MGB-BP-3 is a potential first-in-class antibiotic, a Strathclyde Minor Groove Binder (S-MGB), that has successfully completed Phase IIa clinical trials for the treatment of Clostridioides difficile ...associated disease. Its precise mechanism of action and the origin of limited activity against Gram-negative pathogens are relatively unknown. Herein, treatment with MGB-BP-3 alone significantly inhibited the bacterial growth of the Gram-positive, but not Gram-negative, bacteria as expected. Synergy assays revealed that inefficient intracellular accumulation, through both permeation and efflux, is the likely reason for lack of Gram-negative activity. MGB-BP-3 has strong interactions with its intracellular target, DNA, in both Gram-negative and Gram-positive bacteria, revealed through ultraviolet–visible (UV–vis) thermal melting and fluorescence intercalator displacement assays. MGB-BP-3 was confirmed to bind to dsDNA as a dimer using nano-electrospray ionization mass spectrometry and nuclear magnetic resonance (NMR) spectroscopy. Type II bacterial topoisomerase inhibition assays revealed that MGB-BP-3 was able to interfere with the supercoiling action of gyrase and the relaxation and decatenation actions of topoisomerase IV of both Staphylococcus aureus and Escherichia coli. However, no evidence of stabilization of the cleavage complexes was observed, such as for fluoroquinolones, confirmed by a lack of induction of DSBs and the SOS response in E. coli reporter strains. These results highlight additional mechanisms of action of MGB-BP-3, including interference of the action of type II bacterial topoisomerases. While MGB-BP-3′s lack of Gram-negative activity was confirmed, and an understanding of this presented, the recognition that MGB-BP-3 can target DNA of Gram-negative organisms will enable further iterations of design to achieve a Gram-negative active S-MGB.
Crystallographic and solution studies have shown that IgE molecules are acutely bent in their Fc region. Crystal structures reveal the Cɛ2 domain pair folded back onto the Cɛ3-Cɛ4 domains, but is the ...molecule exclusively bent or can the Cɛ2 domains adopt extended conformations and even 'flip' from one side of the molecule to the other? We report the crystal structure of IgE-Fc captured in a fully extended, symmetrical conformation and show by molecular dynamics, calorimetry, stopped-flow kinetic, surface plasmon resonance (SPR) and Förster resonance energy transfer (FRET) analyses that the antibody can indeed adopt such extended conformations in solution. This diversity of conformational states available to IgE-Fc offers a new perspective on IgE function in allergen recognition, as part of the B-cell receptor and as a therapeutic target in allergic disease.
Background: IgE is now known to upregulate the expression of FcϵRI on human basophils. It is not known which receptor on basophils mediates this process of upregulation.
Objective: We sought to ...determine whether galectin-3, FcϵRII (CD23), or FcϵRI were involved in the upregulation of FcϵRI by IgE.
Methods: The role of galectin-3 was examined by measuring the influence of α-lactose on upregulation. Basophils were examined for expression of FcϵRII (CD23) by flow cytometry and messenger (m)RNA expression. Functional discrimination between binding to FcϵRII or FcϵRI was examined through the use of mutant IgE-Fc fragments or anti-FcϵRII antibody.
Results: Upregulation of FcϵRI on basophils in the presence of IgE was not altered by coincubation with α-lactose, eliminating a role for galectin-3. Basophils were not found to express FcϵRII, as determined by flow cytometry with enriched basophil preparations or RT-PCR with highly purified basophil preparations. A mutant of the Fc fragment of IgE (IgE-Fc), which binds to FcϵRI with a greater than 10-fold lower affinity than IgE or wild-type IgE-Fc but exhibits no change in affinity for FcϵRII, allowed us to distinguish between the functions of the two Fc receptors. The mutant (R334S; Henry et al 1997) was required at about 30-fold higher concentration than the wild-type IgE-Fc for the same stimulation of FcϵRI expression on basophils, thus excluding a role for FcϵRII in the response. In addition, treatment of basophils with anti-FcϵRII antibody (MHM6), which is known to be competitive with IgE, had no effect on the expression of FcϵRI or the ability of IgE to upregulate expression of FcϵRI.
Conclusion: Collectively, these data indicate that IgE interacts with FcϵRI to upregulate its expression on human basophils. (J Allergy Clin Immunol 1999;104:492-8.)
Immunoglobulin E (IgE) plays a central role in the allergic response, in which cross‐linking of allergen by FcϵRI‐bound IgE triggers mast cell and basophil degranulation and the release of ...inflammatory mediators. The high‐affinity interaction between IgE and FcϵRI is a long‐standing target for therapeutic intervention in allergic disease. Omalizumab is a clinically approved anti‐IgE monoclonal antibody that binds to free IgE, also with high affinity, preventing its interaction with FcϵRI. All attempts to crystallize the pre‐formed complex between the omalizumab Fab and the Fc region of IgE (IgE‐Fc), to understand the structural basis for its mechanism of action, surprisingly failed. Instead, the Fab alone selectively crystallized in different crystal forms, but their structures revealed intermolecular Fab/Fab interactions that were clearly strong enough to disrupt the Fab/IgE‐Fc complexes. Some of these interactions were common to other Fab crystal structures. Mutations were therefore designed to disrupt two recurring packing interactions observed in the omalizumab Fab crystal structures without interfering with the ability of the omalizumab Fab to recognize IgE‐Fc; this led to the successful crystallization and subsequent structure determination of the Fab/IgE‐Fc complex. The mutagenesis strategy adopted to achieve this result is applicable to other intractable Fab/antigen complexes or systems in which Fabs are used as crystallization chaperones.
