Hickeydougherty3748

We report here a method for the determination of the pKa of histidine in complex or heterogeneous systems amenable to neither solid-state nor solution NMR spectroscopy. Careful synthesis of a fluorenylmethyloxycarbonyl- and trityl-protected, C2-deuterated histidine produces a vibrational-probe-equipped amino acid that can readily be incorporated into any peptide accessible by standard solid-phase methods. The frequency of the unique, Raman-active stretching vibration of this C2-D probe is a clear reporter of the protonation state of histidine. We investigate here a pH-sensitive peptide that self-assembles to form a hydrogel at neutral pH. The pKa of the lone histidine residue in the peptide, which is likely responsible for this pH-dependent behavior, cannot be investigated by NMR spectroscopy because of the supramolecular, soft nature of the gel. However, after synthesizing a C2-deuterated-histidine-containing peptide, we were able to follow the protonation state of histidine throughout a pH titration using Raman difference spectroscopy, thereby precisely determining the pKa of interest.After surgery or traumatic injury, corneal wound healing can cause a scarring response that stiffens the tissue and impairs ocular function. This fibrosis is caused in part by the activation of corneal keratocytes from a native mechanically quiescent state to an activated myofibroblastic state. This transformation is tied to signaling downstream of transforming growth factor-β1 (TGF-β1). Here, to better understand how biochemical and biophysical cues interact to regulate keratocyte activation and contractility, we cultured primary rabbit corneal keratocytes on flexible substrata of varying stiffness in the presence (or absence) of TGF-β1. Time-lapse fluorescence microscopy was used to assess changes in keratocyte morphology, as well as to quantify the dynamic traction stresses exerted by cells under different experimental conditions. In other experiments, keratocytes were fixed after 5 days of culture and stained for markers of both contractility and myofibroblastic activation. Treatment with TGF-β1 elicited ss can modulate the morphology, cytoskeletal organization, and subcellular pattern of force generation in corneal keratocytes treated with TGF-β1.Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is a recently identified coronavirus that causes the respiratory disease known as coronavirus disease 2019 (COVID-19). Despite the urgent need, we still do not fully understand the molecular basis of SARS-CoV-2 pathogenesis. Here, we comprehensively define the interactions between SARS-CoV-2 proteins and human RNAs. NSP16 binds to the mRNA recognition domains of the U1 and U2 splicing RNAs and acts to suppress global mRNA splicing upon SARS-CoV-2 infection. NSP1 binds to 18S ribosomal RNA in the mRNA entry channel of the ribosome and leads to global inhibition of mRNA translation upon infection. Finally, NSP8 and NSP9 bind to the 7SL RNA in the signal recognition particle and interfere with protein trafficking to the cell membrane upon infection. Disruption of each of these essential cellular functions acts to suppress the interferon response to viral infection. Our results uncover a multipronged strategy utilized by SARS-CoV-2 to antagonize essential cellular processes to suppress host defenses.Childhood obesity, especially in girls, is frequently bound to earlier puberty, which is linked to higher disease burden later in life. The mechanisms underlying this association remain elusive. Here we show that brain ceramides participate in the control of female puberty and contribute to its alteration in early-onset obesity in rats. Postnatal overweight caused earlier puberty and increased hypothalamic ceramide content, while pharmacological activation of ceramide synthesis mimicked the pubertal advancement caused by obesity, specifically in females. Conversely, central blockade of de novo ceramide synthesis delayed puberty and prevented the effects of the puberty-activating signal, kisspeptin. This phenomenon seemingly involves a circuit encompassing the paraventricular nucleus (PVN) and ovarian sympathetic innervation. Early-onset obesity enhanced PVN expression of SPTLC1, a key enzyme for ceramide synthesis, and advanced the maturation of the ovarian noradrenergic system. In turn, obesity-induced pubertal precocity was reversed by virogenetic suppression of SPTLC1 in the PVN. Our data unveil a pathway, linking kisspeptin, PVN ceramides, and sympathetic ovarian innervation, as key for obesity-induced pubertal precocity.cis-9,10-Epoxy-octadecanoic acid (oleic acid epoxide, OAE) and 5α,6α-epoxy-cholesterol (ChE) are endogenous epoxides. Unlike other epoxides, the oxirane groups of OAE and ChE are relatively stable against nucleophiles. OAE lacks toxicity and mutagenicity, while ChE is considered harmful, mutagenic and cancerogenic to animals. In humans, ChE is associated with cancer. The metabolism of OAE and ChE includes hydrolysis by cytosolic and microsomal hydrolases to their diols and glutathione (GSH) conjugation by GSH S-transferases (GST) to form the GSH conjugates (R-SG; R, residue). The GST-catalyzed GSH conjugation of OAE and ChE is poorly investigated. This article reports on the GSH conjugation of OAE, its methyl ester (OAEMe) and of ChE by rat liver homogenate GST. The GSH conjugates of OAE, OAEMe and ChE, i.e., OAE-SG, OAEMe-SG and ChE-SG, respectively, were determined by pre-column derivatization with o-phthaldialdehyde (OPA)/2-mercaptoethanol, high-performance liquid chromatography (HPLC) and fluorescence detection. Complex biphasic kinetics were observed with substrate inhibition of GST activity by OAE, OAEMe and ChE, an optimum pH of about 8.3 for OAE, and no measurable chemical GSH conjugation, underlying the importance of GST for the biotransformation of these epoxides. The results confirm the substrate concentration-dependent kinetic mechanism of GST isoforms first reported by William B. Jakoby (J. Biol. Chem. 1974) for exogenous electrophiles including the epoxide 1,2-epoxy-3-(p-nitrophenoxy)propane and the organic nitrates. read more This mechanism allows for maximal GST activity that can be achieved under given concentrations of GSH, epoxides and other electrophiles.