Genome-wide CRISPR Screens Reveal Host Factors Critical for SARS-CoV-2 Infection

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Expression of sialic acid on the alveolar surface of adult and fetal human lungs

Lung alveoli are coated by a thin layer of extracellular material rich in anionic charges. The nature of this acid layer and its relationship to the phospholipid surfactant are not known. We investigated the possible presence of sialic acid groups by light and electron microscopy in tissues from normal fetal and adult lungs, using neuraminidase treatment followed by staining with the galactose-binding lectin from peanut, labeled with peroxidase. Our results showed that adult lung does not bear peanut lectin-reactive sites but that a very thin and distinct reactive layer becomes evident after neuraminidase treatment, especially on type II pneumocytes. In fetal lung, the entire surface of the developing respiratory tree is outlined by a strongly peanut lectin-reactive layer even if neuraminidase digestion is not performed. We conclude that the acid coat of the alveolar lining is in part composed of sialic acid residues and that sialic acid is added to the fetal lung as the alveoli mature.     

Isolation of succinate dehydrogenase from Desulfobulbus elongatus, a propionate oxidizing, sulfate reducing bacterium

Succinate dehydrogenase was purified from the particulate fraction of Desulfobulbus. The enzyme catalyzed both fumarate reduction and succinate oxidation but the rate of fumarate reduction was 8-times less than that of succinate oxidation. Quantitative analysis showed the presence of 1 mol of covalently bound flavin and 1 mol of cytochrome b per mol of succinate dehydrogenase. The enzyme contained three subunits with molecular mass 68.5, 27.5 and 22 kDa. EPR spectroscopy indicated the presence of at least two iron sulfur clusters. 2-Heptyl-4-hydroxy-quinoline-N-oxide inhibited the electron-transfer between succinate dehydrogenase and a high redox potential cytochrome c3 from Desulfobulbus elongatus.     

Localization of the Miller-Dieker critical region is proximal to locus D17S34 (p144D6) in 17p13.3

A child with normal growth and development and the abnormal karyotype 46,XY,17ps, was analyzed using molecular probes localized to 17p13. The results indicated the presence of two copies of the probes YNZ22.1 (D17S5) and YNH37.3 (D17S28), previously shown to be deleted in all Miller-Dieker (MDS) patients studied. However, the patient was hemizygous for probe p144D6 (D17S34), which is absent in approximately 75% of the MDS patients. As the patient is active at 9 months of age, with no clinical signs of MDS, the results confirm that the absence of locus D17S34 does not lead to the phenotypic expression of MDS. Furthermore, this deletion should assist in defining the distal limits of this contiguous gene syndrome.     

Stoichiometry of interaction between soluble (—)-dopa melanin and enantiomers of ephedrine by NMR spectroscopy

Synthetic soluble (—)-dopa melanin was prepared in deuteriated buffer, pH 8, by autooxidation of the precursor. At 6 mM of the precursor, the incorporation was over 90%. The changes in the line width measurements of N-CH₃ protons of enantiomers of ephedrine in the soluble melanin were quantified by NMR spectroscopy. The dissociation constants of (—)-1R,2S-ephedrine, (+)-1S,2R-ephedrine, (—)-1R,2R-ψ-ephedrine, and (+)-1S,2S-ψ-ephedrine were 11.7, 4.20, 3.60, and 4.80 mM, respectively. Since the concentration of (—)-dopa was known and since the conversion of (—)-dopa to indole units of melanin was considered as 1:1, the stoichiometry of the interaction between the drug and the indole unit was calculated. Based on the dissociation constants of the enantiomers, it appears that up to four molecules of (—)-ephedrine can interact with one indole unit of the melanin, while such a ratio for other isomers appear to be 2:1. The preference by indole units of melanin is stereoselective. © 1992 Wiley-Liss, Inc.     

