Cys-loop receptors are pentameric ligand-gated ion channels (pLGICs) that bind neurotransmitters to open an intrinsic transmembrane ion channel pore. The recent crystal structure of a prokaryotic pLGIC from the cyanobacterium Gloeobacter violaceus (GLIC) revealed that it naturally lacks an N-terminal extracellular ␣ helix and an intracellular domain that are typical of eukaryotic pLGICs. GLIC does not respond to neurotransmitters acting at eukaryotic pLGICs but is activated by protons. To determine whether the structural differences account for functional differences, we used a eukaryotic chimeric acetylcholineglutamate pLGIC that was modified to carry deletions corresponding to the sequences missing in the prokaryotic homolog GLIC. Deletions made in the N-terminal extracellular ␣ helix did not prevent the expression of receptor subunits and the appearance of receptor assemblies on the cell surface but abolished the capability of the receptor to bind ␣-bungarotoxin (a competitive antagonist) and to respond to the neurotransmitter. Other truncated chimeric receptors that lacked the intracellular domain did bind ligands; displayed robust acetylcholine-elicited responses; and shared with the full-length chimeric receptor similar anionic selectivity, effective open pore diameter, and unitary conductance. We suggest that the integrity of the N-terminal ␣ helix is crucial for ligand accommodation because it stabilizes the intersubunit interfaces adjacent to the neurotransmitter-binding pocket(s). We also conclude that the intracellular domain of the chimeric acetylcholine-glutamate receptor does not modulate the ion channel conductance and is not involved in positioning of the pore-lining helices in the conformation necessary for coordinating a Cl ؊ ion within the intracellular vestibule of the ion channel pore.Cys-loop receptors constitute a superfamily of cell surface oligomers that bind neurotransmitters such as acetylcholine (ACh), 3 serotonin (5-hydroxytryptamine (5HT)), ␥-aminobutyric acid (GABA), glycine (Gly), glutamate (Glu), and histamine to open an intrinsic transmembrane ion channel (for reviews, see Refs. 1-8). Hence, Cys-loop receptors mediate the rapid flow of ions such as Na ϩ , K ϩ , Ca 2ϩ , and Cl Ϫ across the cell membrane down their electrochemical gradients to alter the membrane potential or enable Ca 2ϩ influxes and signaling. Cys-loop receptors are known also as pentameric ligand-gated ion channels (pLGICs). The subunits of pLGICs are radially aligned around an axis of 5-fold symmetry, which is the axis of the ion permeation pathway. Each subunit traverses the membrane four times. Upon receptor assembly, the N-terminal extracellular segment forms a ligand-binding domain (LigBD) having neurotransmitterbinding pockets at intersubunit interfaces. The ion channel domain is formed by 20 transmembrane helices, M1-M4 of each subunit (see Fig. 1, A and B, numbered 1-4). All eukaryotic pLGICs have a long intracellular sequence that connects M3 with M4. This sequence (the so-called M3-M4 loop or linker) f...
SummaryCancer stem cell (CSC) identification relies on transplantation assays of cell subpopulations sorted from fresh tumor samples. Here, we attempt to bypass limitations of abundant tumor source and predetermined immune selection by in vivo propagating patient-derived xenografts (PDX) from human malignant rhabdoid tumor (MRT), a rare and lethal pediatric neoplasm, to an advanced state in which most cells behave as CSCs. Stemness is then probed by comparative transcriptomics of serial PDXs generating a gene signature of epithelial to mesenchymal transition, invasion/motility, metastasis, and self-renewal, pinpointing putative MRT CSC markers. The relevance of these putative CSC molecules is analyzed by sorting tumorigenic fractions from early-passaged PDX according to one such molecule, deciphering expression in archived primary tumors, and testing the effects of CSC molecule inhibition on MRT growth. Using this platform, we identify ALDH1 and lysyl oxidase (LOX) as relevant targets and provide a larger framework for target and drug discovery in rare pediatric cancers.
Myelinogenesis in the mammal nervous system occurs predominantly postnatally. Glatiramer acetate (GA), a drug for the treatment for multiple sclerosis (MS), has been shown to induce immunomodulation and neuroprotection in the inflamed CNS in MS and in experimental autoimmune encephalomyelitis (EAE). Here we investigated whether GA can affect myelinogenesis and oligodendrogenesis in the developing nervous system under nonpathological conditions. Towards this end we studied myelination in mice injected daily by GA, at postnatal Days 7-21. Immunohistological and ultrastructural analyses revealed significant elevation in the number of myelinated axons as well as in the thickness of the myelin encircling them and their resulting g-ratios, in spinal cords of GA-injected mice compared with their PBS-injected littermates, at postnatal Day 14. Elevation in myelinated axons was detected also in the peripheral ventral roots of the motor nerves. GA induced also an increase in axonal diameter, implying an effect on the overall development of the nervous system. A prominent elevation in the amount of progenitor oligodendrocytes and their BrdU incorporation, as well as in mature oligodendrocytes indicated that the effect of GA is linked to increased proliferation and differentiation along the oligodendroglial maturation cascade. In addition, elevation in insulin-like growth factor (IGF-1) and brain-derived neurotrophic factor (BDNF) was found in the white matter of the GA-injected mice. Furthermore, a functional advantage in rotating rod test was exhibited by GA-injected mice over their littermates at postnatal Day 21. These cumulative findings corroborate the beneficial effect of GA on oligodendrogenesis and myelination.
