Further biochemical and physicochemical characterization of minor disulfide-bonded (type IX) collagen, extracted from foetal calf cartilage

Minor disulfide-bonded collagen (previously termed X1-X7 and now called type IX collagen) was isolated from foetal calf cartilage after pepsin treatment. At least three native fractions, containing, respectively, the X1X2X3, X4, and X5X6X7 chains, were separated; and from further biochemical and physicochemical experiments (differential scanning calorimetry, electrical birefringence, rotary shadowing), we propose a tentative model for their organization within a parent molecule. X1 and X2 are molecules composed of three chains of apparent Mr 62,000 and 50,000 linked by interchain disulfide bonds and containing pepsin-sensitive regions. The cleavage of at least three of these sites, present within X2, gives rise to the X3 and X5X6X7 fractions composed of molecules 80-100 nm and 40-55 nm in length, respectively. The X5X6X7 fraction is not digested by pepsin at 30 degrees C owing to its high thermal stability (certainly explained by its high hydroxyproline + proline content). This organization is in good accordance with that proposed for chicken cartilage type IX collagen; differences could only exist in the number and (or) the location of the pepsin-sensitive sites.     

The synthesis of magnesium-protoporphyrin IX by etiochloroplast membrane preparations

Magnesium-protoporphyrin IX (or its monomethyl ester) is the first committed intermediate in the biosynthesis of chlorophyll in green plants. Membranes from lysed washed cucumber etiochloroplasts synthesized small amounts of ¹⁴C-labelled magnesium-protoporphyrin IX from [¹⁴C]protoporphyrin IX at the rate of 1–3 pmol/h per mg protein. Maximum activity in these membrane preparations was dependent upon added EDTA, GSH, ATP and MgCl₂. Activity was totally dependent upon added ATP, probably as the species MgATP^2? and there was also a requirement for Mg²⁺ in addition to that used to form the MgATP^2? complex.     

Basis for drug selectivity of plasmepsin IX and X inhibition in Plasmodium falciparum and vivax

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Dissolving microneedles containing aminolevulinic acid improves protoporphyrin IX distribution

One important limitation of topical photodynamic therapy (PDT) is the limited tissue penetration of precursors. Microneedles (MNs) are minimally invasive devices used to promote intradermal drug delivery. Dissolving MNs contain drug-associated to polymer blends, dissolving after insertion into skin, allowing drug release. This study comprises development and characterization of a pyramidal model of dissolving MNs (500 μm) prepared with 5% wt/wt aminolevulinic acid and 20% wt/wt Gantrez AN-139 in aqueous blend. Protoporphyrin IX formation and distribution were evaluated in tumor mice model by using fluorescence widefield imaging, spectroscopy, and confocal microscopy. MNs demonstrated excellent mechanical resistance penetrating about 250 μm with minor size alteration in vitro, and fluorescence intensity was 5-times higher at 0.5 mm on average compared to cream in vivo (being 10 ± 5 a.u. for MNs and 2.4 ± 0.8 a.u. for cream). Dissolving MNs have overcome topical cream application, being extremely promising especially for thicker skin lesions treatment using PDT.     

erythro-β-hydroxyaspartic acid in bovine factor IX and factor X

To localize β-hydroxyaspartic acid in factor IX and factor X the two proteins were cleaved with cyanogen bromide and trypsin, respectively. Peptides containing β-hydroxyaspartic acid were isolated and subjected to Edman degradation. The phenylthiohydantoin derivative of β-hydroxyaspartic acid was identified by HPLC in position 3 in the factor IX fragment and in position 1 in the factor X fragment. This corresponds to position 64 in factor IX and position 63 in the light chain of factor X. The assignments were confirmed by subtractive Edman degradation and with the dansyl method.     

The pH-dependent conformational transition of β-lactoglobulin modulates the binding of protoporphyrin IX

We have investigated the interaction between PPIX and β-lactoglobulin (β-lg) as a function of the pH of the solution. β-lg is a small globular protein (MW ≈18 kDa) with a very well characterized structure that reveals several possible binding sites for ligands. The interaction with β-lg affects the photophysical properties of PPIX. The shift of PPIX emission maximum, excitation maximum and the increase of the fluorescence intensity is an indicator that binding between the porphyrin and β-lg occurs. The binding constant appears to be modulated by the pH of the solution. Spectroscopic measurements do not reveal any significant energy transfer between the Trp residues of β-lg and PPIX, however, fluorescence anisotropy decay measurements confirm the binding and the modulation introduced by the pH of the solution. Since β-lg has been shown to be stable within the range of pH adopted in our experiments (5.0–9.0), the results suggest that PPIX binds a site affected by the pH of the solution. Because of the crystallographic evidence an obvious site is near the aperture of the interior β-barrel however an alternative (or concurrent) binding site may still be present.     

Dual Plasmepsin-Targeting Antimalarial Agents Disrupt Multiple Stages of the Malaria Parasite Life Cycle

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Novel cell lines derived from transgenic mice expressing recombinant human proteins

We have used transgenic mouse technology to establish immortalized hepatoma cell lines stably secreting heterologous proteins, such as human α₁-antitrypsin and human factor IX. Hepatocyte-specific regulatory DNA sequences were used to target both the expression of anonc gene and the gene coding for the human protein to the liver of transgenic mice which eventually developed hepatocellular carcinomas. Tumour cells were subsequently established as permanent cell lines, which maintained a differentiated phenotype under specific culture conditions, being capable of producing biologically active and correctly processed human α₁-antitrypsin and factor IX. Moreover, a preliminary analysis has shown that certain cell lines express elevated total cytochrome P450 activity. These cells could therefore represent a useful alternative to the use of animals or primary cultures in drug safety testing.