Monitoring the autoproteolysis of hiv-1 protease by site-directed spin-labeling and electron paramagnetic resonance spectroscopy

Site-directed spin-labeling with continuous wave electron paramagnetic resonance spectroscopy was used to monitor autoproteolysis of HIV-1 protease, an enzyme essential for viral maturation. Two protein constructs were examined, namely subtype F and the circulating recombinant form CRF01_A/E. As the protease undergoes self-cleavage, protein unfolds and small peptide fragments containing the spin label are generated, which collectively give rise to a sharp spectral component that is easily discernable in the high-field resonance line in the EPR spectrum. By monitoring the intensity of this spectral component over time, the autoproteolytic stability of each construct was characterized under various conditions. Data were collected for samples stored at 4 °C, 25 °C, and 37 °C, and on a subtype F HIV-1 protease sample stored at 25 °C and containing the FDA-approved protease inhibitor Tipranavir. As expected, the rate of autoproteolysis decreased as the storage temperature was lowered. Minimal autoproteolysis was seen for the sample that contained Tipranavir, providing direction for future spectroscopic studies of active protease samples. When compared to standard methods of monitoring protein degradation such as gel electrophoresis or chromatographic analyses, spin-labeling with CW EPR offers a facile, real-time, non-consuming way to monitor autoproteolysis or protein degradation. Additionally, mass spectrometry studies revealed that the N-termini of both constructs are sensitive to degradation and that the sites of specific autoproteolysis vary.

A SPIN-LABELING STUDY OF THE AGGLUTINATION OF CONCANAVALIN A WITH MEMBRANE RECEPTORS IN MOUSE ASCITES HEPATOMA CELLS

We have studied the effect of concanavaline A (Con A) on conformational changes of membrane glycoproteins of mouse ascites hepatoma cells with a maleimide spin label, showing that conformational changes or a physical arrangement of membrane glycoproteins could be induced quickly by the cross-linking of Con A with membrane receptors. We have also studied the effect of Con A on the mobility

Spin-Labelled 1-Ethyl-1-Nitrosourea Prevents Doxorubicin and Bleomycin-Induced Oxidative Stress in Lungs, Hearts and Kidneys of Tumour-Bearing Mice

This study was carried out to determine the possible protective effect of 1-ethyl-3-[4-(2, 2, 6, 6-tetramethylpiperidine-1-oxyl)]-1-nitrosourea (SLENU), recently synthesised in our laboratory on doxorubicin and bleomycin-induced oxidative toxicity in C57 black tumour-bearing mice. Specifically, alterations in some biomarkers of oxidative stress, such as lipid peroxidation products measured as malondialdehyde (MDA) levels and activities of the antioxidant enzymes, superoxide dismutase (SOD) and catalase (CAT), were studied in lung, heart and kidney homogenates isolated from C57 black tumor-bearing mice after i.p. treatment with solutions of DOX (60 mg/kg) and BLM (60 mg/kg). The same biomarkers were also measured after i.p. pretreatment of mice with SLENU (100 mg/kg). After treatment with doxorubicin, heart and kidney homogenates of mice had significantly higher productions of lipid peroxidation compared to lung homogenates. It was accompanied by increased activity of the antioxidant defence enzyme superoxide dismutase and decreased activity of catalase. Bleomycin-induced oxidative stress was confirmed by significantly higher production of lipid peroxidation in lungs compared to heart homogenates, elevation of the anti-oxidant activity of superoxide dismutase and decreased activity of catalase enzymes. After pre-treatment of the mice with SLENU, the levels of all studied oxidative stress biomarkers were significantly improved in comparison with those of the mice treated alone with either bleomycin, or doxorubicin. The present results and those from a previously demonstrated superoxide scavenging activities (SSA) of the nitrosourea SLENU have enabled us to explain the protective effect of the spin-labelled nitrosourea on doxorubicin and bleomycin-induced oxidative stress by scavenging of??O2- and increased NO release.

Synthesis and Cytotoxic Activities of Spin-labeled Derivatives of Cinobufagin

Two series of novel spin-labeled derivatives of Cinobufagin（compounds 5 and 8a--8f in series 1 with five-membered ring nitroxyl free radical and compounds 6 and 9a-9f in series 2 with six-membered ring nitroxyl free radical） were synthesized. The cytotoxic activities in vitro against two tumor cell lines（HepG2 and HeLa） were evaluated, and the results indicate that all compounds display potent cytotoxicity against HepG2 and HeLa cells, and most compounds show better activities on HeLa cells than on HepG2 cells except for compounds 8a and 9d. Gene- rally, the compounds in series 2 have more potent cytotoxic activity against HepG2 than the compounds in series 1. Especially, compounds 6 and 9f in series 2 exhibit even more potent activities against the two tumor cell lines than Cinobufagin. Thus incorPoration of different L-amino acids as the linker changed the cytotoxic profile of the spin-labeled Cinobufagin. In addition, the representative compound 9f significantly changed the cell cycle distribution and led to HeLa cell cycle arrested at G2/M phase.

Effect of arteriosclerotic intracranial arterial vessel wall enhancement on downstream collateral flow

Background:The effect of arteriosclerotic intracranial arterial vessel wall enhancement(IAVWE)on downstream collateral flow found in vessel wall imaging(VWI)is not clear.Regardless of the mechanism underlying IAVWE on VWI,damage to the patient’s nervous system caused by IAVWE is likely achieved by affecting downstream cerebral blood flow.The present study aimed to investigate the effect of arteriosclerotic IAVWE on downstream collateral flow.Methods:The present study recruited 63 consecutive patients at the Second Hospital of Hebei Medical University from January 2021 to November 2021 with underlying atherosclerotic diseases and unilateral middle cerebral artery(MCA)M1-segment stenosis who underwent an magnetic resonance scan within 3 days of symptom onset.The patients were divided into 4 groups according to IAVWE and the stenosis ratio(Group 1,n=17;Group 2,n=19;Group 3,n=13;Group 4,n=14),and downstream collateral flow was analyzed using three-dimensional pseudocontinuous arterial spin labeling(3D-pCASL)and RAPID software.The National Institutes of Health Stroke Scale(NIHSS)scores of the patients were also recorded.Two-factor multivariate analysis of variance using Pillai’s trace was used as the main statistical method.Results:No statistically significant difference was found in baseline demographic characteristics among the groups.IAVWE,but not the stenosis ratio,had a statistically significant significance on the late-arriving retrograde flow proportion(LARFP),hypoperfusion intensity ratio(HIR),and NIHSS scores(F=20.941,P<0.001,Pillai’s trace statistic=0.567).The between-subject effects test showed that IAVWE had a significant effect on the three dependent variables:LARFP(R^(2)=0.088,F=10.899,P=0.002),HIR(R^(2)=0.234,F=29.354,P<0.001),and NIHSS(R^(2)=114.339,F=33.338,P<0.001).Conclusions:Arteriosclerotic IAVWE significantly reduced downstream collateral flow and affected relevant neurological deficits.It was an independent factor affecting downstream collateral flow and NIHSS scores,which should be a focus of future studies.Trial Registration:ChiCTR.org.cn,ChiCTR2100053661.