Physicochemical Aspects of Chitosan Dispersibility in Acidic Aqueous Media: Effects of the Food Acid Counter-Anion

Differences in formation of colloidal dispersions of chitosan in aqueous solutions of citric acid or lactic acid (25, 50 or 100 mM) were quantitatively studied. Protonation enthalpies, electrical conductivity and ζ-potential measurements were additionally undertaken, aiming at better understanding these differences at a molecular level. In dispersion kinetics assays, experimental data were well fitted (R²?>?0.9; MAPE?<?4 %) by a first-order kinetics model with two terms - one accounting for the fast, direct dispersion of biopolymers chains and another accounting for the slow dispersion of chains from lumps. In all cases, maximal dispersibility was reached after about 20?30 min of stirring. For both acids, the higher the acid concentration in the medium, the higher was the chitosan dispersibility. At a given acid concentration, chitosan showed higher dispersibility in lactic acid than in citric acid solutions. Protonation of chitosan -NH₂ groups was strongly exothermic, with ΔH values three times higher for citric acid (triprotic) than lactic acid (monoprotic) (ΔH?=??120 kJ?mol^{- 1} and ΔH?=??40 kJ?mol^{- 1}, respectively), indicating that chitosan -NH₂ protonation itself was not dependent on the type of acid. However, the electrical conductivity of suspensions of powdered chitosan in water evolved differently as these systems were titrated with citric or acid lactic. With citric acid, electrical conductivity remained virtually constant for acid concentration?<?of 15 mM, and then increased linearly as the acid concentration increased until 75 mM. Instead, with lactic acid, electrical conductivity progressively increased with increasing of acid concentration from 0 to 75 mM. The ζ-potential of chitosan dispersed particles was +28.5 mV and +52.1 mV in dispersions containing 10 mM of citric and lactic acids, respectively. The conjoint analysis of data from physicochemical analyses suggested that, contrarily to lactate anions, citrate anions bind more strongly on the electrical double layer of protonated, positively charged chains of chitosan, diminishing the inter-chains electrostatic repulsion, thus leading to a lower dispersibility of this polysaccharide in aqueous solutions of citric acid, compared to equimolar solutions of lactic acid.     

Human enzyme polymorphism on the Canary Islands. I. Gran Canaria Island population

Genetic polymorphism of seven red cell enzymes was examined in two Canary Island populations and in an Iberian peninsula sample. The more isolated insular population shows a considerable gene frequency heterogeneity when compared with the peninsular sample but both are within the range of European populations. The presence of certain rare alleles normally associated with blacks suggests an African component in the Canarian populations.     

Further evidence on the photodynamic and the novel non-photodynamic inactivation of uroporphyrinogen decarboxylase by uroporphyrin I.

The action of uroporphyrin I (URO I) on the activity of red cell uroporphyrinogen decarboxylase (URO-D) in the dark and under UV light was studied. Light-dependent-and light-independent inactivation was observed. Both effects increased at increasing concentrations of URO I, the former reached its maximum at 150 microM of sensitizer. At 100 microM of URO I, both light and dark inactivation were temperature dependent amounting to about 50% at 30-37 degrees C. The velocity of dark inactivation increased with increasing temperature in the range of 0 to 45 degrees C. Photoinactivation can be ascribed to primary oxidation of essential amino acids, very likely histidyl residues, followed by secondary inter or intrapeptide cross-linking. Dark inactivation could be the result of both oxidation and cross-linking (although to a less degree than that produced by light) and also direct inhibition of the enzyme by induced conformational changes at its active site through binding of the porphyrin to the protein. When the action of URO I was tested on partially purified URO-D, the enzyme appeared to be more susceptible to the dark than to the light effect.     

Multi-target novel neuroprotective compound ITH33/IQM9.21 inhibits calcium entry, calcium signals and exocytosis

