Cell volume measurement as an estimation of mammalian cell biomass

Measurements of volume distributions and dry weight are made on hybridoma cells in culture. The volume of viable hybridoma cells is significantly larger than that of nonviable cells. During exponential growth, the volume of the viable hybridoma cells is found to be significantly larger than that during other stages of batch culture. Proportionality is found between the volume of the cells and their dry weight, indicating that the volume data can be used in conjunction with cell concentration data as a practical technique for indirect measurement of the biomass concentration present in a culture. Comparison of dry weight concentrations in continuous culture to predictions from the volume data shows very good agreement.     

Cytostatic and growth-stimulating effects of alveolar macrophages of rats on tumor cells

Cytostatic and growth-stimulating effects of alveolar macrophages (AM) of rats on tumor cells were studied. The experimental results are summarized as follows. 1. The cytotoxicity of AM activated with BCG to tumor cells was increasing with the increase of effector cells/target cells (E/T) ratio. AM without the treatment with BCG expressed slight cytotoxicity to tumor cells at a high E/T, and growth-stimulating effect on tumor cells, at a low E/T. 2. AM after 24-hour culture had a lower manifestation of cytotoxicity to human lung adenocarcinoma cell line than that of AM without 24-hour culture, and had a growth-stimulating effect on B-16 cell line. 3. Cytostatic and growth-stimulating effects of AM without or with 24-hour culture were decreasing with the increase of irradiation doses.     

Enumeration and identification of IS3 elements in Escherichia coli strains.

Escherichia coli K-12 strains ordinarily contain five IS3 elements. Three of these correspond to previously mapped IS3 elements (R. C. Deonier, G. R. Oh, and M. Hu, J. Bacteriol. 129:1129--1140, 1977; S. Hu, E. Ohtsubo, and N. Davidson, J. Bacteriol. 122:749--763, 1975), and two additional IS3 elements are identified. The distribution of IS3 elements among deoxyribonucleic acid fragments generated by digestion with EcoRI indicates a basic pattern from which deviation is detected.     

Characterization of the plasmid DNAs of Rhodopseudomonas capsulata

Presence of extrachromosomal DNa in Rhodopseudomonas capsulata strain BH9 was shown by the appearance of a satellite band in a dye-buoyant density gradient. Radioactively labelled DNA was prepared from this satellite band and examined on a 5–20% sucrose gradient. Three radioactive peaks with sedimentation coefficients of 100 S, 94 S, and 58–64 S, respectively, were consistently observed. Analysis of these sedimentation coefficients suggested that there are two species of plasmid DNA with molecular sizes of 94×10⁶ daltons (named pBH91) and 74×10⁶ daltons (named pBH92). The 58–64 S peak is attributed to open circular molecules. DNAs from each peak of the sucrose gradient were examined by electronmicroscopy, and the results agree closely with those of the sucrose gradient analysis. Reassociation kinetics of the plasmid DNA was also followed. Addition of total DNA of strain BH9 increased the renaturation rate of the plasmid DNA. It was calculated from the magnitude of the increase that approximately 10% of the BH9 total DNA may hybridize with the plasmid sequences. DNA prepared from the gene transfer agent (GTA) produced by R. capsulata increases the renaturation rate of the plasmid to the same extent as total DNA isolated from the GTA producing strain, Y262.     

Kinetics of glycogen phosphorylase a with a series of semisynthetic, branched saccharides. A model for binding of polysaccharide substrates.

The requirement of muscle phosphorylase for branched polysaccharide substrates was investigated by kinetic studies on semisynthetic branched saccharides. One series of saccharides was prepared from maltoheptose by oxidizing the reducing group to a carboxyl group and coupling this with an amino group of ethylenediamine. The resulting aminooligosaccharide was coupled with p-nitrophenyl esters of mono-, di-, tetra-, and polycarboxylic aicds to produce saccharides containing one, two, four, and approximately 52 maltodextrin chains per molecule. A similar series of saccharides was prepared from a heterogeneous maltodextrin of average chain length 11.7. Kinetic constants were determined for the reaction with phoshorylase a in the direction of chain elongation. Michaelis constants are equilibrium constants for dissociation of saccharide from the enzyme-AMP-glucose-1P-saccharide complex. The Michaelis constants, expressed in terms of the concentration of nonreducing end groups, are independent of maltodextrin chain length but decrease considerably as the number of chains per molecule increases. Maximum velocities do not differ greatly from that for glycogen. Among the synthetic saccharides, only the polymer behaves similarly to glycogen in exhiiting a decreasing reaction rate as the chains are elongated. The kinetic constants are quantitatively consistent with a model in which two chain termini from the same saccharide molecule bind to the phosphorylase molecule simultaniously, Differences in binding between saccharides having different numbers of equally accessible chains are caused solely by statistical factors in the equilibrium. Highly branched substrates bind better because of their greater multiplicity of two end-group pairs.     

