SP-A, SP-B, and SP-C in surfactant subtypes around birth: reexamination of alveolar life cycle of surfactant.

none7Transformations of surfactant after secretion are incompletely understood. To clarify them, we lavaged lungs in fetuses and in newborn rabbits, fractionated the lavage fluid by differential and density gradient centrifugation, and analyzed the distribution of surfactant protein (SP) phospholipids, SP-A, SP-B, and SP-C. Furthermore, we administered into trachea of newborn rabbits labeled surfactant and compared the alveolar clearance of SP-A, SP-B, SP-C and saturated phosphatidylcholine. We found that, in the fetus, secreted lamellar bodies contain all components of surfactant, except a small amount of SP-A. As breathing starts and new surfactant subtypes are generated, the proteins are mostly associated with dense subtypes, but SP-B and SP-C are especially concentrated in dense materials that contain minor amounts of phospholipids and SP-A. Furthermore, we found that, during breathing, alveolar surfactant is degraded into more than one type of remnant, that the lavage fluid contains a pool of SP-A not associated with membranes, and that SP-A, SP-B, and SP-C are all turned over at a faster rate than saturated phosphatidylcholine.noneBARITUSSIO A; ALBERTI A; QUAGLINO D; PETTENAZZO A; DALZOPPO D; SARTORI L; PASQUALIRONCHETTI IBaritussio, Aldo; Alberti, Antonella; Quaglino, D; Pettenazzo, Andrea; Dalzoppo, Daniele; Sartori, Leonardo; Pasqualironchetti, I

Synthesis and conformational studies of peptides encompassing the carboxy-terminal helix of thermolysin.

The 21-residue fragment Tyr-Gly-Ser-Thr-Ser-Gln-Glu-Val-Ala-Ser-Val-Lys-Gln-Ala-Phe-Asp-Ala-Val- Gly-Val-Lys, corresponding to sequence 296-316 of thermolysin and thus encompassing the COOH-terminal helical segment 301-312 of the native protein, was synthesized by solid-phase methods and purified to homogeneity by reverse-phase high performance liquid chromatography. The peptide 296-316 was then cleaved with trypsin at Lys307 and Staphylococcus aureus V8 protease at Glu302, producing the additional fragments 296-307, 308-316, 296-302, and 303-316. All these peptides, when dissolved in aqueous solution at neutral pH, are essentially structureless, as determined by circular dichroism (CD) measurements in the far-ultraviolet region. On the other hand, fragment 296-316, as well as some of its proteolytic fragments, acquires significant helical conformation when dissolved in aqueous trifluoroethanol or ethanol. In general, the peptides mostly encompassing the helical segment 301-312 in the native thermolysin show helical conformation in aqueous alcohol. In particular, quantitative analysis of CD data indicated that fragment 296-316 attains in 90% aqueous trifluoroethanol the same percentage (approximately 58%) of helical secondary structure of the corresponding chain segment in native thermolysin. These results indicate that peptide 296-316 and its subfragments are unable to fold into a stable native-like structure in aqueous solution, in agreement with predicted location and stabilities of isolated subdomains of the COOH-terminal domain of thermolysin based on buried surface area calculations of the molecule

Thiol-activated anticancer agents: The state of the art

The thiol or sulfhydryl group, as part of low molecular weight non-peptide biomolecules, as well as part of the cysteine residues in peptides and proteins, is known to play extremely important roles in several aspects of cellular function. Glutathione (γ-glu-cys-gly; GSH) is the most abundant thiolcontaining peptide in mammals, being present intracellularly in the low millimolar concentration range, but only in the low micromolar concentration range in the majority of extracellular fluids. Notably, intracellular levels of GSH have been found to be significantly upregulated in a number of human cancers, a phenomenon thought to contribute, in concert with overexpression of some GSH-associated enzymes, to the development of tumor cell chemo- and radioresistance. On the other hand, various natural and synthetic chemical entities of different sizes show significant cytotoxic activity only upon interaction with a thiol, and can therefore exploit the GSH-rich intracellular environment of tumors. This review article attempts to summarize the current structural and pharmacological knowledge in the field of thiol-activated anticancer agents, with a focus on the mechanism(s) of their activation. Even thought a great part of the available thiol-activated anticancer compounds is still in the preclinical phase of testing, some of them are undergoing trials in cancer patients

