Chondroitin-4-sulphate reduced oxidative injury in caerulein-induced pancreatitis in mice: the involvement of NF-kappaB translocation and apoptosis activation

Activation of nuclear factor kappaB (NF-kappaB) and caspases may greatly amplify inflammation and cell damage in addition to that directly exerted by free radicals. Since reactive oxygen species (ROS) are involved in acute pancreatitis, we studied whether the administration of chondroitin-4-sulphate (C4S), in addition to its antioxidant activity, was able to modulate NF-kappaB and caspase activation in an experimental model of caerulein-induced acute pancreatitis in mice. Hyperstimulating doses of caerulein (50 microg/ kg), five injections per mouse given at hourly intervals produced the following: high serum lipase and amylase activity; lipid peroxidation, evaluated by 8-isoprostane concentrations; loss of antioxidant defenses such as glutathione reductase (GR) activity; NF-kappaB activation and loss of cytoplasmic IkappaBalpha protein; increases in tumor necrosis factor-alpha (TNF-alpha), interleukin-6 (IL-6), caspase-3, and caspase-7 gene expression and their related protein; accumulation and activation of neutrophils in the damaged tissue, evaluated by elastase (ELA) determination; and pancreatic injury, evaluated by histologic analysis. Pretreatment of mice with different doses of C4S, given 1 hr before caerulein injections and 1 and 2 hrs after the last caerulein injection, reduced lipid peroxidation, inhibited NF-kappaB translocation and cytoplasmic IkappaBalpha protein loss, decreased TNF-alpha, IL-6, and caspase gene expression and their related protein levels, limited endogenous antioxidant depletion, and reduced tissue neutrophils accumulation and tissue damage. Since molecules with antioxidant activity can block NF-kappaB and apoptosis activation, we suggest that C4S administration is able to block NF-kappaB and caspase activation by reducing the oxidative burst.     

7alpha- and 12alpha-Hydroxysteroid dehydrogenases from Acinetobacter calcoaceticus lwoffii: a new integrated chemo-enzymatic route to ursodeoxycholic acid

We report the very efficient biotransformation of cholic acid to 7-keto- and 7,12-diketocholic acids with Acinetobacter calcoaceticus lwoffii. The enzymes responsible of the biotransformation (i.e. 7alpha- and 12alpha-hydroxysteroid dehydrogenases) are partially purified and employed in a new chemo-enzymatic synthesis of ursodeoxycholic acid starting from cholic acid. The first step is the 12alpha-HSDH-mediated total oxidation of sodium cholate followed by the Wolf-Kishner reduction of the carbonyl group to chenodeoxycholic acid. This acid is then quantitatively oxidized with 7alpha-HSDH to 7-ketochenodeoxycholic acid, that was chemically reduced to ursodeoxycholic acid (70% overall yield).     

Development and characterization of microsatellite markers in <Emphasis Type="Italic">Castanea sativa</Emphasis> (Mill.)

Thirty-three simple sequence repeat (SSR) markers were isolated andcharacterized in Castanea sativa (Mill.) from the cultivarGarrone Nero. For the identification of SSR loci, primers were designed on eachside flanking the repeat region and they were initially tested on 5 chestnutsamples using chemiluminescence detection. Twenty four loci where shown to bepolymorphic and the number of alleles detected per locus varied from 2 to 7.Fourteen loci were chosen for the analysis of 20 cultivars grown in North Italyusing the semi-automatic system ABI PRISM 377. These 14 markers showed a highlevel of genetic polymorphism with a total of 90 alleles; the number of allelesranged from 4 to 10 per locus, with an average level of 6.4. The mean expectedand observed heterozygosity were 0.724 (range: 0.649–0.835) and 0.793(range: 0.350–0.950) respectively. The estimated frequency of nullallelesshowed a positive value for 3 loci, but except for 1 locus, the values wereverylow. The total value for the probability of identity was 7.04 ×10^–11. Paternity exclusion probability was very high (0.999),sufficiently high to study pollen flow.     

On the relationship between Pielou’s evenness and landscape dominance within the context of Hill’s diversity profiles

Entropy-related biodiversity indices deriving their conceptual basis from Shannon’s information theory have a long history of use in ecology for quantifying community structure and diversity. In addition, in the last two decades, numerous information–theoretical indices, such as the landscape dominance index, have been extensively applied to characterize landscape diversity in space and time. In this contribution, we offer a simple analytical relation between Pielou’s evenness J and landscape dominance D within the broader context of Hill’s parametric diversity family. Within this context, we recommend the use of Hill’s diversity number evenness E_1,0 to overcome the shortcomings both of Pielou’s evenness J and the landscape dominance index D.     

