Squash trypsin inhibitors from Momordica cochinchinensis exhibit an atypical macrocyclic structure

Three trypsin inhibitors (TIs), from the seeds of the squash Momordica cochinchinensis (MCo), have been isolated and purified using gel filtration, ion exchange chromatography, and reverse-phase HPLC. Their sequences could be determined only after proteolytic cleavages. In the case of MCoTI-I and -II, it was shown that their polypeptide backbones are cyclic, a structure that has never been described in squash TIs. They contain 34 amino acid residues with 3 disulfide bridges and measured molecular masses of 3453.0 and 3480.7, respectively. They are the largest known macrocyclic peptides containing disulfide bridges. Their sequences show strong homology to other squash TIs, suggesting a similar three-dimensional structure and an analogous mechanism of action. A model of MCoTI-II was constructed by analogy to the crystal structure of the complex between bovine trypsin and CMTI-I, indicating that the linker connecting the two termini is flexible and does not impose significant geometrical constraints. This flexibility allows an Asp-Gly peptide bond rearrangement to occur in this region, giving rise to two isoforms of MCoTI-II. Although the importance of cyclization is not clear, it might confer increased stability and resistance to proteolysis. A minor species, MCoTI-III, was also characterized as containing 30 amino acid residues with a molecular mass of 3379.6. This component possesses a linear backbone with a blocked N-terminus. MCoTIs represent interesting candidates for drug design, either by changing their specificity of inhibition or by using their structure as natural scaffolds bearing new binding activities.     

Changes in the metabolism of the microalga Chlorella vulgaris when coimmobilized in alginate with the nitrogen-fixing Phyllobacterium myrsinacearum

In an agroindustrial wastewater pond, a naturally occurring unicellular microalga, Chlorella vulgaris, was closely associated with the terrestrial plant-associative N2-fixing bacterium Phyllobacterium myrsinacearum. When the two microorganisms were artificially coimmobilized in alginate beads, they shared the same internal bead cavities, and the production of five microalgal pigments increased, but there were no effects on the number of the cells or the biomass of the microalga. The association, however, reduces the ability of C. vulgaris to remove ammonium ions and phosphorus from water. The bacterium produced nitrate from ammonium in synthetic wastewater with or without the presence of the microalga, and fixed nitrogen in two culture media. Our results suggest that interactions between microalgae and associative bacteria should be considered when cultivating microalgae for wastewater treatment.     

Proteoglycan metabolism during repair of the ruptured medial collateral ligament in skeletally mature rabbits

The metabolism of the chondroitin/dermatan sulfate (CS/DS) proteoglycans (PGs) decorin and biglycan is markedly altered during short-term (3-6 weeks) and long-term (40 weeks-2 years) repair of surgically ruptured medial collateral ligaments from mature rabbits. A PG-rich extracellular matrix accumulates in injury gaps by 3 weeks postsurgery and extends into tissue regions containing the original ligaments, and elevated PG levels remain apparent up to 2 years postinjury. CS/DS PGs were prepared from such ligaments and identified after SDS-polyacrylamide gel electrophoresis by Alcian blue staining or immunoblotting. In normal ligaments, decorin is the most abundant proteoglycan (accounting for approximately 80% of the total); the remainder is biglycan and a large PG, possibly versican. In repairing ligaments, decorin is barely detected, but instead a large proteoglycan and abundant amounts of biglycan accumulate. Biglycan is present in two forms in repairing ligaments, and they can be separated on SDS-PAGE into 200- and 140-kDa forms. The slower migrating species is absent in normal ligaments and may represent a different glycoform (containing either a single or two short chondroitin/dermatan sulfate chains) of biglycan. Alteration in PG expression and posttranslational processing during medial collateral ligament repair are similar to those reported for repair and scar formation of other connective tissues. The accumulation of biglycan observed here may interfere with proper collagen network remodeling and may lead to persistent inflammatory and matrix turnover processes, thus preventing restoration of a long-term functional ligament tissue.     

Effects of different diets and dietary restriction on perinatal endogenous DNA adducts. Time dependence of oxidative and presumptive nonoxidative lesions

Type II I-compounds (indigenous DNA adducts) denote a class of bulky oxidative DNA lesions that are detectable by 32P-postlabeling and represent useful biomarkers of DNA damage induced by oxidative stress. Their levels are increased in tissue DNA under pro-oxidant conditions, for example, as previously shown, in newborn rat organs. Here we have investigated whether the maternal diet affects perinatal type II I-compound levels. Pregnant F344 rats were fed Purina-5001 natural-ingredient or AIN-93G purified diet from day 11 of gestation. Type II I-compounds were measured in liver DNA at three different developmental stages, i.e., fetus, and 24 h and 9 days postnatally. Higher adduct levels were detected in the Purina-5001 group at each stage. In a second experiment, pregnant F344 rats were subjected to dietary restriction (DR) (by 40%; Purina-5001) from day 12 of gestation. At 24 h postpartum hepatic type II I-compound levels were decreased compared to parallel ad libitum (AL) fed controls. As an unrelated observation, fetal lung, but not liver, kidney, and skin DNA contained a different pattern of nonpolar, apparently nonoxidative adducts, which were not diet-dependent. These spots were not detectable 24 h after birth and were observed at much reduced levels and only in a few samples at 9 days. The main results show for the first time that the maternal nutrition modulated levels of oxidative lesions in fetal and neonatal DNA, but the underlying mechanisms (e.g., differences in metal or caloric content of the diets) still need to be determined. The dietary effects were apparently transmitted through both placenta and the mother's milk.     

Coordination and speciation of cadmium in corn seedlings and its effects on macro- and micronutrients uptake

The effect of cadmium (Cd) on both the absorption of important nutrients and the synthesis of low molecular weight thiols (LMWTs) was investigated in corn plants. The inductively coupled plasma-optical emission spectroscopy results demonstrated that the concentration of Cd in tissues (mainly in roots) increased as the concentration in the medium increased. In addition, the concentration of phosphorus increased in roots of Cd treated plants but remained at normal concentration in shoots. On the other hand, the uptake of sulfur (S) followed a similar trend as the Cd uptake. The concentration of S and the production of LMWT were found to increase significantly upon exposure to Cd. The results of the X-ray absorption spectroscopy analyses indicated that Cd within tissues was bound to S ligands with interatomic distances of 2.51–2.52 Å. These results confirm a strong linkage between S uptake and the production of LMWT upon exposure to Cd.