Novel monodisperse PEG - Dendrons as new tools for targeted drug delivery: Synthesis, characterization and cellular uptake

Dendrimers, dendrons, and hyperbranched polymers are gaining popularity as novel drugs, imaging agents, and drug delivery systems. They present advantages of well-defined molecular weight, multivalent surfaces, and high drug carrying capacity. Moreover, it is emerging that such architectures can display unique endocytic properties. As poly(ethylene glycol) (PEG) is widely used for protein and drug conjugation, the aim of this study was for the first time to synthesize novel, branched PEG-based architectures, to define their cytotoxicity and, via preparation of Oregon green (OG) conjugates define the effect of structure on their cellular uptake. Five PEG-based dendrons were synthesized using monodisperse Fmoc-amino PEG propionic acid (Mw = 840) as a monomer, and cadaverine, tris(2-aminoethyl)amine or lysine as the branching moieties. These were diamino,bisPEG (Mw = 1300); triamino,trisPEG (Mw = 1946); tetraamino,tetraPEG (Mw = 3956); monocarboxy,diamino,bisPEG (Mw = 1346); and monocarboxy,tetraamino,tetraPEG (Mw = 3999). These products had NH2 or both NH2 and COOH terminal groups and the identity was verified by amino group analysis and ESI-TOF mass spectroscopy. Purity was determined by HPLC. Representative structures were not toxic towards an endothelial-like cell line (ECV304) at concentrations up to 4 mg/mL (over 72 h). At 37 \ub0C, all of the OG-labeled PEG dendrons showed progressive uptake by ECV304 cells, but tetraamino,tetraPEG showed the greatest rate of internalization over the first 20 min. Cellular uptake was inhibited at 4 \ub0C, and PEG dendron localization to perinuclear vesicles was confirmed by fluorescence microscopy. These well-defined novel architectures have potential for further development as targetable drug delivery systems or tools for construction of structurally defined modified surfaces. \ua9 2006 American Chemical Society

Halogen-Mediated Partial Oxidation of Polyvinyl Alcohol for Tissue Engineering Purposes

Partial oxidation of polyvinyl alcohol (PVA) with potassium permanganate turned out to be an efficient method to fabricate smart scaffolds for tissue engineering, endowed with biodegradation and protein delivery capacity. This work considered for the first time the use of halogens (bromine, chlorine and iodine) as less aggressive agents than potassium permanganate to perform controlled PVA oxidation, in order to prevent degradation of polymer molecular size upon chemical modification. Oxidized PVA solutions were chemically characterized (i.e., dinitrophenylhydrazine assay, viscosity measurements, molecular size distribution) before preparing physically cross-linked hydrogels. Scaffolds were assessed for their mechanical properties and cell/tissue biocompatibiliy through cytotoxic extract test on IMR-90 fibroblasts and subcutaneous implantation into BALB/c mice. According to chemical investigations, bromine and iodine allowed for minor alteration of polymer molecular weight. Uniaxial tensile tests demonstrated that oxidized scaffolds had decreased mechanical resistance to deformation, suggesting tunable hydrogel stiffness. Finally, oxidized hydrogels exhibited high biocompatibility both in vitro and in vivo, resulting neither to be cytotoxic nor to elicit severe immunitary host reaction in comparison with atoxic PVA. In conclusion, PVA hydrogels oxidized by halogens were successfully fabricated in the effort of adapting polymer characteristics to specific tissue engineering applications