Compound ITH33/IQM9.21 (ITH/IQM) belongs to a new family of l-glutamic acid derivatives with antioxidant and neuroprotective properties on in vitro and in vivo models of stroke. Because neuronal damage after brain ischemia is tightly linked to excess Ca²⁺ entry and neuronal Ca²⁺ overload, we have investigated whether compound ITH/IQM antagonises the elevations of the cytosolic Ca²⁺ concentrations ([Ca²⁺]_c) and the ensuing exocytotic responses triggered by depolarisation of bovine chromaffin cells. In fluo-4-loaded cell populations, ITH/IQM reduced the K⁺-evoked [Ca²⁺]_c transients with an IC₅₀ of 5.31 μM. At 10 μM, the compound decreased the amplitude and area of the Ca²⁺ transient elicited by challenging single fura-2-loaded cells with high K⁺, by 40% and 80%, respectively. This concentration also caused a blockade of K⁺-induced catecholamine release at the single-cell level (78%) and cell populations (55%). These effects are likely due to blockade of the whole-cell inward Ca²⁺ currents (IC₅₀ = 6.52 μM). At 10 μM, ITH/IQM also inhibited the Ca²⁺-dependent outward K⁺ current, leaving untouched the voltage-dependent component of I_K. The inward Na⁺ current was unaffected. Inhibition of depolarisation-elicited Ca²⁺ entry, [Ca²⁺]_c elevation and exocytosis could contribute to the neuroprotective effects of ITH/IQM in vulnerable neurons undergoing depolarisation during brain ischemia.     

Diabetes-induced increase of renal medullary hydrogen peroxide and urinary angiotensinogen is similar in normotensive and hypertensive rats

Aims

Activation of renal renin–angiotensin system (RAS) and reactive oxygen species (ROS) are the main pathophysiological mechanisms associated with kidney injury both in diabetes and hypertension. However, the contribution to medullary damage when the two pathologies coexist has seldom been explored.

Main methods

Diabetes was induced with streptozotocin in twelve week-old male Wistar and spontaneously hypertensive rats (SHR) rats; controls received vehicle. Three weeks later, systolic blood pressure (SBP), plasma and urinary angiotensinogen (AGT), renal oxidative stress and metabolic status were evaluated.

Key findings

SBP was higher in SHR-controls than in Wistar-controls (200 ± 6 and 127 ± 3 mmHg, respectively) and decreased in SHR-diabetics but not in Wistar-diabetics (143 ± 8 and 122 ± 6 mmHg, respectively). Renal medullary hydrogen peroxide (H₂O₂) production was similarly increased in diabetics (Wistar: 0.32 ± 0.04 and 1.11 ± 0.10 nmol/mg protein, respectively; SHR: 0.40 ± 0.05 and 0.90 ± 0.14 nmol/mg protein, respectively) and positively correlated with glycemia (Wistar: r = 0.7166, SHR: r = 0.7899, p < 0.05) and urinary AGT excretion (Wistar: r = 0.8333; SHR: r = 0.8326, p < 0.05). Cortical H₂O₂ production was higher in SHR-controls than in Wistar-controls (1.10 ± 0.09 and 0.26 ± 0.04 nmol/mg protein, respectively) and diabetes induction decreased it in SHR (0.70 ± 0.09 nmol/mg protein). Diabetes increased urinary AGT excretion by more than 7-fold and decreased plasma AGT concentration by more than 1.5-fold in both strains.

Significance

Our results show that STZ-induced diabetes increases medullary H₂O₂ production and urinary AGT excretion with similar magnitude in normotensive and hypertensive animals. Reducing blood pressure attenuates hypertension-associated cortical damage but does not prevent medullary dysfunction.     

Control and expression of cystatin C by mouse decidual cultures.

During mouse embryo implantation, trophoblast invasion is controlled in part by a balance of trophoblast-derived proteinases and uterine decidual proteinase inhibitors. Our work has focused on cystatin C, the secreted inhibitor of cathepsins B and L. We have previously shown that cystatin C is synthesized by the uterine decidua and localized to the cells in close contact with the trophoblast during implantation in vivo. In the work reported here we have established that decidualizing cultures show a similar upregulation of cystatin C. Using Northern and Western blotting and immunolocalization techniques both cystatin C mRNA and secreted protein increased with the morphological differentiation of stromal or decidual capsule cultures. In an effort to understand the regulation of cystatin C expression, decidual cells were analyzed under various culture conditions. Cystatin C expression was upregulated by increased cell density and by the presence of serum in the media. The growth factors TGF-beta(1) and EGF were found to induce cystatin C to levels comparable to serum stimulation. Co-culture with ectoplacental cones (EPCs) likewise induced expression and resulted in the localization of cystatin at the decidua:trophoblast interface. This work shows that decidualizing cultures are a good system to study cystatin C expression and that the expression is controlled in part by TGF-beta(1) and EGF signaling.     

