGE2 or ISO but less so than that induced by DEX.