Regulation of the sodium/sulfate co-transporter by farnesoid X receptor alpha

Fxralpha is known to regulate a variety of metabolic processes, including bile acid, cholesterol, and carbohydrate metabolism. In this study, we show direct evidence that Fxralpha is a key player in maintaining sulfate homeostasis. We identified and characterized the sodium/sulfate co-transporter (NaS-1; Slc13a1) as an Fxralpha target gene expressed in the kidney and intestine. Electromobility shift assays, chromatin immunoprecipitation, and promoter reporter studies identified a single functional Fxralpha response element in the second intron of the mouse Slc13a1 gene. Treatment of wild-type mice with GW4064, a synthetic Fxralpha agonist, induced Slc13a1 mRNA in the intestine and kidney. Slc13a1 mRNA was also induced in the kidney and intestine of wild-type, but not Fxralpha-/- mice, after treatment with the hepatotoxin alpha-naphthylisothiocyanate, which is known to result in elevated blood bile acid levels. Finally, we observed a decrease in Slc13a1 mRNA in the kidney and intestine of Fxralpha-/- mice and a corresponding increase in urinary excretion of free sulfates as compared with wild-type mice. These results demonstrate that mouse Slc13a1 is a novel Fxralpha target gene expressed in the kidney and intestine and that in the absence of Fxralpha, mice waste sulfate into the urine. Thus, Fxralpha is necessary for normal sulfate homeostasis in vivo.     

Control of Echinothrips americanus on lima bean by Franklinothrips vespiformis crawford using supplemental food

BioControl - Supplemental food application to crops as a resource for generalist predators has shown utility for promoting their establishment and persistence leading to enhanced biological control...     

Transcriptomic analysis of IgG4 Fc-fusion protein degradation in a panel of clonally-derived CHO cell lines using RNASeq

In this study, we report an investigation of a panel of clonally-derived Chinese hamster ovary (CHO) cell lines exhibiting variability in the proportion of full-length IgG4 Fc-fusion protein produced. The recombinant protein was found to be degraded during cell culture into four shorter “clipped” species (three of the four cleavage sites occurred at arginine residues) and preliminary analyses suggested that a host cell enzyme was responsible for proteolysis. To identify the specific enzyme responsible, RNA sequencing was used to identify gene expression differences between the cell lines with a “high” and “low” clipping phenotype. From this analysis, six protease-encoding genes were found to be significantly upregulated in those cell lines yielding the lowest proportion of full-length IgG4 Fc-fusion protein. Four of these protease candidates were deprioritized after examination of their cleavage site specificity. The remaining enzymes, Adam19 and Furin, were found to be capable of cleavage at arginine residues, and inhibitors for both proteases were added to cell-free media to determine if the product degradation could be reduced. While the Adam19 inhibitor had no impact, Furin inhibitor I (specific for the proprotein convertase family of enzymes) was found to result in a 33–39% increase in complete IgG4 Fc-fusion protein when compared with untreated samples.     

68Ga-PSMA-HBED-CC PET/MRI is superior to multiparametric magnetic resonance imaging in men with biochemical recurrent prostate cancer: A prospective single-institutional study

BackgroundThe primary objective was to compare the overall diagnostic performance, presented as detection rate of ⁶⁸Ga-PSMA-HBED-CC positron emission tomography/magnetic resonance imaging (PSMA PET/MRI) versus conventional, multiparametric MRI (mpMRI) in a population of patients with biochemically recurrent prostate cancer. In conjunction with this analysis, secondary objectives included the evaluation of the detection rate stratified by PSA levels and primary treatment modality.MethodsA total of 165 PSMA PET MRI were performed from April 2018 to May 2021, of whom 108 were presenting for biochemical recurrent disease. The PSMA PET vertex to thigh were read by two different board-certified nuclear medicine physicians while the MRI head and neck, chest, abdomen, and pelvis (with dedicated, PI-RADS compliant multiparametric prostate MRI) were read by two board certified diagnostic radiologists.AnalysisPSMA PET/MRI had a higher detection rate than mpMRI when evaluating patients with biochemical recurrence (BCR) with similar results demonstrated when sub-analysis was performed using PSA levels, primary treatment modality, and time since androgen deprivation therapy. Our study also showed PSMA PET/MRI had a higher sensitivity than mpMRI.DiscussionOur findings demonstrate that PSMA PET/MRI is a better imaging modality in the detection of disease in the setting of BCR when compared to MRI alone. Combined utility with PSMA PET/MRI is a powerful tool which can aid in not only the detection of disease, but also guide in treatment planning for prostate cancer patients.     

