Isolation, molecular and biological properties of a lectin from rice embryo: relationship with wheat germ agglutinin properties

Rice lectin (Oryza sativa, var. Balilla 28) was purified from defatted embryos by aqueous acid extraction at pH 1.3 followed by ammonium sulfate precipitation between 2 and 4 M, affinity chromatography on agarose-p-aminophenyl-beta-D-N-acetylglucosamine, and gel filtration on AcA 54. The homogeneity of the lectin was checked by polyacrylamide gel electrophoresis, gel filtration, and immunodiffusion. The amino acid analysis revealed a high half-cystine content (9%) and a low aromatic and hydrophobic amino acid content. The lectin contained neither neutral carbohydrates nor amino sugars. The isoelectric point was estimated to be 8.1. The molecular weight of rice lectin was estimated to be 38,000. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis under reducing conditions showed two polypeptides with Mr 19,000 and 15,000. The circular dichroism spectrum of rice lectin in far ultraviolet was characterized by a positive maximum at 228 nm and a negative band at 203 nm suggesting the presence of a beta-pleated sheet and the absence of alpha-helix. Rice lectin had no human blood group specificity and agglutinated rabbit erythrocytes more efficiently than erythrocytes from other animal species. Furthermore, agglutination was enhanced by trypsin treatment of erythrocytes. The erythroagglutinating activity was very high since the minimal concentration needed to agglutinate erythrocytes was 0.05 micrograms/ml. Although [methyl-3H]thymidine incorporation was stimulated in human lymphocytes, rice lectin could not be considered as a mitogenic lectin since it stimulated neither blast transformation nor lymphocyte proliferation. The saccharide specificity of rice lectin was related to N-acetylglucosamine and its oligomers: N,N',N"-triacetylchitotriose was the most powerful inhibitor. Furthermore, the N-acetylneuraminic acid was not a specific rice determinant. Finally, the double immunodiffusion method revealed a cross-reactivity between rice lectin and wheat germ agglutinin, indicating that these lectins were closely antigenically related. The analogies and differences between biological and immunological properties of rice lectin and wheat germ agglutinin are discussed and the possibility of their evolution from a common ancestor is put forward.     

The relevance of imidazole tautomerism for the hormonal activity of histidine-containing peptides

Substitution of 5-nitro- -histidine for -histidine is proposed as a useful tool to study the relationships among tautomerism, acid-base properties, and biological activity of peptide hormones. This approach is illustrated by an analog of the tripeptide thyroliberin, [5-nitro- -histidine]²-thyroliberin, which has been prepared by solid-phase peptide synthesis. The acid-base properties of the hormone analog and the position of the imidazole ring tautomeric equilibrium have been investigated by spectroscopic methods. Correlation of these properties with the biological activity of the nitrated tripeptide strongly supports the idea that imidazole ring tautomerism is a key factor for hormonal activity and that the N^τ-H tautomer must be considered the biologically active form of thyroliberin.     

Analysis of the pathways of nitric oxide utilization in mitochondria

The regulatory role that mitochondria play in cell dysfunction and cell-death pathways involves the concept of a complex and multisite regulation of cellular respiration and energy production signaled by cellular and intercellular messengers. Hence, the role of nitric oxide, as a physiological regulator acting directly on the mitochondrial respiratory chain acquires further relevance. This article provides a survey of the major regulatory roles of nitric oxide on mitochondrial functions as an expression of two major metabolic pathways for nitric oxide consumption: a reductive pathway, involving mitochondrial ubiquinol and yielding nitroxyl anion and an oxidative pathway involving superoxide anion and yielding peroxynitrite. The modulation of the decay pathways for nitrogen-and oxygen-centered radicals is further analyzed as a function of the redox transitions of mitochondrial ubiquinol. The interplay among these redox processes and its implications for mitochondrial function is discussed in terms of the mitochondrial steady-state levels (and gradients) of nitric oxide and superoxide anion.     

Differential gene expression in whitefly Bemisia tabaci-infested tomato (Solanum lycopersicum) plants at progressing developmental stages of the insect's life cycle

A suppression-subtractive-hybridization (SSH) strategy was used to identify genes whose expression was modified in response to virus-free whitefly Bemisia tabaci ( Bt , biotype A) infestation in tomato ( Solanum lycopersicum ) plants. Thus, forward and reverse SSH gene libraries were generated at four points in the whitefly's life cycle, namely at (1) 2 days (adult feeding and oviposition: phase I); (2) 7 days (mobile crawler stage: phase II); (3) 12 days (second to third instar nymphal transition: phase III) and (4) 18 days (fourth instar nymphal stage: phase IV). The 169 genes with altered expression (up and downregulated) that were identified in the eight generated SSH libraries, together with 75 additional genes that were selected on the basis of their involvement in resistance responses against phytofagous insects and pathogens, were printed on a Nexterion^® Slide MPX 16 to monitor their pattern of expression at the above phases. The results indicated that Bt infestation in tomato led to distinctive phase-specific expression/repression patterns of several genes associated predominantly with photosynthesis, senescence, secondary metabolism and (a)biotic stress. Most of the gene expression modifications were detected in phase III, coinciding with intense larval feeding, whereas fewer changes were detected in phases I and IV. These results complement previously reported gene expression profiles in Bt -infested tomato and Arabidopisis, and support and expand the opinion that Bt infestation leads to the downregulation of specific defense responses in addition to those controlled by jasmonic acid.     