EPR-detectable redox centers of the periplasmic hydrogenase from Desulfovibrio vulgaris

The periplasmic hydrogenase of Desulfovibrio vulgaris (Hildenbourough NCIB 8303) belongs to the category of [Fe] hydrogenase which contains only iron-sulfur clusters as its prosthetic groups. Amino acid analyses were performed on the purified D. vulgaris hydrogenase. The amino acid composition obtained compared very well with the result derived from the nucleotide sequence of the structural gene (Voordouw, G., Brenner, S. (1985) Eur. J. Biochem. 148, 515-520). Detailed EPR reductive titration studies on the D. vulgaris hydrogenase were performed to characterize the metal centers in this hydrogenase. In addition to the three previously observed EPR signals (namely, the "isotropic" 2.02 signal, the rhombic 2.10 signal, and the complex signal of the reduced enzyme), a rhombic signal with resonances at the g-values of 2.06, 1.96, and 1.89 (the rhombic 2.06 signal) was detected when the samples were poised at potentials between 0 and -250 mV (with respect to normal hydrogen electrode). The midpoint redox potentials for each of the four EPR-active species were determined, and the characteristics of each EPR signal are described. Both the rhombic 2.10 and 2.06 signals exhibit spectral properties that are distinct from a ferredoxin-type [4Fe-4S] cluster and are proposed to originate from the same H2-binding center but in two different conformations. The complex signal of the reduced hydrogenase has been shown to represent two spin-spin interacting ferredoxin-type [4Fe-4S]1+ clusters (Grande, H. J., Dunham, W. R., Averill, B., Van Dijk, C., and Sands, R. H. (1983) Eur. J. Biochem. 136, 201-207). The titration data indicated a strong cooperative effect between these two clusters during their reduction. In an effort to accurately estimate the number of iron atoms/molecule of hydrogenase, plasma emission and chemical methods were used to determine the iron contents in the samples; and four different methods, including amino acid analysis, were used for protein determination. The resulting iron stoichiometries were found to be method-dependent and vary over a wide range (+/- 20%). The uncertainties involved in the determination of iron stoichiometry are discussed.     

Evidence that centre 2 in Escherichia coli fumarate reductase is a [4Fe-4S]cluster

Redox titrations of the iron-sulphur clusters in fumarate reductase purified from Escherichia coli, monitored by ESR spectroscopy, identified three redox events, similar to those observed in other fumarate reductases and succinate dehydrogenases: Centre 1, a [2Fe-2S] cluster, at g = 2.03, 1.93, appeared on reduction with Em = -20 mV. Centre 3, probably a [3Fe-xS] cluster, at g = 2.02 appeared in the oxidized state with Em = -70 mV. Centre 2 has been observed as an increase in the electron-spin relaxation of Centre 1. It titrates as an n = 1 species with Em = -320 mV, but in our hands did not appear to contribute significant intensity to the g = 2.03, 1.93 signal. It therefore appears to be an additional centre which undergoes spin-spin interaction with Centre 1. The reduction of Centre 2 coincided with the appearance of an extremely broad ESR spectrum, observed at temperatures below 20 K, with features at g = 2.17, 1.9, 1.68. The broad signal was observed in both soluble and membrane-bound preparations. Its midpoint potential was -320 mV. Its integrated intensity was approximately equal to that of Centre 1, if its broad outer wings were taken into account. Consideration of the ESR properties of this signal, together with the amino acid sequence of the frdB subunit of the enzyme, indicates that Centre 2 is a [4Fe-4S] cluster which, in its reduced state, enhances the spin relaxation of the [2Fe-2S] Centre 1.     

Preliminary characterization of an antibiotic produced by Xanthomonas albilineans which inhibits DNA synthesis in Escherichia coli

Chlorosis-inducing isolates of Xanthomonas albilineans, the sugarcane leaf scald pathogen, produced a mixture of antibacterial compounds in culture. The antibiotic mixture, which eluted as a single strongly retarded peak from Sephadex LH-20 in methanol, was bactericidal to Escherichia coli. Inhibition of E. coli was not reversed by added nutrients, and affected cells were not lysed but many accumulated polyphosphate granules. The major antibacterial component, isolated in crystalline form after HPLC, is given the trivial name albicidin. Near the minimum inhibitory concentration, albicidin caused a complete block to DNA synthesis, followed by partial inhibition of RNA and protein synthesis, as assessed by incorporation of radioactive precursors. Spontaneous antibiotic-resistant mutants of E. coli showed no cross-resistance between albicidin and inhibitors of either subunit of DNA gyrase. Mixing albicidin with purified DNA from E. coli did not alter the thermal denaturation behaviour of the DNA, or the absorption spectrum of the antibiotic. PolA+ and PolA - strains of E. coli were equally sensitive to albicidin, indicating that the antibiotic does not bind to or modify DNA. Selective inhibition of DNA synthesis without evidence of DNA binding suggests a specific interaction of albicidin with an essential replication protein.