Establishment of a monoclonal antibody for human LXRα: Detection of LXRα protein expression in human macrophages

Liver X activated receptor alpha (LXRα) forms a functional dimeric nuclear receptor with RXR that regulates the metabolism of several important lipids, including cholesterol and bile acids. As compared with RXR, the LXRα protein level in the cell is low and the LXRα protein itself is very hard to detect. We have previously reported that the mRNA for LXRα is highly expressed in human cultured macrophages. In order to confirm the presence of the LXRα protein in the human macrophage, we have established a monoclonal antibody against LXRα, K-8607. The binding of mAb K-8607 to the human LXRα protein was confirmed by a wide variety of different techniques, including immunoblotting, immunohistochemistry, and electrophoretic mobility shift assay (EMSA). By immunoblotting with this antibody, the presence of native LXR protein in primary cultured human macrophage was demonstrated, as was its absence in human monocytes. This monoclonal anti-LXRα antibody should prove to be a useful tool in the analysis of the human LXRα protein

Expression and localization of P1 promoter-driven hepatocyte nuclear factor-4α (HNF4α) isoforms in human and rats

BACKGROUND: Hepatocyte nuclear factor-4α (HNF4α; NR2A1) is an orphan member of the nuclear receptor superfamily involved in various processes that could influence endoderm development, glucose and lipid metabolism. A loss-of-function mutation in human HNF4α causes one form of diabetes mellitus called maturity-onset diabetes of the young type 1 (MODY1) which is characterized in part by a diminished insulin secretory response to glucose. The expression of HNF4α in a variety of tissues has been examined predominantly at the mRNA level, and there is little information regarding the cellular localization of the endogenous HNF4α protein, due, in part, to the limited availability of human HNF4α-specific antibodies. RESULTS: Monoclonal antibodies have been produced using baculovirus particles displaying gp64-HNF4α fusion proteins as the immunizing agent. The mouse anti-human HNF4α monoclonal antibody (K9218) generated against human HNF4α1/α2/α3 amino acids 3–49 was shown to recognize not only the transfected and expressed P1 promoter-driven HNF4α proteins, but also endogenous proteins. Western blot analysis with whole cell extracts from Hep G2, Huh7 and Caco-2 showed the expression of HNF4α protein, but HEK293 showed no expression of HNF4α protein. Nuclear-specific localization of the HNF4α protein was observed in the hepatocytes of liver cells, proximal tubular epithelial cells of kidney, and mucosal epithelial cells of small intestine and colon, but no HNF4α protein was detected in the stomach, pancreas, glomerulus, and distal and collecting tubular epithelial cells of kidney. The same tissue distribution of HNF4α protein was observed in humans and rats. Electron microscopic immunohistochemistry showed a chromatin-like localization of HNF4α in the liver and kidney. As in the immunohistochemical investigation using K9218, HNF4α mRNA was found to be localized primarily to liver, kidney, small intestine and colon by RT-PCR and GeneChip analysis. CONCLUSION: These results suggest that this method has the potential to produce valuable antibodies without the need for a protein purification step. Immunohistochemical studies indicate the tissue and subcellular specific localization of HNF4α and demonstrate the utility of K9218 for the detection of P1 promoter-driven HNF4α isoforms in humans and in several other mammalian species

Amino acid catabolite markers for early prognostication of pneumonia in patients with COVID-19

Abstract Effective early-stage markers for predicting which patients are at risk of developing SARS-CoV-2 infection have not been fully investigated. Here, we performed comprehensive serum metabolome analysis of a total of 83 patients from two cohorts to determine that the acceleration of amino acid catabolism within 5 days from disease onset correlated with future disease severity. Increased levels of de-aminated amino acid catabolites involved in the de novo nucleotide synthesis pathway were identified as early prognostic markers that correlated with the initial viral load. We further employed mice models of SARS-CoV2-MA10 and influenza infection to demonstrate that such de-amination of amino acids and de novo synthesis of nucleotides were associated with the abnormal proliferation of airway and vascular tissue cells in the lungs during the early stages of infection. Consequently, it can be concluded that lung parenchymal tissue remodeling in the early stages of respiratory viral infections induces systemic metabolic remodeling and that the associated key amino acid catabolites are valid predictors for excessive inflammatory response in later disease stages

The generation of monoclonal antibodies against human peroxisome proliferator-activated receptors (PPARs)

Monoclonal antibodies (Mabs) are valuable reagents for the purification, characterization and immunolocalization of proteins.In this study, we raised Mabs against human peroxisome proliferator-activated receptors (PPARs) using baculovirus particles displaying surface glycoprotein gp64-fusion proteins as the immunizing agent.In this system, to display fusion proteins on the viral surface, the amino terminal sequences of human PPARd and PPARg2 are inserted in-frame between the signal sequence and the mature domain of the gp64 nucleotide sequence.Mabs were raised by immunization with whole virus without a purification of the target antigens.The Mabs generated by this novel method were shown to recognize not only the gp64-PPARs fusion protein, but also mature, expressed proteins by a wide variety of techniques, including immunohistochemistry, immunoblotting, and electrophoretic mobility shift assays (EMSAs).Transfection of the transfer vector containing a nucleotide sequence encoding less than 30 amino acids along with linearized baculovirus DNA allows for the production of a high affinity antibody against the corresponding mature form.This method is of potential utility in that it allows the production of valuable antibodies without the requirement of a protein purification step