We have shown that ouabain activates Src, resulting in subsequent tyrosine phosphorylation of multiple effectors. Here, we tested if the Na ؉ /K ؉ -ATPase and Src can form a functional signaling complex. In LLC-PK1 cells the Na ؉ /K ؉ -ATPase and Src colocalized in the plasma membrane. Fluorescence resonance energy transfer analysis indicated that both proteins were in close proximity, suggesting a direct interaction. GST pulldown assay showed a direct, ouabain-regulated, and multifocal interaction between the ␣1 subunit of Na ؉ /K ؉ -ATPase and Src. Although the interaction between the Src kinase domain and the third cytosolic domain (CD3) of ␣1 is regulated by ouabain, the Src SH3SH2 domain binds to the second cytosolic domain constitutively. Functionally, binding of Src to either the Na ؉ /K ؉ -ATPase or GST-CD3 inhibited Src activity. Addition of ouabain, but not vanadate, to the purified Na ؉ /K ؉ -ATPase/Src complex freed the kinase domain and restored the Src activity. Consistently, exposure of intact cells to ouabain apparently increased the distance between the Na ؉ /K ؉ -ATPase and Src. Concomitantly, it also stimulated tyrosine phosphorylation of the proteins that are associated with the Na ؉ /K ؉ -ATPase. These new findings illustrate a novel molecular mechanism of signal transduction involving the interaction of a P-type ATPase and a nonreceptor tyrosine kinase.

To explore the clinical value of immune-inflammatory markers to assess the severity of coronavirus disease 2019 . Methods: 127 consecutive hospitalized patients with confirmed COVID-19 were enrolled in this study, and classified into non-severe and severe groups. Demographics, symptoms, underlying diseases and laboratory data were collected and assessed for predictive value. Results: Of 127 COVID-19 patients, 16 cases (12.60%) were classified into the severe group. High level of interleukin-6 (IL-6), C-reaction protein (CRP) and hypertension were independent risk factors for the severity of COVID-19. The risk model based on IL-6, CRP and hypertension had the highest area under the receiver operator characteristic curve (AUROC). Additionally, the baseline IL-6 was positively correlated with other immune-inflammatory parameters and the dynamic change of IL-6 in the severe cases were parallel to the amelioration of the disease. Conclusion: Our study showed that high level of IL-6, CRP and hypertension were independent risk factors for assessing the severity of COVID-19. The risk model established upon IL-6, CRP and hypertension had the highest predictability in this study. Besides, IL-6 played a pivotal role in the severity of COVID-19 and had a potential value for monitoring the process of severe cases.

The Na+-K+--ATPase, or Na+ pump, is a member of the P-type ATPase superfamily. In addition to pumping ions, Na+-K+--ATPase is engaged in assembly of multiple protein complexes that transmit signals to different intracellular compartments. The signaling function of the enzyme appears to have been acquired through the evolutionary incorporation of many specific binding motifs that interact with proteins and ligands. In some cell types the signaling Na+ --ATPase and its protein partners are compartmentalized in coated pits (i.e., caveolae) the plasma membrane. Binding of ouabain to the signaling Na+-K+--ATPase activates the cytoplasmic tyrosine kinase Src, resulting in the formation of an active "binary receptor" that phosphorylates and assembles other proteins into different signaling modules. This in turn activates multiple protein kinase cascades including mitogen-activated protein kinases and protein kinase C isozymes in a cell-specific manner. It also increases mitochondrial production of reactive oxygen species (ROS)and regulates intracellular calcium concentration. Crosstalk among the activated pathways eventually results in changes in the expression of a number of genes. Although ouabain stimulates hypertrophic growth in cardiac myocytes and proliferation in smooth muscle cells, it also induces apoptosis in many malignant cells. Finally, the signaling function of the enzyme is also pivotal to ouabain-induced nongenomic effects on cardiac myocytes.

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Human hepatocellular carcinoma (HCC) is one of the most common cancers worldwide. In this work, we report on a comprehensive characterization of gene expression profiles of hepatitis B viruspositive HCC through the generation of a large set of 5-read expressed sequence tag (EST) clusters (11,065 in total) from HCC and noncancerous liver samples, which then were applied to a cDNA microarray system containing 12,393 genes͞ESTs and to comparison with a public database. The commercial cDNA microarray, which contains 1,176 known genes related to oncogenesis, was used also for profiling gene expression. Integrated data from the above approaches identified 2,253 genes͞ESTs as candidates with differential expression. A number of genes related to oncogenesis and hepatic function͞differentiation were selected for further semiquantitative reverse transcriptase-PCR analysis in 29 paired HCC͞noncancerous liver samples. Many genes involved in cell cycle regulation such as cyclins, cyclin-dependent kinases, and cell cycle negative regulators were deregulated in most patients with HCC. Aberrant expression of the Wnt-␤-catenin pathway and enzymes for DNA replication also could contribute to the pathogenesis of HCC. The alteration of transcription levels was noted in a large number of genes implicated in metabolism, whereas a profile change of others might represent a status of dedifferentiation of the malignant hepatocytes, both considered as potential markers of diagnostic value. Notably, the altered transcriptome profiles in HCC could be correlated to a number of chromosome regions with amplification or loss of heterozygosity, providing one of the underlying causes of the transcription anomaly of HCC.