Cryo-EM structure of the fatty acid reductase LuxC–LuxE complex provides insights into bacterial bioluminescence

The discovery of reduced flavin mononucleotide and fatty aldehydes as essential factors of light emission facilitated study of bacterial luminescence. Although the molecular mechanisms underlying bacterial luminescence have been studied for more than 60 years, the structure of the bacterial fatty acid reductase complex remains unclear. Here, we report the cryo-EM structure of the Photobacterium phosphoreum fatty acid reductase complex LuxC–LuxE to a resolution of 2.79 Å. We show that the active site Lys238/Arg355 pair of LuxE is >30 Å from the active site Cys296 of LuxC, implying that catalysis relies on a large conformational change. Furthermore, mutagenesis and biochemical experiments support that the L-shaped cleft inside LuxC plays an important role in substrate binding and reaction. We obtained a series of mutants with significantly improved activity as measured by in vitro bioluminescence assays and demonstrated that the double mutant W111A/F483K displayed the highest activity (370% of the WT). Our results indicated that the activity of LuxC significantly affects the bacterial bioluminescence reaction. Finally, we expressed this mutated lux operon in Escherichia coli but observed that the in vivo concentrations of ATP and NADPH limited the enzyme activity; thus, we conclude that the luminous intensity mainly depends on the level of metabolic energy.     

SARS-CoV-2 envelope protein causes acute respiratory distress syndrome (ARDS)-like pathological damages and constitutes an antiviral target

Cytokine storm and multi-organ failure are the main causes of SARS-CoV-2-related death. However, the origin of excessive damages caused by SARS-CoV-2 remains largely unknown. Here we show that the SARS-CoV-2 envelope (2-E) protein alone is able to cause acute respiratory distress syndrome (ARDS)-like damages in vitro and in vivo. 2-E proteins were found to form a type of pH-sensitive cation channels in bilayer lipid membranes. As observed in SARS-CoV-2-infected cells, heterologous expression of 2-E channels induced rapid cell death in various susceptible cell types and robust secretion of cytokines and chemokines in macrophages. Intravenous administration of purified 2-E protein into mice caused ARDS-like pathological damages in lung and spleen. A dominant negative mutation lowering 2-E channel activity attenuated cell death and SARS-CoV-2 production. Newly identified channel inhibitors exhibited potent anti-SARS-CoV-2 activity and excellent cell protective activity in vitro and these activities were positively correlated with inhibition of 2-E channel. Importantly, prophylactic and therapeutic administration of the channel inhibitor effectively reduced both the viral load and secretion of inflammation cytokines in lungs of SARS-CoV-2-infected transgenic mice expressing human angiotensin-converting enzyme 2 (hACE-2). Our study supports that 2-E is a promising drug target against SARS-CoV-2.Subject terms: Cell death, Molecular biology     

TEB/POLQ plays dual roles in protecting Arabidopsis from NO-induced DNA damage

Nitric oxide (NO) is a key player in numerous physiological processes. Excessive NO induces DNA damage, but how plants respond to this damage remains unclear. We screened and identified an Arabidopsis NO hypersensitive mutant and found it to be allelic to TEBICHI/POLQ, encoding DNA polymerase θ. The teb mutant plants were preferentially sensitive to NO- and its derivative peroxynitrite-induced DNA damage and subsequent double-strand breaks (DSBs). Inactivation of TEB caused the accumulation of spontaneous DSBs largely attributed to endogenous NO and was synergistic to DSB repair pathway mutations with respect to growth. These effects were manifested in the presence of NO-inducing agents and relieved by NO scavengers. NO induced G2/M cell cycle arrest in the teb mutant, indicative of stalled replication forks. Genetic analyses indicate that Polθ is required for translesion DNA synthesis across NO-induced lesions, but not oxidation-induced lesions. Whole-genome sequencing revealed that Polθ bypasses NO-induced base adducts in an error-free manner and generates mutations characteristic of Polθ-mediated end joining. Our experimental data collectively suggests that Polθ plays dual roles in protecting plants from NO-induced DNA damage. Since Polθ is conserved in higher eukaryotes, mammalian Polθ may also be required for balancing NO physiological signaling and genotoxicity.