Effective inhibition of protein aggregation is a major goal in biopharmaceutical production processes optimized for product quality. To examine the characteristics of process-stress-dependent aggregation of human granulocyte colony-stimulating factor (G-CSF), we applied controlled stirring and bubble aeration to a recombinant non-glycosylated preparation of the protein produced in Escherichia coli. We characterized the resulting denaturation in a time-resolved manner using probes for G-CSF conformation and size in both solution and the precipitate. G-CSF was precipitated rapidly from solutions that were aerated or stirred; only small amounts of soluble aggregates were found. Exposed hydrophobic surfaces were a characteristic of both soluble and insoluble G-CSF aggregates. Using confocal laser scanning microscopy, the aggregates presented mainly a circular shape. Their size varied according to incubation time and stress applied. The native intramolecular disulfide bonds in the insoluble G-CSF aggregates were largely disrupted as shown by mass spectrometry. New disulfide bonds formed during aggregation. All involved Cys(18) , which is the only free cysteine in G-CSF; one of them had an intermolecular Cys(18(A)) -Cys(18(B)) crosslink. Stabilization strategies can involve external addition of thiols and extensive reduction of surface exposition during processing.
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http://dx.doi.org/10.1002/biot.201100436 | DOI Listing |
Apoptosis
December 2024
Department of Biochemistry and Molecular Biophysics, Kansas State University, 141 Chalmers Hall, Manhattan, KS, 66506, USA.
Mol Neurobiol
November 2024
Department of Pediatrics, Infants Hospital of Rhode Island, The Alpert Medical School of Brown University, Women &101 Dudley Street, Providence, RI, 02905-2499, USA.
Inter-alpha inhibitor proteins (IAIPs) are neuroprotective and attenuate lipopolysaccharide (LPS)-mediated blood-brain barrier (BBB) disruption in neonatal rodents. We investigated some mechanism(s) fundamental to neuroprotection by IAIPs including changes in cerebral endothelial components and inflammation. Postnatal day-7 rats exposed to sham surgery and placebo or carotid ligation plus 8% FiO (90 min) were given IAIPs (30 or 60 mg/kg) or placebo and were killed 6, 12, 24, or 36 h after hypoxia-ischemia (HI).
View Article and Find Full Text PDFBrain Behav Immun
January 2025
Molecular Neuropathology Unit, Laboratory of Neurogenetics, National Institute on Aging, National Institutes of Health, Bethesda, MD 20892, USA. Electronic address:
Synucleinopathies are age-related neurological disorders characterized by the abnormal accumulation of α-synuclein (α-syn) in neuronal and non-neuronal cells. It has been proposed that microglial cells play an important role in synucleinopathy neuroinflammation, as well as homeostatically, such as in the clearance of α-syn aggregates in the brain. Here, we examined the effects of microglia on the pathogenesis of synucleinopathies by cell depletion in a mouse model of synucleinopathies.
View Article and Find Full Text PDFMed J Armed Forces India
November 2022
Assistant Professor (Radiology), Army Institute of Cardio Thoracic Sciences (AICTS), Pune, India.
Pulmonary alveolar proteinosis (PAP) is a rare disease which involves the accumulation of insoluble lipoproteinaceous material in the alveoli leading to impaired gas exchange and even respiratory failure. Autoimmune PAP is the most common type and is characterized by the presence of anti-granulocyte-monocyte colony stimulating factor (anti GM-CSF) antibody. Whole lung lavage has been traditionally used as first-line management of PAP but there is a lack of clarity especially in the treatment of relapsing cases of PAP.
View Article and Find Full Text PDFFood Funct
June 2024
State Key Laboratory of Food Science and Resources, China-Canada Joint Laboratory of Food Science and Technology (Nanchang), Nanchang University, 235 Nanjing East Road, Nanchang 330047, China.
Our laboratory previously extracted bound polyphenols (BPP) in insoluble dietary fiber from navel orange peel (NOP-IDF), and the aim of this study was to investigate the anti-inflammatory activity and potential molecular mechanisms of BPP by establishing an LPS-induced intestinal-like Caco-2/RAW264.7 co-culture inflammation model. The results demonstrated that BPP reduced the expression of cyclooxygenase-2 (COX-2) and inducible nitric oxide synthase (iNOS), as well as the production of pro-inflammatory cytokines, nitric oxide (NO), and reactive oxidative species (ROS) during the inflammatory damage process.
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