A detailed study of the periodate oxidation of sialic acids in glycoproteins

Periodate oxidation of terminalN-acetyl- andN-glycoloylneuraminic acid residues in the mucins from edible bird nest substance and pig submandibular gland, respectively, can be carried out under conditions which exclusively give rise to the formation of the C-7 analogues of these sialic acids. In contrast, the C-8 compounds can be obtained in a maximum yield of about 40%. Under identical conditions,N-glycoloylneuraminic acid is oxidized about 1.5 times faster than theN-acetylated derivative. After release of the sialic acids by acid hydrolysis, the characterization of the oxidation products was carried out by TLC, by GLC and GLC-MS of the corresponding pertrimethylsilyl derivatives, and by 500-MHz¹H-NMR spectroscopy. In addition, molar response factors for GLC analysis and extinction coefficients in the orcinol/Fe³⁺/HCl assay were determined.     

Peripheral benzodiazepine binding sites: Effect of PK 11195, 1-(2-chlorophenyl)-n-methyl-n-(1-methylpropyl)-3-isoquinolinecarboxamide: I. In vitro studies

[³H] R05-4864 binding sites have been characterized in kidney, heart, brain, adrenals and platelets in the rat. In all these organs the following order of potency in the R05-4864 displacement was found : R05-4864 > diazepam > clonazepam indicating that they correspond to the “peripheral type” of benzodiazepine binding sites. PK 11195, an isoquinoline carboxamide derivative, displaces [³H] R05-4864 from its binding sites in all the organs. PK 11195 was as potent as R05-4864 in the platelets, heart, adrenals, kidney and several brain regions (midbrain, hypothalamus, medulla + pons and hippocampus. However it was 5 to 10 times more effective in cortex and striatum. In conclusion PK 11195 might represent a new tool to elucidate the physiological relevance of “peripheral type” benzodiazepine binding sites and might help to discriminate the hypothetical subclasses of these binding sites.     

Patterns of divergence during evolution of alpha 1-proteinase inhibitors in mammals

alpha 1-Proteinase inhibitor (alpha 1-PI), a member of the serine proteinase inhibitor superfamily, has a primary role in controlling neutrophil elastase activity within the mammalian circulation. Several studies have indicated that the reactive center region of alpha 1-PI, the amino acid sequence of which is critical to recognition of and binding to target proteinases, is highly divergent within and among species. This appears to be a consequence of accelerated rates of evolution that may have been driven by positive Darwinian selection. In order to examine this and other features of alpha 1-PI evolution in more detail, we have isolated and sequenced cDNAs representing alpha 1- PI mRNAs of the mouse species Mus saxicola and Mus minutoides and have compared these with a number of other mammalian alpha 1-PI mRNAs. Relative to other mammalian mRNAs, the extent of nonsynonymous substitution is generally high throughout the alpha 1-PI mRNA molecule, indicating greater overall rates of amino acid substitution. Within and among mouse species, the 5'-half of the mRNA, but not the 3'-half, has been homogenized by concerted evolution. Finally, the reactive center is under diversifying or positive Darwinian selection in murid rodents (rats, mice) and guinea pigs yet is under purifying selection in primates and artiodactyls. The significance of these findings to alpha 1-PI function and the possible selective forces driving evolution of serpins in general are discussed.      

Scaling-up of a B-phycoerythrin production and purification bioprocess involving aqueous two-phase systems: Practical experiences

One of the most attractive segments in food and cosmetic industries is that of natural pigments. Since some synthetic pigments have been reported to be hazardous for humans, natural pigments obtained through biotechnological processes represent an attractive alternative. Our research group has previously worked on the development of an aqueous two-phase system (ATPS)-based prototype process for the recovery of B-phycoerythrin (BPE), a natural high-value pigment obtained from Porphyridium cruentum. Detailed studies describing the scaling up of ATPS processes from bench scale to pilot plant facilities are not common. In this paper experiences derived from the scale-up of a previously developed process for production and recovery of highly purified (purity defined as the absorbance ratio A₅₄₅/A₂₈₀ > 4) BPE are described, where a scale-up factor of 850× was implemented. Characterization of cell disruption with a pilot-scale bead mill allowed efficient BPE release at 2900 rpm, 10% (w/v) sample load, 60% (v/v) bead load and 0.5 mm glass beads and 22 min of residence time with a yield of 1.35 mg BPE/g of wet biomass. BPE was recovered and purified using a strategy comprising isoelectric precipitation, aqueous two-phase fractionation and ultrafiltration. A 54% global BPE recovery yield, with final purity of 4.1, was achieved under optimal process conditions. Considering total costs for raw materials and energy expenditures for one batch, it was determined that the production cost of BPE was of $1.17 USD/mg, which is underneath the commercial price of a BPE standard (>$30 USD/mg).     

