Lysosomal-type PLA2 and turnover of alveolar DPPC

This study evaluated the role of a lysosomal-type phospholipase A2 (aiPLA(2)) in the degradation of internalized dipalmitoylphosphatidylcholine (DPPC) and in phospholipid synthesis by the rat lung. Uptake and degradation of DPPC were measured in isolated perfused rat lungs over 3 h following endotracheal instillation of [(3)H]DPPC in mixed unilamellar liposomes plus or minus MJ33, a specific inhibitor of lung aiPLA(2). Uptake of DPPC was calculated from total tissue-associated radiolabel, and degradation was calculated from the sum of radiolabel in degradation products. Both uptake and degradation were markedly stimulated by addition of 8-bromo-cAMP to the perfusate. MJ33 had no effect on DPPC uptake but decreased DPPC degradation at 3 h by approximately 40-50%. The effect of MJ33 on lung synthesis of DPPC was evaluated with intact rats over a 12- to 24-h period following intravenous injection of radiolabeled palmitate and choline. MJ33 treatment decreased palmitate incorporation into disaturated phosphatidylcholine of lamellar bodies and surfactant by approximately 65% at 24 h but had no effect on choline incorporation. This result is compatible with inhibition of the deacylation/reacylation pathway for DPPC synthesis. These results obtained with intact rat lungs indicate that aiPLA(2) is a major enzyme for degradation of internalized DPPC and also has an important role in DPPC synthesis.     

1-Cys peroxiredoxin, a bifunctional enzyme with glutathione peroxidase and phospholipase A2 activities

This report provides definitive evidence that the protein 1-Cys peroxiredoxin is a bifunctional ("moonlighting") enzyme with two distinct active sites. We have previously shown that human, rat, and bovine lungs contain an acidic Ca(2+)-independent phospholipase A(2) (aiPLA(2)). The cDNA encoding aiPLA(2) was found to be identical to that of a non-selenium glutathione peroxidase (NSGPx). Protein expressed using a previously reported E. coli construct which has a His-tag and 50 additional amino acids at the NH(2) terminus, did not exhibit aiPLA(2) activity. A new construct which contains the His-tag plus two extra amino acids at the COOH terminus when expressed in Escherichia coli generated a protein that hydrolyzed the sn-2 acyl chain of phospholipids at pH 4, and exhibited NSGPx activity with H(2)O(2) at pH 8. The expressed 1-Cys peroxiredoxin has identical functional properties to the native lung enzyme: aiPLA(2) activity is inhibited by the serine protease inhibitor, diethyl p-nitrophenyl phosphate, by the tetrahedral mimic 1-hexadecyl-3-trifluoroethylglycero-sn-2-phosphomethanol (MJ33), and by 1-Cys peroxiredoxin monoclonal antibody (mAb) 8H11 but these agents have no effect on NSGPx activity; NSGPx activity is inhibited by mercaptosuccinate and by 1-Cys peroxiredoxin mAb 8B3 antibody which have no effect on aiPLA(2) activity. Mutation of Ser(32) to Ala abolishes aiPLA(2) activity, yet the NSGPx activity remains unaffected; a Cys(47) to Ser mutant is devoid of peroxidase activity but aiPLA(2) activity remains intact. These results suggest that Ser(32) in the GDSWG consensus sequence provides the catalytic nucleophile for the hydrolase activity of aiPLA(2), while Cys(47) in the PVCTTE consensus sequence is at the active site for peroxidase activity. The bifunctional catalytic properties of 1-Cys peroxiredoxin are compatible with a simultaneous role for the protein in the regulation of phospholipid turnover as well as in protection against oxidative injury.     

Thermo-optic measurements and their inter-dependencies for delineating cancerous breast biopsy tissue from adjacent normal

The histopathological diagnosis of cancer is the current gold standard to differentiate normal from cancerous tissues. We propose a portable platform prototype to characterize the tissue's thermal and optical properties, and their inter-dependencies to potentially aid the pathologist in making an informed decision. The measurements were performed on 10 samples from five subjects, where the cancerous and adjacent normal were extracted from the same patient. It was observed that thermal conductivity (k) and reduced-scattering-coefficient (μ'_s) for both the cancerous and normal tissues reduced with the rise in tissue temperature. Comparing cancerous and adjacent normal tissue, the difference in k and μ'_s (at 940 nm) were statistically significant (p = 7.94e-3), while combining k and μ'_s achieved the highest statistical significance (6.74e-4). These preliminary results promise and support testing on a large number of samples for rapidly differentiating cancerous from adjacent normal tissues.