Antifibrotic effect of a proline analogue delivered in liposomes to cells in culture

Summary Proline analogues inhibit procollagen triple helix formation and are antifibrotic in vivo. Efficacy of the proline analoguecis-4-hydroxy-L-proline (cHyp) on vascular collagen accumulation is improved by in vivo delivery in liposomes. This effect may be due to local release of drug from liposomes taken up by vascular endothelium. To test this postulate, we used a co-culture system to assess the antifibrotic effect of cHyp in liposomes taken up by endothelium (upper well) by measuring inhibition of growth of smooth muscle cells and fibroblasts (lower well). We also studied whether release of cHyp was prolonged in poly(ethyleneglycol) (PEG)-conjugated liposomes compared to liposomes not conjugated with PEG (control liposomes). In fibroblasts, free (unencapsulated) cHyp (1 mg/ml) added to the upper well inhibited growth for 3 days; an equivalent dose of cHyp in control liposomes inhibited growth for 4 days. cHyp in PEG-liposomes produced greater growth inhibition than cHyp in control liposomes. cHyp in liposomes did not inhibit growth of smooth muscle cells more than free cHyp. Washing free cHyp from endothelium after 1 day incubation restored growth of smooth muscle cells whereas washing liposomes containing cHyp failed to restore cell growth. These results suggest that liposomes enhance drug efficacy of cHyp by prolonging the release of drug from endothelium.     

Protein methylation as a marker of aspartate damage in glucose-6-phosphate dehydrogenase-deficient erythrocytes: role of oxidative stress.

The 'Mediterranean' variant of glucose-6-phosphate dehydrogenase (G6PD) deficiency is due to the C563CT point mutation, leading to replacement of Ser with Phe at position 188, resulting in acute haemolysis triggered by oxidants. Previous work has shown increased formation of altered aspartate residues in membrane proteins during cell ageing and in response to oxidative stress in normal erythrocytes. These abnormal residues are specifically recognized by the repair enzyme L-isoaspartate (d-aspartate) protein O-methyltransferase (PCMT; EC 2.1.1.77). The aim of this work was to study the possible involvement of protein aspartate damage in the mechanism linking the G6PD defect and erythrocyte injury, through oxidative stress. Patients affected by G6PD deficiency (Mediterranean variant) were selected. In situ methylation assays were performed by incubating intact erythrocytes in the presence of methyl-labelled methionine. Altered aspartate residues were detected in membrane proteins by methyl ester quantification. We present here evidence that, in G6PD-deficient erythrocytes, damaged residues are significantly increased in membrane proteins, in parallel with the decay of pyruvate kinase activity, used as a cell age marker. Erythrocytes from patients were subjected to oxidative stress in vitro, by treatment with t-butylhydroperoxide, monitored by a rise in concentration of both methaemoglobin and thiobarbituric acid-reactive substances. L-Isoaspartate residues increased dramatically in G6PD-deficient erythrocytes in response to such treatment, compared with baseline conditions. The increased susceptibility of G6PD-deficient erythrocytes to membrane protein aspartate damage in response to oxidative stress suggests the involvement of protein deamidation/isomerization in the mechanisms of cell injury and haemolysis.     

Oxygen and carbon isotope variations in Chamelea gallina shells: Environmental influences and vital effects

Stable isotopes in mollusc shells, together with variable growth rates and other geochemical properties, can register different environmental clues, including seawater temperature, salinity and primary productivity. However, the strict biological control over the construction of biominerals exerted by many calcifying organisms can constrain the use of these organisms for paleoenvironmental reconstructions. Biologically controlled calcification is responsible for the so called vital effects that cause a departure from isotopic equilibrium during shell formation, resulting in lower shell oxygen and carbon compared to the equilibrium value. We investigated shell oxygen and carbon isotopic composition of the bivalve Chamelea gallina in six sites along with a latitudinal gradient on the Adriatic Sea (NE Mediterranean Sea). Seawater δ¹⁸O and δ¹³C_DIC varied from North to South, reflecting variations in seawater temperature, salinity, and chlorophyll concentration among sites. Shell δ¹⁸O and δ¹³C differed among sites and exhibited a wide range of values along with the ~400 km latitudinal gradient, away from isotopic equilibrium for both isotopes. These results hampered the utilization of this bivalve as a proxy for environmental reconstructions, in spite of C. gallina showing promise as a warm temperature proxy. Rigorous calibration studies with a precise insight of environment and shell growth are crucial prior to considering this bivalve as a reliable paleoclimatic archive.     

Molecular analysis of hyperoxaluria type 1 in Italian patients reveals eight new mutations in the alanine : glyoxylate aminotransferase gene

Systematic screening using the SSCP technique followed by sequencing of bands with abnormal mobility derived from the AGXT exons of 15 unrelated Italian patients with primary hyperoxaluria type 1 (PH1) allowed us to characterize both the mutant alleles in each individual. Eight new mutations were identified: C155del, C156ins, G244T, C252T, GAG408ins, G468A, G588A and G1098del. This study demonstrates both the effectiveness of the screening strategy chosen to identify all the mutant alleles and the high degree of allelic heterogeneity in PH1. Received: 5 January 1999 / Accepted: 12 April 1999