Notes

Visualization involving helical buildings of poly(diphenylacetylene)utes having chiral amide jewellery by fischer force microscopy.
5 pg/ml), and successfully employed this assay in the quantification of extracellular β-1,6-glucan released by >250 patient-derived strains of different Candida species (including Candida auris) in culture supernatant in vitro. We also used this assay to measure β-1,6-glucan in vivo in the serum and several organs in a mouse model of systemic candidiasis. Our work describes a reliable method for β-1,6-glucan detection, which may prove useful for the diagnosis of invasive fungal infections. Published under license by The American Society for Biochemistry and Molecular Biology, Inc.Increased plasma concentrations of lipoprotein(a) (Lp(a)) are associated with an increased risk for cardiovascular disease. Lp(a) is composed of apolipoprotein(a) (apo(a)) covalently bound to apolipoprotein B of low-density lipoprotein (LDL). Many of apo(a)'s potential pathological properties, such as inhibition of plasmin generation, have been attributed to its main structural domains, the kringles, and have been proposed to be mediated by their lysine-binding sites. However, available small-molecule inhibitors, such as lysine analogs, bind unselectively to kringle domains and are therefore unsuitable for functional characterization of specific kringle domains. Here, we discovered small molecules that specifically bind to the apo(a) kringle domains KIV-7, KIV-10, and KV. Chemical synthesis yielded compound AZ-05, which bound to KIV-10 with a Kd of 0.8 μM and exhibited more than 100-fold selectivity for KIV-10, compared with the other kringle domains tested, including plasminogen kringle 1. To better understand and further improve ligand selectivity, we determined the crystal structures of KIV-7, KIV-10, and KV in complex with small-molecule ligands at 1.6-2.1 Å resolutions. Furthermore, we used these small molecules as chemical probes to characterize the roles of the different apo(a) kringle domains in in vitro assays. These assays revealed the assembly of Lp(a) from apo(a) and LDL, as well as potential pathophysiological mechanisms of Lp(a), including (i) binding to fibrin, (ii) stimulation of smooth-muscle cell proliferation, and (iii) stimulation of LDL uptake into differentiated monocytes. Our results indicate that a small-molecule inhibitor targeting the lysine-binding site of KIV-10 can combat the pathophysiological effects of Lp(a). Published under license by The American Society for Biochemistry and Molecular Biology, Inc.Obesity and elevation of circulating free fatty acids are associated with an accumulation and pro-inflammatory polarization of macrophages within metabolically active tissues such as adipose tissue, muscle, liver, and pancreas. Beyond macrophages, neutrophils also accumulate in adipose and muscle tissues during high fat diets and contribute to a state of local inflammation and insulin resistance. However, the mechanisms by which neutrophils are recruited to these tissues are largely unknown. Here, we used a cell culture system as proof-of-concept to show that upon exposure to a saturated fatty acid, palmitate, macrophages release nucleotides that attract neutrophils. Moreover, we find that palmitate upregulates pannexin-1 channels in macrophages that mediate the attraction of neutrophils, previously shown to allow transfer of nucleotides across membranes. These findings suggest that pro-inflammatory macrophages release nucleotides through pannexin-1, a process that may facilitate neutrophil recruitment into metabolic tissues during obesity. Published under license by The American Society for Biochemistry and Molecular Biology, Inc.Following its evoked release, DA signaling is rapidly terminated by presynaptic reuptake, mediated by the cocaine-sensitive DAT. DAT surface availability is dynamically regulated by endocytic trafficking, and direct PKC activation acutely diminishes DAT surface expression by accelerating DAT internalization. Previous cell line studies demonstrated that PKC-stimulated DAT endocytosis requires both Ack1 inactivation, which releases a DAT-specific endocytic brake, and the neuronal GTPase, Rit2, which binds DAT. However, it is unknown whether Rit2 is required for PKC-stimulated DAT endocytosis in DAergic terminals, or whether there are region- and/or sex-dependent differences in PKC-stimulated DAT trafficking. Moreover, the mechanisms by which Rit2 controls PKC-stimulated DAT endocytosis are unknown. Here, we directly examined these important questions. Ex vivo studies revealed that PKC activation acutely decreased DAT surface expression selectively in ventral, but not dorsal, striatum. AAV-mediated, conditional Rit2 knockdown in DAergic neurons impacted baseline DAT surfaceintracellular distribution in DAergic terminals from female ventral, but not dorsal, striatum. this website Further, Rit2 was required for PKC-stimulated DAT internalization in both male and female ventral striatum. FRET and surface pulldown studies in cell lines revealed that PKC activation drives DAT-Rit2 surface dissociation, and that the DAT N-terminus is required for both PKC-mediated DAT-Rit2 dissociation and DAT internalization. Finally, we found that Rit2 and Ack1 independently converge on DAT to facilitate PKC-stimulated DAT endocytosis. Together, our data provide greater insight into mechanisms that mediate PKC-regulated DAT internalization, and reveal unexpected region-specific differences in PKC-stimulated DAT trafficking in bona fide DAergic terminals. Published under license by The American Society for Biochemistry and Molecular Biology, Inc.A homozygous mutation in growth hormone 1 (GH1) was recently identified in an individual with growth failure. This mutation, c.705G>C causes the replacement of cysteine at position 53 of the 191 amino acid sequence of 22 kDa human GH (hGH) with serine (p.Cys53Ser). This hGH molecule (further referred to as GH-C53S) lacks the disulfide bond between p.Cys53 and p.Cys165, which is highly conserved among species. It has been previously reported that monomeric GH-C53S has reduced bioactivity compared with wild-type GH (GH-wt) because of its decreased ability to bind and activate the GH receptor in vitro. In this study, we discovered that the substitution of p.Cys53 in hGH significantly increased formation of hGH-dimers in pituitary cells. We expressed his-tagged hGH variants in the cytoplasm of genetically modified Rosetta gami B DE3 Escherichia coli cells, facilitating high yield production. We observed that the bioactivity of monomeric GH-C53S is 25.2% of that of wild-type GH and that dimeric GH-C53S-his has no significant bioactivity in cell proliferation assays.
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