Evidence for the presence of cholinergic muscarinic receptors negatively linked to adenylate cyclase in the iris-ciliary body

Adenylate cyclase activity can be stimulated in the rabbit iris-ciliary body directly by forskolin or through receptor-mediated mechanisms by vasoactive intestinal peptide (VIP) and the β-adrenoreceptor agonists isoproterenol and salbutamol. Increases in the level of c-AMP observed following application of forskolin, isoproterenol and VIP are decreased by carbachol in a dose-dependent manner. The carbachol response is blocked by pertussis toxin and is insensitive to the phosphodiesterase inhibitor theophyline suggesting the involvement of a Gi-protein. Carbachol attenuation of elevated c-AMP levels can be inhibited by the muscarinic antagonist atropine but not by the specific muscarinic receptor antagonist pirenzepine. This is in contrast to carbachol stimulation of inositol phosphate accumulation, where both atropine and pirenzepine inhibit the muscarinic response. Thus there exist two distinct muscarinic receptors in the iris-ciliary body, one linked to adenylate cyclase and the other to the hydrolysis of phosphoinositides.     

Cu XAS shows a change in the ligation of CuB upon reduction of cytochrome bo3 from Escherichia coli

Copper X-ray absorption spectroscopy (XAS) has been used to examine the structures of the Cu(II) and Cu(I) forms of the cytochrome bo3 quinol oxidase from Escherichia coli. Cytochrome bo3 is a member of the superfamily of heme-copper respiratory oxidases. Of particular interest is the fact that these enzymes function as redox-linked proton pumps, resulting in the net translocation of one H+ per electron across the membrane. The molecular mechanism of how this pump operates and the manner by which it is linked to the oxygen chemistry at the active site of the enzyme are unknown. Several proposals have featured changes in the coordination of CuB during enzyme turnover that would result in sequential protonation or deprotonation events that are key to the functioning proton pump. This would imply lability of the ligands to CuB. In this work, the structure of the protein in the immediate vicinity of CuB, in both the fully oxidized and fully reduced forms of the enzyme, has been examined by Cu XAS, a technique that is particularly sensitive to changes in metal coordination. The results show that in the oxidized enzyme, CuB(II) is four-coordinate, consistent with three imidazoles and one hydroxyl (or water). Upon reduction of the enzyme, the coordination of CuB(I) is significantly altered, consistent with the loss of one of the histidine imidazole ligands in at least a substantial fraction of the population. These data add to the credibility that changes in the ligation of CuB might occur during catalytic turnover of the enzyme and, therefore, could, in principle, be part of the mechanism of proton pumping.     

A novel method for analysis of nuclear receptor function at natural promoters: peroxisome proliferator-activated receptor gamma agonist actions on aP2 gene expression detected using branched DNA messenger RNA quantitation

Peroxisome proliferator-activated receptor-gamma (PPARgamma), a member of the nuclear hormone receptor superfamily, plays an essential role in the mediation of the actions of antidiabetic drugs known as thiazolidinediones (TZDs). PPARgamma activates many target genes involved in lipid anabolism including the adipocyte fatty acid binding protein (aP2). In this study, induction of aP2 gene expression by PPARgamma agonists was examined in both cultured cells and diabetic mice using branched DNA (bDNA)-mediated mRNA quantitation. bDNA technology allows for the direct measurement of a particular mRNA directly within cellular lysate using a 96-well plate format in a time frame comparable to a reporter gene assay. In cultured human subcutaneous preadipocytes, the TZDs, troglitazone and BRL-49653, both rapidly induced aP2 mRNA as detected with the bDNA method. In these cells, the effect of BRL-49653 on aP2 mRNA levels was detectable as early as 30 min after treatment (47% increase) and was maximal after 24 h of treatment (12-fold increase). The effects of troglitazone on aP2 mRNA induction were similar to those of BRL-49653 except that the maximal level of induction was consistently lower (e.g. 24 h treatment = 4-fold increase). Dose-response relationships for both of the TZDs were also determined using the 24-h treatment time point. EC50s for both BRL-49653 and troglitazone were estimated to be 80 nM and 690 nM, respectively. A natural PPARgamma ligand, 15-deoxy-delta12,14-PGJ2, was also active in this assay with a maximal induction of aP2 mRNA of approximately 5-fold when tested at 1 microM. Since the PPARgamma:retinoid X receptor (RXR) heterodimer has been characterized as a permissive heterodimer with respect to RXR ligands, the ability of 9-cis-retinoic acid (9-cis-RA) to induce aP2 mRNA was examined. Although 9-cis-RA had very low efficacy (2-fold induction), the maximal effect was reached at 100 nM. No synergism or additivity in aP2 mRNA induction was detected when 9-cis-RA was included with either of the TZDs used in this study. Significant induction of aP2 mRNA in bone marrow of db/db mice treated with either troglitazone or BRL-49653 was also detected, indicating that the bDNA assay may be a simple method to monitor nuclear receptor target gene induction in vivo.