Protein carbonylation: 2,4-dinitrophenylhydrazine reacts with both aldehydes/ketones and sulfenic acids

Most of the assays for detection of carbonylated proteins, the most general and widely used marker of severe protein oxidation, involve derivatization of the carbonyl group with 2,4-dinitrophenylhydrazine (DNPH), which leads to formation of a stable dinitrophenyl hydrazone product. Here, by using a Cys-containing model peptide and high-resolution mass spectrometry, we demonstrate that DNPH is not exclusively selective for carbonyl groups, because it also reacts with sulfenic acids, forming a DNPH adduct, through the acid-catalyzed formation of a thioaldehyde intermediate that is further converted to an aldehyde. β-Mercaptoethanol prevents the formation of the DNPH derivative because it reacts with the oxidized Cys residue, forming the corresponding disulfide.     

Synthesis,cannabinoid receptor affinity,molecular modeling studies and in vivo pharmacological evaluation of new substituted 1-aryl-5-(1H-pyrrol-1-yl)-1H-pyrazole-3-carboxamides. 2. Effect of the 3-carboxamide substituent on the affinity and selectivity profile

New substituted 1-aryl-5-(1H-pyrrol-1-yl)-1H-pyrazole-3-carboxamides were synthesized by replacing the 2,4-dichlorobenzyl and cyclohexyl moieties at the 3-carboxamide nitrogen of the previously reported CB₁ receptor antagonists/inverse agonists 4 and 5. Several ligands showed potent affinity for the hCB₁ receptor, with K_i concentrations comparable to the reference compounds 1, 4 and 5, and exhibited CB₁ selectivity comparable to 1 and 2. Docking experiments and molecular dynamics (MD) simulations explained the potent hCB₁ binding affinity of compounds 31 and 37. According to our previous studies, 31 and 37 formed a H-bond with K3.28(192), which accounted for the high affinity for the receptor inactive state and the inverse agonist activity. The finding of inhibition of food intake following their acute administration to rats, supported the concept that the CB₁ selective compounds 4 and 52 act as antagonists/inverse agonists.     

Yeast-secreted Recombinant Extracellular Domain of Human CD105 Antigen is Able to Bind TGF-beta Type II Receptor In Vitro

Human CD105 antigen, a type I integral membrane glycoprotein, is expressed as homodimer and oligomer by human endothelial cells, and forms a heteromeric association with TGF-β signaling receptors I and II. Several mutations of CD105 antigen gene are involved in a vascular disorder known as hereditary hemorrhagic telangiectasia type 1. The proposed mechanism by which CD105 is involved in said disorder is haploinsufficiency. We report expression and characterization of human CD105 antigen extracellular domain in yeast Saccharomyces cerevisiae. Different strategies to influence the release of heterologous proteins in the medium, such as alteration of cell wall integrity or coexpression of protein disulfide isomerase, were addressed. Purified extracellular domain of human CD105 antigen retains capacity to bind human TGF-β receptor II in vitro.     

Distinct contractile and cytoskeletal protein patterns in the Antarctic midge are elicited by desiccation and rehydration

Desiccation presents a major challenge for the Antarctic midge, Belgica antarctica. In this study, we use proteomic profiling to evaluate protein changes in the larvae elicited by dehydration and rehydration. Larvae were desiccated at 75% relative humidity (RH) for 12 h to achieve a body water loss of 35%, approximately half of the water that can be lost before the larvae succumb to dehydration. To evaluate the rehydration response, larvae were first desiccated, then rehydrated for 6 h at 100% RH and then in water for 6 h. Controls were held continuously at 100% RH. Protein analysis was performed using 2‐DE and nanoscale capillary LC/MS/MS. Twenty‐four identified proteins changed in abundance in response to desiccation: 16 were more abundant and 8 were less abundant; 84% of these proteins were contractile or cytoskeletal proteins. Thirteen rehydration‐regulated proteins were identified: 8 were more abundant and 5 were less abundant, and 69% of these proteins were also contractile or cytoskeletal proteins. Additional proteins responsive to desiccation and rehydration were involved in functions including stress responses, energy metabolism, protein synthesis, glucogenesis and membrane transport. We conclude that the major protein responses elicited by both desiccation and rehydration are linked to body contraction and cytoskeleton rearrangements.