The omalizumab Fab was engineered to disrupt recurring crystal packing interactions in Fab crystal structures; this led to the eventual structure determination of an omalizumab‐derived Fab in complex with its target, IgE‐Fc.
In recent years it has become evident that tissue cyclooxygenase-2 (COX-2) may play a role in carcinogenesis and tumor malignancy. There is now a mounting body of information that strongly implies ...that COX-2 inhibitors may be of some value in the management of patients with carcinomas, and most recently several similar reports have appeared relating to sarcomas.
The authors studied 32 samples of cartilage tumors from our tumor tissue bank for the presence of COX-2 by a Western blot technique. There were 29 patients from whom the samples were obtained, including 8 with enchondromas and 21 with chondrosarcomas.
Thirteen of the 24 chondrosarcoma samples and none of the 8 enchondromas were positive for COX-2. An attempt was made to correlate these results with clinical data including age, gender, staging according to the Musculoskeletal Tumor Society, anatomical site, ploidic pattern, presence of metastases and death rate but no statistically valid correlation could be found.
It is evident that COX-2 may play some role in chondrosarcoma but not in the benign enchondroma and that further studies with COX-2 inhibitors are warranted.
The main host carbon energy source transferred from wheat leaves (Triticum aestivum L.) to wheat powdery mildew (Erysiphe graminis f.sp. tritici) has been investigated in three ways. When the uptake ...of sugars by isolated mycelial suspensions was examined, the uptake rate for glucose was considerably higher than that for a range of other solutes. Analysis by high-performance liquid chromatography of leaf and mycelial extracts following uptake of sugars into infected leaf pieces confirmed that sucrose was rapidly hydrolyzed in the leaf; no sucrose or fructose could be detected in mycelial extracts. Furthermore, studies of the uptake of asymmetrically labelled sucrose indicated that this sugar is cleaved prior to uptake by the pathogen. Thus several lines of evidence show that glucose, and not sucrose, is the major carbon energy source transferred from host to fungal mycelium.
The high-affinity receptor for immunoglobulin E (IgE), FcεRI, is an αβγ2 tetramer found on mast cells, basophils, and several other types of immune effector cells. The interaction of IgE with the ...α-subunit of FcεRI is central to the pathogenesis of allergy. Detailed knowledge of the mode of interaction of FcεRI with IgE may facilitate the development of inhibitors for general use in the treatment of allergic disease. To this end we have performed site-directed mutagenesis on a soluble form of the FcεRI α-chain (sFcεRIα). The effects of four mutations in the second immunoglobulin-like domain of sFcεRIα upon the kinetics of binding to IgE and fragments of IgE have been analyzed using surface plasmon resonance. As described in the preceding paper of this issue Henry, A. J., et al. (1997) Biochemistry 36, 15568−15578, biphasic binding kinetics was observed. Two of the mutations had significant effects on binding: K117D reduced the affinity of sFcεRIα for IgE by a factor of 30, while D159K increased the affinity for IgE by a factor of 7, both principally through changes in the rates of dissociation of the slower phase of the interaction. Circular dichroism spectra of sFcεRIα incorporating either of these mutations were indistinguishable from those of wild-type sFcεRIα, demonstrating that the native conformation had not been disrupted. Our results, together with those from site-directed mutagenesis on fragments of IgE presented in the accompanying paper, define the contact surfaces in the IgE:sFcεRIα complex.
‘FLLA09015‐U1’ (Reg. no. CV‐387, PI 699117) is a new facultative oat (Avena sativa L.) cultivar that was co‐developed by the University of Florida and Louisiana State University Agricultural Center ...and was released in 2019. This line was derived from a single cross of FL0210‐J1/MN06203. FLLA09015‐U1 has considerable potential for grain and forage yield and for conservation tillage purposes in the southern United States. Exclusive marketing rights for FLLA09015‐U1 has been granted to JoMar Seeds and is currently commercialized under the name of Juggernaut. FLLA09015‐U1 was developed using selected bulk breeding method and was selected as an F5:6 head row. The line was evaluated in advanced, regional, and state grain and forage yield trials from 2015 to 2021. FLLA09015‐U1 was observed to be uniform and stable across environments in the southern United States from 2015 to present. The line possesses a semi‐prostrate growth habit and has large leaves that are dark green in color. It is a mid‐maturing, medium to mid‐tall height with excellent grain yield and good forage yield and test weight. It has excellent crown rust resistance and very good resistance to Barley yellow dwarf virus and stem rust and demonstrated moderate lodging resistance. It has performed very well in both grain and forage trials. FLLA09015‐U1 has broad environmental adaptation and has performed well in Louisiana, Florida, Georgia, Texas, Alabama, and South Carolina. We consider FLLA09015‐U1 to be a good dual‐purpose type of oat because of its high grain yield potential and vigorous growth and high tillering capacity.
Core Ideas
FLLA09015‐U1 a facultative oat cultivar that was co‐developed by the University of Florida and Louisiana State University.
FLLA09015‐U1 has broad adaptation and has performed well in Louisiana, Florida, Georgia, Texas, and Alabama.
FLLA09015‐U1 is a good dual‐purpose type of oat and has high grain yield potential and vigorous growth and high tillage.
FLLA09015‐U1 has good crown and stem rust and Barley yellow dwarf virus resistance.
FLLA09015‐U1 has considerable potential for grain, forage, conservation tillage, and wildlife use in the southern USA.
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