Magnetic Cross-Linked Enzyme Aggregates (mCLEAs) of Candida antarctica Lipase: An Efficient and Stable Biocatalyst for Biodiesel Synthesis

Enzyme-catalyzed production of biodiesel is the object of extensive research due to the global shortage of fossil fuels and increased environmental concerns. Herein we report the preparation and main characteristics of a novel biocatalyst consisting of Cross-Linked Enzyme Aggregates (CLEAs) of Candida antarctica lipase B (CALB) which are covalently bound to magnetic nanoparticles, and tackle its use for the synthesis of biodiesel from non-edible vegetable and waste frying oils. For this purpose, insolubilized CALB was covalently cross-linked to magnetic nanoparticles of magnetite which the surface was functionalized with –NH₂ groups. The resulting biocatalyst combines the relevant catalytic properties of CLEAs (as great stability and feasibility for their reutilization) and the magnetic character, and thus the final product (mCLEAs) are superparamagnetic particles of a robust catalyst which is more stable than the free enzyme, easily recoverable from the reaction medium and reusable for new catalytic cycles. We have studied the main properties of this biocatalyst and we have assessed its utility to catalyze transesterification reactions to obtain biodiesel from non-edible vegetable oils including unrefined soybean, jatropha and cameline, as well as waste frying oil. Using 1% mCLEAs (w/w of oil) conversions near 80% were routinely obtained at 30°C after 24 h of reaction, this value rising to 92% after 72 h. Moreover, the magnetic biocatalyst can be easily recovered from the reaction mixture and reused for at least ten consecutive cycles of 24 h without apparent loss of activity. The obtained results suggest that mCLEAs prepared from CALB can become a powerful biocatalyst for application at industrial scale with better performance than those currently available.     

Restoration and management for cliff‐nesting birds in Mediterranean mining sites: the Sand Martin case study

Habitat enhancement for birds is frequently implemented during mine site restoration. Cliff‐nesting birds often colonize anthropogenic environments such as mining areas (aggregate sites and quarries for aggregate and cement production). Mining activity can compromise breeding success, causing cliff‐nesting birds to depend on the management and restoration of mining areas. The objective of our study is to assess the importance of mine site habitats for Sand Martin conservation and reconcile mining activity with breeding success in Mediterranean environments. We studied Sand Martin breeding habitat preferences in mining areas at three spatial scales. At the mining site scale, we compared 10 mining sites with Sand Martin burrows with 19 mining sites without burrows. At the colony scale (vertical structures with colonies), we evaluated the relationships between the number of breeding pairs, number of burrows, and colony characteristics within 30 distinct Sand Martin colonies. At the burrow scale, we compared the characteristics of the available vertical structure with the areas used by Sand Martins. At the mining site scale, Sand Martins preferred more surface of water bodies, shorter distances to flowing water, older sites, and mining sites which produce aggregates instead of cement. At the colony scale, Sand Martins preferred southwest orientations and stockpiles to vertical extraction faces. At the burrow scale, birds preferred the most vertical areas of the face. Our results support the need for effective habitat restoration and improved management for more effective Sand Martin conservation within mining areas. Simple interventions can enhance habitat quality and conservation of cliff‐nesting birds.     

Helping decision making for reliable and cost‐effective 2b‐RAD sequencing and genotyping analyses in non‐model species

High‐throughput sequencing has revolutionized population and conservation genetics. RAD sequencing methods, such as 2b‐RAD, can be used on species lacking a reference genome. However, transferring protocols across taxa can potentially lead to poor results. We tested two different IIB enzymes (AlfI and CspCI) on two species with different genome sizes (the loggerhead turtle Caretta caretta and the sharpsnout seabream Diplodus puntazzo) to build a set of guidelines to improve 2b‐RAD protocols on non‐model organisms while optimising costs. Good results were obtained even with degraded samples, showing the value of 2b‐RAD in studies with poor DNA quality. However, library quality was found to be a critical parameter on the number of reads and loci obtained for genotyping. Resampling analyses with different number of reads per individual showed a trade‐off between number of loci and number of reads per sample. The resulting accumulation curves can be used as a tool to calculate the number of sequences per individual needed to reach a mean depth ≥20 reads to acquire good genotyping results. Finally, we demonstrated that selective‐base ligation does not affect genomic differentiation between individuals, indicating that this technique can be used in species with large genome sizes to adjust the number of loci to the study scope, to reduce sequencing costs and to maintain suitable sequencing depth for a reliable genotyping without compromising the results. Here, we provide a set of guidelines to improve 2b‐RAD protocols on non‐model organisms with different genome sizes, helping decision‐making for a reliable and cost‐effective genotyping.     

Diagnosis,Burden and Mortality of Pneumocystis jirovecii Pneumonia in Venezuela

Current Fungal Infection Reports - The aim of this work is to contribute to the knowledge of diagnosis, burden, and mortality of pneumocystosis or Pneumocystis jirovecii pneumonia (PCP) in...