The genome of a very virulent Marek's disease virus

Here we present the first complete genomic sequence, with analysis, of a very virulent strain of Marek's disease virus serotype 1 (MDV1), Md5. The genome is 177,874 bp and is predicted to encode 103 proteins. MDV1 is colinear with the prototypic alphaherpesvirus herpes simplex virus type 1 (HSV-1) within the unique long (UL) region, and it is most similar at the amino acid level to MDV2, herpesvirus of turkeys (HVT), and nonavian herpesviruses equine herpesviruses 1 and 4. MDV1 encodes 55 HSV-1 UL homologues together with 6 additional UL proteins that are absent in nonavian herpesviruses. The unique short (US) region is colinear with and has greater than 99% nucleotide identity to that of MDV1 strain GA; however, an extra nucleotide sequence at the Md5 US/short terminal repeat boundary results in a shorter US region and the presence of a second gene (encoding MDV097) similar to the SORF2 gene. MD5, like HVT, encodes an ICP4 homologue that contains a 900-amino-acid amino-terminal extension not found in other herpesviruses. Putative virulence and host range gene products include the oncoprotein MEQ, oncogenicity-associated phosphoproteins pp38 and pp24, a lipase homologue, a CxC chemokine, and unique proteins of unknown function MDV087 and MDV097 (SORF2 homologues) and MDV093 (SORF4). Consistent with its virulent phenotype, Md5 contains only two copies of the 132-bp repeat which has previously been associated with viral attenuation and loss of oncogenicity.     

Schistosoma haematobium total antigen induces increased proliferation, migration and invasion, and decreases apoptosis of normal epithelial cells

Schistosome worms are blood-dwelling flukes that cause chronic infection in more than 200 million people and are thought to be responsible for 500,000 deaths annually. During infection with Schistosoma haematobium, eggs are deposited in the mucosa and submucosa of the bladder and lower ureters. Squamous cell carcinoma (SCC) of the bladder is a long-term sequela of chronic infection. The mechanisms underlying the association between S. haematobium and SCC of the bladder are largely unknown, with all reports to date exclusively demonstrating epidemiological evidence linking S. haematobium infection with SCC of the bladder. We hypothesised that the parasite antigens might induce alterations in epithelial cells towards cancer. For this we used Chinese Hamster Ovary (CHO) cells and treated the cells in culture with S. haematobium total antigen (Sh). Our results showed increased proliferation, increased S-phase and decreased apoptosis, as well as down-regulation of tumour suppressor p27 and up-regulation of anti-apoptotic molecule Bcl-2 in Sh-treated cells compared with controls. We also found increased migration and invasion. To our knowledge, this is the first report demonstrating alterations of normal epithelial cells as a direct effect of S. haematobium antigens.     

Genetic parameters and genotype–environment interactions for skeleton deformities and growth traits at different ages on gilthead seabream (Sparus aurata L.) in four Spanish regions

One of the most important problems of fish aquaculture is the high incidence of fish deformities, which are mainly skeletal. In this study, genetic parameters on gilthead seabream (Sparus aurata L.) for skeleton deformities at different ages (179, 269, 389, 539 and 689 days) and their correlations with growth traits were estimated, as were as their genotype × environment interactions (G × E) at harvesting age. A total of 4093 offspring from the mass spawning of three industrial broodstocks belonging to the PROGENSA^® breeding programme were mixed and on‐grown by different production systems in four Spanish regions: Canary Islands (tanks and cage), Andalusia (estuary), Catalonia (cage) and Murcia (cage). Parental assignment was inferred using the standardized SMsa1 microsatellite multiplex PCR. From three broodstocks, 139 breeders contributed to the spawn and a total of 297 full‐sibling families (52 paternal and 53 maternal half‐sibling families) were represented. Heritabilities at different ages were medium for growth traits (0.16–0.48) and vertebral deformities (0.16–0.41), and low for any type of deformity (0.07–0.26), head deformities (0.00–0.05) and lack of operculum (0.06–0.11). The genetic correlations between growth and deformity traits were medium and positive, suggesting that to avoid increasing deformities they should be taken into account in breeding programmes when growth is selected. The G × E interactions among the different facilities were weak for length and deformity and strong for growth rate during this period. These results highlight the potential for the gilthead seabream industry to reduce the prevalence of deformities by genetic improvement tools.