The severe motor impairment in the MS animal model experimental autoimmune encephalomyelitis (EAE) obstructs the assessment of cognitive functions. We developed an experimental system that evaluates memory faculties in EAE-affected mice, irrespective of their motor performance, enabling the assessment of cognitive impairments along the disease duration, the associated brain damage, and the consequences of glatiramer acetate (GA) treatment on these manifestations. The delayed-non-matching to sample (DNMS) T-maze task, testing working and long term memory was adapted and utilized. Following the appearance of clinical manifestations task performances of the EAE-untreated mice drastically declined. Cognitive impairments were associated with disease severity, as indicated by a significant correlation between the T-maze performance and the clinical symptoms in EAE-untreated mice. GA-treatment conserved cognitive functions, so that despite their exhibited mild motor impairments, the treated mice performed similarly to naïve controls. The cognitive deficit of EAE-mice coincided with inflammatory and neurodegenerative damage to the frontal cortex and the hippocampus; these damages were alleviated by GA-treatment. These combined findings indicate that in addition to motor impairment, EAE leads to substantial impairment of cognitive functions, starting at the early stages and increasing with disease aggravation. GA-treatment, conserves cognitive capacities and prevents its disease related deterioration.
Angiomyolipoma (AML), the most common benign renal tumor, can result in severe morbidity from hemorrhage and renal failure. While mTORC1 activation is involved in its growth, mTORC1 inhibitors fail to eradicate AML, highlighting the need for new therapies. Moreover, the identity of the AML cell of origin is obscure. AML research, however, is hampered by the lack of in vivo models. Here, we establish a human AML‐xenograft (Xn) model in mice, recapitulating AML at the histological and molecular levels. Microarray analysis demonstrated tumor growth in vivo to involve robust PPARG‐pathway activation. Similarly, immunostaining revealed strong PPARG expression in human AML specimens. Accordingly, we demonstrate that while PPARG agonism accelerates AML growth, PPARG antagonism is inhibitory, strongly suppressing AML proliferation and tumor‐initiating capacity, via a TGFB‐mediated inhibition of PDGFB and CTGF. Finally, we show striking similarity between AML cell lines and mesenchymal stem cells (MSCs) in terms of antigen and gene expression and differentiation potential. Altogether, we establish the first in vivo human AML model, which provides evidence that AML may originate in a PPARG‐activated renal MSC lineage that is skewed toward adipocytes and smooth muscle and away from osteoblasts, and uncover PPARG as a regulator of AML growth, which could serve as an attractive therapeutic target.
The neuromuscular acetylcholine receptor (AChR) alternatively switches between an inactive (closed) and active (open) conformation. during the gating isomerization (R4R*) process. The 'gate' region is formed by the equatorial residues (9'-16'). We and others have speculated that channel gating involves
Pleuropulmonary blastoma (PPB) is a rare pediatric lung neoplasm that recapitulates developmental pathways of early embryonic lungs. As lung development proceeds with highly regulated mesenchymal-epithelial interactions, a DICER1 mutation in PPB generates a faulty lung differentiation program with resultant biphasic tumors composed of a primitive epithelial and mesenchymal stroma with early progenitor blastomatous cells. Deciphering of PPB progression has been hampered by the difficulty of culturing PPB cells, and specifically progenitor blastomatous cells. Here, we show that in contrast with in-vitro culture, establishment of PPB patient-derived xenograft (PDX) in NOD-SCID mice selects for highly proliferating progenitor blastoma overexpressing critical regulators of lung development and multiple imprinted genes. These stem-like tumors were sequentially interrogated by gene profiling to show a FGF module that is activated alongside Neural cell adhesion molecule 1 (NCAM1). Targeting the progenitor blastoma and these transitions with an anti-NCAM1 immunoconjugate (Lorvotuzumab mertansine) inhibited tumor growth and progression providing new paradigms for PPB therapeutics. Altogether, our novel in-vivo PPB xenograft model allowed us to enrich for highly proliferating stem-like cells and to identify FGFR and NCAM1 as two key players that can serve as therapeutic targets in this poorly understood and aggressive disease.
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