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Endotoxin, Antibiotics, and Inflammation in Gram-Negative Infections
Published in Helmut Brade, Steven M. Opal, Stefanie N. Vogel, David C. Morrison, Endotoxin in Health and Disease, 2020
Penicillin-binding proteins (PBPs) are the primary biochemical targets of β-lactam antibiotics in bacteria. These PBPs catalyze terminal stages in the assembly of the peptidoglycan network of the bacterial cell wall (47). Whereas the older penicillins (penicillin G) aspecifically bind to all of these PBPs, the newer β-lactams often specifically bind to only one or two of the PBPs. Treatment of Enterobacteriaceae with (β-lactam antibiotics that have a high selective affinity for PBP 1a and especially 1b causes rapid and extensive killing of the bacteria, with degradation of cell wall material and cellular lysis. Antibiotics with selective affinity for PBP 2 cause conversion of the bacilli to round-shaped cells (also called spheroplasts). Inhibitors of PBP 3 cause selective inhibition of bacterial septation, which leads to the formation of long filaments, but initially only limited bactericidal activity and lysis takes place (47).
Physiology and Growth
Published in Paul Pumpens, Single-Stranded RNA Phages, 2020
A special procedure was necessary for the large-scale isolation in milligram quantities of the caulophage φCB5 (Leffler et al. 1971). Isolation of the φCB5 by conventional procedures was impossible because of its extreme sensitivity to high ionic strength that excluded salt precipitation or the CsCl gradient fractions. To achieve the goal, the infected cells containing mature virions were concentrated 5.5 hours after infection before the cells begin to disintegrate. Then, the concentrated infected cells were converted to spheroplasts by lysozyme treatment in the presence of an osmotically protective concentration of polyethylene glycol (Leffler et al. 1971).
Unexplained Fever in Infectious Diseases Section 1: Viruses, Chlamydia, Mycoplasma, Rickettsiae, Higher Bacteria, Cell-Wall Deficient Bacteria, And Fungi
Published in Benedict Isaac, Serge Kernbaum, Michael Burke, Unexplained Fever, 2019
Cell wall-deficient bacteria (C.W.D.), or L-forms, or microbial spheroplasts, have atypical morphologic forms because they are derived from parent (classical) bacteria that have lost their rigidity owing to defective cell wall.106 They are very sensitive to osmotic forces: they swell and often burst in media of low osmolality and can be isolated in hypertonic ones.106
The force-from-lipid principle and its origin, a ‘what is true for E. coli is true for the elephant’ refrain
Published in Journal of Neurogenetics, 2022
Spheroplasts are live cells and can grow normally after the removal from cephalexin and lysozyme. Patch-clamping E. coli spheroplasts is therefore an exercise in vivo. There were methods to study ion-channel activities in artificial lipid bilayer (the ‘black lipid membrane’) even before the invention of the patch clamp. Modifying existing methods, Ann and Boris generated E. coli liposomes by mixing membrane fractions with exogenous phospholipids (azolectin). They then induced blisters from them and sampled patches from these blisters. There, they encountered at least three types of channel activities, including that of the MS channels (Delcour, Martinac, Adler, & Kung, 1989). Such reconstitutions were also successful in the laboratory of Alexandre Ghazi independently (Berrier, Coulombe, Houssin, & Ghazi, 1989). The fact that these channel activities survive these treatments in vitro is key to the eventual biochemical identification of the material behind the activity.
Yeast-inspired drug delivery: biotechnology meets bioengineering and synthetic biology
Published in Expert Opinion on Drug Delivery, 2019
Chinnu Sabu, Panakkal Mufeedha, Kannissery Pramod
Yeast cells are 2–3 µm ovoid or ellipsoidal structures covered by a thick cell wall. Saccharomyces is a Latin word which means sugar fungus, and cerevisiae is derived from two Latin words ceres and vise which means grain and strength. Cell envelope constitutes 15% of the total cell volume and is responsible for regulating the osmotic and permeability characteristics of the cell. The cytosol is further enveloped by a plasma membrane, periplasmic space, and the cell wall. Yeast cell wall which constitutes 25% of the total dry mass of cell is a rigid structure with a thickness of 200 nm. The four major macromolecules that constitute the cell wall are highly glycosylated glycoprotein or mannoproteins, two types of β-glucans and chitin. The cell wall constitution varies in accordance with growth condition of the cell. Spheroplasts or naked cells are formed by removing cell wall by reaction with a lytic enzyme in presence of osmotic stabilizers without causing any harm. They are susceptible to intergeneric and intrageneric cell fusions [8].
Engineered aglycosylated full-length IgG Fc variants exhibiting improved FcγRIIIa binding and tumor cell clearance
Published in mAbs, 2018
Migyeong Jo, Hyeong Sun Kwon, Kwang-Hoon Lee, Ji Chul Lee, Sang Taek Jung
For displaying full-length IgG Fc variants, Jude1 cells that harbored two plasmids, pPelB-AglycoT(H)-Fc variant-FLAG and pBADAglycoT(L)-His,21 were cultured at 37°C for 16 h with shaking at 250 rpm in 25 ml of TB containing 2% (wt/vol) glucose, chloramphenicol (40 µg/ml), and kanamycin (50 µg/ml) in a 250-ml Erlenmeyer flask. The overnight cultured cells were diluted 1:100 in 110 ml of fresh TB media supplemented with chloramphenicol (40 µg/ml) and kanamycin (50 µg/ml) in a 2-L Erlenmeyer flask. After incubation at 37°C until the optical density at 600 nm reached 0.6, the cells were cooled at 25°C for 20 min and induced with 1 mM of isopropyl-1-thio-D-galactopyranoside and 0.2% arabinose for protein expression. After incubating the cells at 25°C for 20 h, the cells were pelleted by centrifugation at 14,000 rpm and washed twice with 1 ml of cold 10 mM Tris‐HCl (pH 8.0). The outer membrane was removed by resuspending in 1 ml of cold STE solution (0.5 M sucrose, 10 mM Tris‐HCl, 10 mM EDTA; pH 8.0) and incubating at 37°C for 30 min on a rotator. The cells, pelleted by centrifugation at 14,000 rpm for 1 min, were washed in 1 ml of cold Solution A (0.5 M sucrose, 20 mM MgCl2, 10 mM MOPS; pH 6.8) and incubated in 1 ml of Solution A with 1 mg/ml of chicken egg lysozyme at 37°C for 15 min. After centrifugation at 14,000 rpm for 1 min, spheroplasts were recovered in the pellet.