The enhancement of electrostriction caused by lowering the solvent dielectric constant leads to the decrease of activation energy in trypsin catalysis.

The contribution of electrostriction of the solvent to the stabilization of the negatively charged tetrahedral transition state of a trypsin-catalyzed reaction was probed by means of kinetic studies involving high-pressure and solvent dielectric constant. A good correlation was observed between the increased catalytic efficiency of trypsin and the decreased solvent dielectric constant. When the dielectric constant of the solvents was lowered by 4.68 units, the loss of activation energy and that of free energy of activation were 2.26 kJ/mol and 3.09 kJ/mol, respectively. The activation volume for k(cat) decreased significantly as the dielectric constant of the solvent decreased, indicating that the degree of electrostriction of the solvent around the charged tetrahedral transition state has been enhanced. These observations demonstrate that the increase in the catalytic efficiency of the trypsin reaction with decreasing dielectric constant resulted from the stabilization of electrostatic energy for the formation of an oxyanion hole, and this stabilization was caused by the increase of electrostricted water around the charged tetrahedral transition state. Therefore, we conclude that control of the solvent dielectric constant can stabilize the tetrahedral transition state, and this lowers the activation energy.     

Changes in lung volume and deflation stability in hyaline membrane disease

Total lung capacity (TLC), inspiratory capacity, functional residual capacity, and deflation stability of prematurely delivered Macaca nemestrina primates were measured serially during development of, and recovery from, hyaline membrane disease (HMD) to relate changes in lung volumes to changes in deflation stability. Gestational age-matched primates that did not develop HMD served as controls. TLC, measured by N2 washout, fell at 2-12 h of age (P less than 0.0001) in animals with HMD and remained lower than controls for at least 48 h (P less than 0.005). However, deflation stability, defined as the fraction of TLC remaining upon deflation to 10 cm H2O, improved from 2 to 12 h of age (P less than 0.001). Postmortem studies confirm the measurements of TLC and deflation stability and provide evidence that interstitial thickening and obstruction of air spaces with debris may be partially responsible for the observed changes in TLC in primates that develop HMD. It has been assumed that TLC is reduced in HMD because of atelectasis from elevated alveolar surface tension, but the sequential measurements in these animals suggest that other mechanisms also contribute.     

Mouse blastocyst immunosurgery with commercial antiserum to mouse erythrocytes

Summary Immunosurgery is a useful technique for the isolation of inner cell masses from murine blastocysts. Conventionally, rabbit antisera made ad hoc against murine splenic or fetal cells or fibroblasts have been used as antibody sources. We investigated the feasibility of using commercially available rabbit antiserum to murine erythrocytes (anti-RBC) and compared it with rabbit antiserum generated ad hoc to murine L-cells (anti-L-cell). Our results indicate that anti-RBC is at least as effective as anti-L-cell serum for the immunosurgical isolation of inner cell masses, which became either miniblastocysts (later forming outgrowths) or embryoid bodies (undergoing ectoderm-endodermlike differentiation within 48 h). Because anti-RBC is commercially available, the technical modification described herein increases the accessibility of the immunosurgical protocol for the isolation of murine inner cell masses.     

An improved enzymatic cycle for nicotinamide-adenine dinucleotide phosphate.

We describe the use of column chromatography on the nonpolar adsorbent. Amberlite XAD-2, and on silanized silica gel in the desalting and partial purification of cobalamins. These techniques are both simpler and more versatile than phenol extraction, without sacrificing efficiency. In addition, a solvent system for thin-layer chromatography on silanized silica gel is described which rapidly separates naturally occurring cobalamins.     

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     

LGR5 regulates osteogenic differentiation of human thoracic ligamentum flavum cells by Wnt signalling pathway

Thoracic ossification of the ligamentum flavum (TOLF) is ectopic ossification of the spinal ligaments. Histologically, the development of TOLF can be described as the process of endochondral ossification. However, the underlying aetiology has not been completely clarified. In this investigation, the gene expression profile associated with leucine‐rich repeat‐containing G‐protein‐coupled receptors (LGR) and Wnt signalling pathway in the thoracic ligamentum flavum cells (TLFCs) of different ossification stages was analysed via RNA sequencing. We further confirmed the significant differences in the related gene expression profile by Gene Ontology (GO) enrichment analysis. LGR5 was first identified in primary human TLFCs during osteogenic differentiation. To evaluate the effect of LGR5 on osteogenic differentiation, LGR5 has been knocked down and overexpressed in human TLFCs. We observed that the knockdown of LGR5 inhibited the activity of Wnt signalling and attenuated the potential osteogenic differentiation of TLFCs, while overexpression of LGR5 activated the Wnt signalling pathway and increased osteogenic differentiation. Our results provide important evidence for the potent positive mediatory effects of LGR5 on osteogenesis by enhancing the Wnt signalling pathway in TOLF.     

mTORC1-independent translation control in mammalian cells by methionine adenosyltransferase 2A and S-adenosylmethionine

Methionine adenosyltransferase (MAT) catalyzes the synthesis of S-adenosylmethionine (SAM). As the sole methyl-donor for methylation of DNA, RNA, and proteins, SAM levels affect gene expression by changing methylation patterns. Expression of MAT2A, the catalytic subunit of isozyme MAT2, is positively correlated with proliferation of cancer cells; however, how MAT2A promotes cell proliferation is largely unknown. Given that the protein synthesis is induced in proliferating cells and that RNA and protein components of translation machinery are methylated, we tested here whether MAT2 and SAM are coupled with protein synthesis. By measuring ongoing protein translation via puromycin labeling, we revealed that MAT2A depletion or chemical inhibition reduced protein synthesis in HeLa and Hepa1 cells. Furthermore, overexpression of MAT2A enhanced protein synthesis, indicating that SAM is limiting under normal culture conditions. In addition, MAT2 inhibition did not accompany reduction in mechanistic target of rapamycin complex 1 activity but nevertheless reduced polysome formation. Polysome-bound RNA sequencing revealed that MAT2 inhibition decreased translation efficiency of some fraction of mRNAs. MAT2A was also found to interact with the proteins involved in rRNA processing and ribosome biogenesis; depletion or inhibition of MAT2 reduced 18S rRNA processing. Finally, quantitative mass spectrometry revealed that some translation factors were dynamically methylated in response to the activity of MAT2A. These observations suggest that cells possess an mTOR-independent regulatory mechanism that tunes translation in response to the levels of SAM. Such a system may acclimate cells for survival when SAM synthesis is reduced, whereas it may support proliferation when SAM is sufficient.