Comparison between the enzymatic activity, structure and substrate binding of mouse and human lecithin retinol acyltransferase.

Lecithin retinol acyltransferase (LRAT) is involved in the visual cycle where it catalyzes the formation of all-trans retinyl ester. The mouse animal model has been widely used to study LRAT. Primary sequence alignment shows 80% identity and 90% similarity between human and mouse LRAT. However, human LRAT has a proline at position 173 (hLRAT (P173)) while an arginine can be found at this position for the mouse protein (mLRAT (R173)). Moreover, residue 173 is important for the human protein since a substitution mutation of this residue to a leucine (P173L-hLRAT) caused night blindness in a patient. The present study was thus undertaken to determine whether mouse and human LRAT have a similar enzymatic activity, structure and substrate binding affinity using a truncated form of LRAT (tLRAT). The enzymatic activity and binding affinity to the substrate, all-trans retinol, of mtLRAT (R173) were found to be 2.7- and 3.9-fold lower, respectively, than that of htLRAT (P173). Moreover, the enzymatic activity of P173L-htLRAT is 6.3-fold lower compared to that of htLRAT (P173). Furthermore, a significant difference was observed between the intrinsic fluorescence emission as well as between the circular dichroism spectra of mtLRAT (R173) and htLRAT (P173). In addition, mtLRAT proteins are less thermostable than htLRAT proteins, which suggests that structural differences exist between the mouse and human proteins. Altogether, these data strongly suggest that the much lower catalytic activity of mtLRAT (R173) compared to that of htLRAT (P173) mostly results from differences between their structure, predominantly revealed by their dissimilar thermal stability, as well as their efficiency to bind all-trans retinol. Therefore, conclusions regarding the behavior of human LRAT based on measurements performed with mouse LRAT must be made with caution. Also, the much lower enzymatic activity of P173L-htLRAT compared to that of htLRAT (P173) might explain the night blindness of a patient carrying this mutation.

Quantitative gated PET for the assessment of left ventricular function in small animals.

UNLABELLED: 18F-FDG PET can identify areas of myocardial viability and necrosis and provide useful information on the effectiveness of experimental techniques designed to improve contractile function and myocardial vascularization in small animals. The left ventricular volume (LVV) and left ventricular ejection fraction (LVEF) in normal and diseased rats were measured in vivo using the high-resolution avalanche photodiode (APD) small-animal PET scanner of the Université de Sherbrooke. The measurements obtained by PET were compared with those obtained by high-resolution echocardiography and with known values obtained from a small, variable-volume cardiac phantom. METHODS: List-mode gated (18)F-FDG PET studies were performed using the APD PET scanner on 30 rats: 11 healthy, 4 under septic shock, and 15 with heart failure induced by ligature of the left coronary artery. PET images were resized to match human-scale pixels and analyzed using a standard clinical cardiac software program. The LVV and LVEF from the same animals were also evaluated by echocardiography. RESULTS: Agreement was excellent between the endocardial volumes determined by PET and the actual volumes of the cardiac phantom (r(2) = 0.96). Agreement between PET and echocardiography for LVV ranged from good in healthy rats (r(2) = 0.89) to fair in diseased rats (r(2) = 0.49). Agreement was fair between LVEF values measured by the 2 methods (r(2) = 0.56). Normal rats had an average LVEF of 83.2% +/- 8.0% using PET and 81.6% +/- 6.0% using echocardiography. In rats with heart failure, LVEF was 54.6% +/- 15.9% using PET and 54.2% +/- 13.3% using echocardiography. CONCLUSION: Both PET and echocardiography clearly differentiated normal rats from rats with heart failure. Echocardiography is fast and convenient, whereas list-mode PET is also able to assess defect size, myocardial viability, and metabolism.