Optically detected magnetic resonance studies of porcine pancreatic phospholipase A2 binding to a negatively charged substrate analogue

The direct binding of porcine pancreatic phospholipase A2 and its zymogen to 1,2-bis(heptanylcarbamoyl)-rac-glycerol 3-sulfate was studied by optical detection of triplet-state magnetic resonance spectroscopy in zero applied magnetic field. The zero-field splittings of the single Trp3 residue undergo significant changes upon binding of phospholipase A2 to lipid. Shifts in zero-field splittings, characterized mainly by a reduction of the E parameter from 1.215 to 1.144 GHz, point to large changes in the Trp3 local environment which accompany the complexing of phospholipase A2 with lipid. This may be attributed to Stark effects caused by the binding of a charged group near Trp3 in the enzyme-lipid complex. The cofactor, Ca2+, which is strongly bound to the enzyme active site, has an influence on the bonding, as reflected by smaller zero-field splitting shifts. A relatively small change in the Trp environment was observed for the interaction of the zymogen with lipid.     

Investigation of phospholipase-lipid interactions by optical detection of triplet state magnetic resonance spectroscopy

We have investigated the binding of porcine pancreatic phospholipase A2 (PA2) to n-hexadecylphosphocholine (C16PN) micelles using optical detection of triplet state magnetic resonance (ODMR) spectroscopy. The zero field splittings (zfs) of the single Trp3 residue undergo significant changes upon binding of PA2 to C16PN micelles. Zfs titrations of PA2 vs C16PN indicate that the binding stoichiometry is C16PN:PA2 approximately 25. A reduction of the (E) parameter from 1.227 to 1.135 GHz is postulated to result from Stark effects caused by the binding of a polar group (possibly phosphocholine) near Trp3 in the PA2-C16PN micelle complex.     

The Combination of SMAD4 Expression and Histological Grade of Dysplasia Is a Better Predictor for the Malignant Transformation of Oral Leukoplakia

Oral leukoplakia (OL) is the most common premalignancy in the oral cavity and can progress to oral squamous cell carcinoma (OSCC). SMAD4 is a tumor suppressor implicated in multiple cancer types including OSCC. To assess the role of SMAD4 in oral leukoplakia malignant transformation, the authors investigated SMAD4 expression patterns in OL and OSCC using a highly specific antibody and correlated the patterns with the risk of malignant transformation oral leukoplakia. Immunohistochemistry and a quantitative imaging system were used to measure SMAD4 expression in OL from 88 OL patients, including 22 who later went through malignant transformation, and their OSCC counterpart. Forty-three (48.9%) of the 88 OL patients had strong SMAD4 expression. SMAD4 expression had no significant correlation with patients'' clinicopathological parameters. Interestingly, 17 (39.5%) of the 43 OL lesions with strong SMAD4 expression went through malignant transformation whereas only 5 (11.1%) of the 45 OL lesions with weak SMAD4 expression did so (p = 0.002). The SMAD4 expression in OL was much higher than that in their OSCC counterpart. Kaplan-Meier analysis revealed that the combination of SMAD4 expression and histological grade of dysplasia (p = 0.007) is a better predictor for the malignant transformation of oral leukoplakia. In the multivariate analysis, both SMAD4 expression and grade of dysplasia were identified as independent factors for OL malignant transformation risk (p = 0.013 and 0.021, respectively). It was concluded that high SMAD4 expression may be indicative of an early carcinogenic process in OL and serve as an independent biomarker in assessing malignant transformation risk in patients with OL, and the combination of SMAD4 expression and histological grade of dysplasia is a better predictor for the malignant transformation of oral leukoplakia.     

MARCH5 restores endothelial cell function against ischaemic/hypoxia injury via Akt/eNOS pathway

MARCH5 is a critical regulator of mitochondrial dynamics, apoptosis and mitophagy. However, its role in cardiovascular system remains poorly understood. This study aimed to investigate the role of MARCH5 in endothelial cell (ECs) injury and the involvement of the Akt/eNOS signalling pathway in this process. Rat models of myocardial infarction (MI) and human cardiac microvascular endothelial cells (HCMECs) exposed to hypoxia (1% O₂) were used in this study. MARCH5 expression was significantly reduced in ECs of MI hearts and ECs exposed to hypoxia. Hypoxia inhibited the proliferation, migration and tube formation of ECs, and these effects were aggravated by knockdown of MARCH5 but antagonized by overexpressed MARCH5. Overexpression of MARCH5 increased nitric oxide (NO) content, p-eNOS and p-Akt, while MARCH5 knockdown exerted the opposite effects. The protective effects mediated by MARCH5 overexpression on ECs could be inhibited by eNOS inhibitor L-NAME and Akt inhibitor LY294002. In conclusion, these results indicated that MARCH5 acts as a protective factor in ischaemia/hypoxia-induced ECs injury partially through Akt/eNOS pathway.