Botulinum neurotoxins (BoNTs) are used in a wide variety of medical applications, but there is limited pharmacokinetic data on active BoNT. Monitoring BoNT activity in the circulation is challenging because BoNTs are highly toxic and are rapidly taken up by neurons and removed from the bloodstream. Previously we reported a sensitive BoNT "Assay with a Large Immunosorbent Surface Area" that uses conversion of fluorogenic peptide substrates to measure the intrinsic endopeptidase activity of bead-captured BoNT. However, in complex biological samples, protease contaminants can also cleave the substrates, reducing sensitivity and specificity of the assay. Here, we present a novel set of fluorogenic peptides that serve as BoNT-specific substrates and protease-sensitive controls. BoNT-cleavable substrates contain a C-terminal Nle, while BoNT-noncleavable controls contain its isomer ε-Ahx. The substrates are cleaved by BoNT subtypes A1-A3 and A5. Substrates and control peptides can be cleaved by non-BoNT proteases (e.g., trypsin, proteinase K, and thermolysin) while obeying Michaelis-Menten kinetics. Using this novel substrate/control set, we studied BoNT/A1 activity in two mouse models of botulism. We detected BoNT/A serum activities ranging from ~3600 to 10 amol/L in blood of mice that had been intravenously injected 1 h prior with BoNT/A1 complex (100 to 4 pg/mouse). We also detected the endopeptidase activity of orally administered BoNT/A1 complex (1 μg) in blood 5 h after administration; activity was greatest 7 h after administration. Redistribution and elevation rates for active toxin were measured and are comparable to those reported for inactive toxin.
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http://dx.doi.org/10.1021/ac4008418 | DOI Listing |
Vet Res Commun
January 2025
Biochemistry, Veterinary Biosciences Department, Veterinary Faculty, Universidad de la República, Ruta 8, Km 18 y Ruta 102, Montevideo, 13000, Uruguay.
The aim was to study the effect of long-acting analogue of oxytocin (Carbetocin) on cervical collagenolysis of MAP-eCG synchronized ewes. At the expected time of artificial insemination, five ewes were slaughtered (n = 5) and their cervical explants (100-200 mg) were incubated during 12 h with MEM supplemented with 0, 8, 16, 32 and 64 ng/mL of Cb. Activities of activated and latent forms of matrix metalloproteinases-2 and - 9 (MMP-2 and MMP-9, respectively) in the supernatant were determined by a SDS-PAGE zymography and prostaglandin E2 concentration by immunoassay.
View Article and Find Full Text PDFMol Biol Rep
January 2025
College of Life Sciences, Liaoning Normal University, Dalian, Liaoning, 116029, China.
Background: High temperature is a critical environmental factor leading to mass mortality in oyster aquaculture in China. Recent advancements highlight the physiological regulation function of γ-aminobutyric acid (GABA) in the adaptation of environmental stress.
Methods And Results: This study examined the physiological responses of the Pacific oyster (Crassostrea gigas) upon high temperature exposure, focusing on the histopathological changes in gill, the GABA concentration, the mRNA expression and activities of apoptosis-related genes.
Acta Neuropathol
January 2025
Department of Clinical Sciences, Lund Brain Injury Laboratory for Neurosurgical Research, Lund University, 222 20, Lund, Sweden.
Traumatic brain injury (TBI) often leads to impaired regulation of cerebral blood flow, which may be caused by pathological changes of the vascular smooth muscle cells (VSMCs) in the arterial wall. Moreover, these cerebrovascular changes may contribute to the development of various neurodegenerative disorders such as Alzheimer's-like pathologies that include amyloid beta aggregation. Despite its importance, the pathophysiological mechanisms responsible for VSMC dysfunction after TBI have rarely been evaluated.
View Article and Find Full Text PDFProc Natl Acad Sci U S A
January 2025
Department of Biological Sciences, College of Natural Sciences, Sungkyunkwan University, Suwon 16419, Republic of Korea.
Bacterial cell wall assembly and remodeling require activities of peptidoglycan (PG) hydrolases as well as PG synthases. In particular, the activity of DD-endopeptidases, which cleave the 4-3 peptide crosslinks in PG, is essential for PG expansion in gram-negative bacteria. Maintaining optimal levels of DD-endopeptidases is critical for expanding PG without compromising its integrity.
View Article and Find Full Text PDFMalar J
January 2025
Department of Pharmacology, Kangwon National University School of Medicine, Chuncheon, 24341, Republic of Korea.
Background: The Plasmodium proteasome emerges as a promising target for anti-malarial drug development due to its potential activity against multiple life cycle stages.
Methods: In this investigation, a comparative analysis was conducted on the structural features of the β5 subunit in the 20S proteasomes of both Plasmodium and humans.
Results: The findings underscore the structural diversity inherent in both proteasomes.
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