Direct and Indirect Effects of Climate on Demography and Early Growth of Pinus sylvestris at the Rear Edge: Changing Roles of Biotic and Abiotic Factors

Global change triggers shifts in forest composition, with warming and aridification being particularly threatening for the populations located at the rear edge of the species distributions. This is the case of Scots pine (Pinus sylvestris) in the Mediterranean Basin where uncertainties in relation to its dynamics under these changing scenarios are still high. We analysed the relative effect of climate on the recruitment patterns of Scots pine and its interactions with local biotic and abiotic variables at different spatial scales. Number of seedlings and saplings was surveyed, and their annual shoot growth measured in 96 plots located across altitudinal gradients in three different regions in the Iberian Peninsula. We found a significant influence of climate on demography and performance of recruits, with a non-linear effect of temperature on the presence of juveniles, and a positive effect of precipitation on their survival. Abundance of juveniles of P. sylvestris that underwent their first summer drought was skewed towards higher altitudes than the altitudinal mean range of the conspecific adults and the optimum elevation for seedlings'' emergence. At local level, light availability did not influence juveniles'' density, but it enhanced their growth. Biotic interactions were found between juveniles and the herb cover (competition) and between the number of newly emerged seedlings and shrubs (facilitation). Results also highlighted the indirect effect that climate exerts over the local factors, modulating the interactions with the pre-existing vegetation that were more evident at more stressful sites. This multiscale approach improves our understanding of the dynamics of these marginal populations and some management criteria can be inferred to boost their conservation under the current global warming.     

Properties of Confined Square-well Fluids using the Gibbs Ensemble Simulation Technique

In this work we have used the extension of the Gibbs ensemble simulation technique to inhomogeneous fluids [Panagiotopoulos, A.Z. (1987) "Adsorption and capillary condensation of fluid in cylindrical pores by Monte Carlo simulation in the Gibbs ensemble", Mol. Phys. , 62 (3), 701-719], which has been applied to adsorption phenomena of confined fluids. Fluid molecules are described by spherical particles interacting via a square-well potential. The fluid is confined in two types of walls: symmetrical (two hard walls) and non-symmetrical (one square-well wall and one hard wall). In order to analyze the behavior of the confined fluid by varying the potential parameters, we evaluated the bulk and confined densities, the internal energies and the density profiles for different supercritical temperatures. A variety of adsorption profiles can be obtained by using this model. The simulation data reported here complements the available simulation data for this system and can be useful in the development of inhomogeneous fluid theories. Since the square-well parameters can be related to real molecules this system can also be used to understand real adsorption systems.     

Bioprocess intensification: a potential aqueous two-phase process for the primary recovery of B-phycoerythrin from Porphyridium cruentum

A process for the primary recovery of B-phycoerythrin from Porphyridium cruentum exploiting aqueous two-phase systems (ATPS) was developed in order to reduce the number of unit operations and benefit from an increased yield of the protein product. The evaluation of system parameters such as poly(ethylene glycol) (PEG) molecular mass, concentration of PEG as well as salt, system pH and volume ratio was carried out to determine under which conditions the B-phycoerythrin and contaminants concentrate to opposite phases. PEG 1450-phosphate ATPS proved to be suitable for the recovery of B-phycoerythrin because the target protein concentrated to the top phase whilst the protein contaminants and cell debris concentrated in the bottom phase. An extraction ATPS stage comprising volume ratio (Vr) equal to 1.0, PEG 1450 24.9% (w/w), phosphate 12.6% (w/w) and system pH of 8.0 allowed B-phycoerythrin recovery with a purity of 2.9 (estimated as the relation of the 545-280 nm absorbances). The use of ATPS resulted in a primary recovery process that produced a protein purity of 2.9 +/- 0.2 and an overall product yield of 77.0% (w/w). The results reported demonstrated the practical implementation of ATPS for the design of a primary recovery process as a first step for the commercial purification of B-phycoerythrin produced by P. cruentum.     

Abundance and morphometry of tuberculosa Anadara and A. similis (Mollusca: Bivalvia) in the Manglar de Purruja, Dulce Gulf, Costa Rica

The density, population, length, yield and sex proportion of the mollusks Anadara tuberculosa and A. similis were studied in Bahía de Golfito, Golfo Dulce, Pacific coast of Costa Rica from February 1998 to February 1999. A. tuberculosa was more abundant (0.9 units m(-2)), than A. similis (0.2 units m(-2)); the highest abundance was found at the canal mouths. The average lengths were 43.3 mm for A. tuberculosa and 42.8 mm for A. similis (both under the Costa Rican legal minimal length for exploitation: 47 mm). Maximum lengths were measured in the middle and upstream Canal Mayor, respectively: 43.0 mm and 43.4 mm. The correlation between length and fresh weight was 0.81 (Pearson). The average total weights were 26.2 g for A. tuberculosa and 19.1 g for A. similis. The condition index (a meat yield measurement) was higher in A. similis (21.2%) than in A. tuberculosa (17.2%). The maximum yield for both species lies in the 31-35 mm range. The male ratio was 43.7%. A species recovery plan is urgent because these results suggest both a decrease in density and illegal exploitation.