Proteomics of the aqueous humor in healthy New Zealand rabbits

There are several physiological roles postulated for aqueous humor, a liquid located in the anterior and posterior chamber of the eye, such as maintenance of the intraocular pressure, provision of nutrients, and removal of metabolic waste from neighboring tissues and provision of an immune response and protection during inflammation and infection. To link these function to specific or classes of proteins, identification of the aqueous humor proteome is essential. Aqueous humor obtained from healthy New Zealand white rabbits was analyzed using three synergistic protein separation methods: 1-D gel electrophoresis, 2-DE, and 1-DLC (RPLC) prior to protein identification by MS. As each of these separation methods separates intact proteins based on different physical properties (pIs, molecular weights, hydrophobicity, solubility, etc.) the proteome coverage is expanded. This was confirmed, since overlap between all three separation technologies was only about 8.2% with many proteins found uniquely by a single method. Although the most dominant protein presented in normal aqueous humor is albumin, by using this extensive separation/MS strategy, additional proteins were identified in total amount of 98 nonredundant proteins (plus an additional ten proteins for consideration). This expands the current protein identifications by approximately 65%. The aqueous humor proteome comprises a specific selection of cellular and plasma based proteins and can almost exclusively be divided into four functional groups: cell-cell interactions/wound healing, proteases and protease inhibitors, antioxidant protection, and antibacterial/anti-inflammatory proteins.     

Changes in antioxidants and cell damage in heterotrophic maize seedlings differing in drought sensitivity after exposure to short-term osmotic stress

Seedlings of two cultivars of maize (Zea mays L.) differing in their drought sensitivity were exposed to osmotic stress (0.3 M sorbitol, −1.4 MPa) for 4, 8, 12, 24 and 48 h during their heterotrophic stage of development. Alterations in their antioxidant pools combined with the activities of enzymes involved in defence against oxidative stress were investigated. Significant activation of antioxidative defence mechanisms correlated with drought-induced oxidative stress tolerance, and this phenomenon was shown to be characteristic of the drought-tolerant cv. Nova. Activities of some ROS-scavenging enzymes, superoxide dismutase (SOD), guaiacol peroxidase (POX), catalase (CAT) and ascorbate peroxidase (APX) were already enhanced significantly 4 h after the start of drought exposure in the drought-tolerant cv. Nova. Furthermore, a significant increase in the ascorbate pool was observed in this cultivar. On the other hand, in the drought-sensitive cv. Ankora only SOD and POD activities and the thiol pool were increased. No changes in APX activity or the level of ascorbate were recorded in cv. Ankora. Studies of root cell viability indicated that marked oxidative damage appeared only in cv. Ankora. These results, together with our previous observations, confirmed the higher ability of cv. Nova to tolerate drought stress and cope effectively with oxidative damage.     

Total synthesis of a protected form of sphingofungin E using the [3,3]-sigmatropic rearrangement of an allylic thiocyanate as the key reaction

An approach to the stereocontrolled synthesis of the protected form of sphingofungin E (32) starting from the known protected d-glucose derivative 3 is described herein. For the construction of a tetrasubstituted carbon atom that is substituted with nitrogen, the [3,3]-sigmatropic rearrangement of thiocyanate 8 was employed. Subsequent functional group interconversions afforded the highly functionalized fragment, allylic bromide 26. Its coupling reaction with the known C₁₂ hydrophobic segment 2, followed by further manipulation, completed the total synthesis.     

Cytotaxonomic study of theVicia cracca complex 1. Czechoslovak taxa

Three races ofVicia cracca L. have been found on the territory of Czechoslovakia, two with the chromosome number 2n=14 and one with 2n=28. Diploid races seem to be more primitive and are less widely distributed. They occur mostly in the primary communities while the tetraploid race is very plastic in the ecological respect and is common both in primary and secondary communities. These races are characterized by some morphological and ecological features. As far as the vertical distribution is concerned, one of the diploid races and the tetraploid occur mostly in the lowland and in the colline zone, the second diploid race has a mountain character. The related tetraploid speciesVicia oreophila ?ertová, with the chromosome number 2n=28 is distributed at similar altitudes.     

A modified procedure for the detection of microbial producers of extracellular proteolytic enzymes

Summary A simple procedure for the detection of microbial producers of proteolytic enzymes using dyed gelatin microcarrier particles incorporated into appropriate nutrient agar is described. Extracellular proteinases produced by the tested microbial strains hydrolyzed the substrate and clear dyed zones around and under the colonies were formed.     

New and critical species ofVicia in Iran and neighbouring countries 2. TheVicia ciceroidea group

In Iran, Iraq, Turkey and adjacent southern parts of the U.S.S.R., four species of the genusVicia, group ofV. ciceroidea, occur, namelyV. multijuga (Boiss.)Rech. f.,V. ciceroidea Boiss.,V. rafigae Tamam?jan and the new speciesV. sojakii described here. This group of species is distinguished by its tendency to reduction of leaflets, broadening of leaf rhachis and dense branching of stems. With respect to these particular properties it is now convenient to esparate these species as a seriesCiceroideae in the sectionCracca.     

Differential effect of phosphatidylethanolamine depletion on raft proteins: Further evidence for diversity of rafts in Saccharomyces cerevisiae

A considerable amount of evidence supports the idea that lipid rafts are involved in many cellular processes, including protein sorting and trafficking. We show that, in this process, also a non-raft lipid, phosphatidylethanolamine (PE), has an indispensable function. The depletion of this phospholipid results in an accumulation of a typical raft-resident, the arginine transporter Can1p, in the membranes of Golgi, while the trafficking of another plasma membrane transporter, Pma1p, is interrupted at the level of the ER. Both these transporters associate with a Triton (TX-100) resistant membrane fraction before their intracellular transport is arrested in the respective organelles. The Can1p undelivered to the plasma membrane is fully active when reconstituted to a PE-containing vesicle system in vitro. We further demonstrate that, in addition to the TX-100 resistance at 4 °C, Can1p and Pma1pa exhibit different accessibility to nonyl glucoside (NG), which points to distinct intimate lipid surroundings of these two proteins. Also, at 20 °C, these two proteins are extracted by TX-100 differentially. The features above suggest that Pma1p and Can1p are associated with different compartments. This is independently supported by the observations made by confocal microscopy. In addition we show that PE is involved in the stability of Can1p-raft association.     

Structural basis of interactions between human glutamate carboxypeptidase II and its substrate analogs

Human glutamate carboxypeptidase II (GCPII) is involved in neuronal signal transduction and intestinal folate absorption by means of the hydrolysis of its two natural substrates, N-acetyl-aspartyl-glutamate and folyl-poly-γ-glutamates, respectively. During the past years, tremendous efforts have been made toward the structural analysis of GCPII. Crystal structures of GCPII in complex with various ligands have provided insight into the binding of these ligands, particularly to the S1′ site of the enzyme. In this article, we have extended structural characterization of GCPII to its S1 site by using dipeptide-based inhibitors that interact with both S1 and S1′ sites of the enzyme. To this end, we have determined crystal structures of human GCPII in complex with phosphapeptide analogs of folyl-γ-glutamate, aspartyl-glutamate, and γ-glutamyl-glutamate, refined at 1.50, 1.60, and 1.67 Å resolution, respectively. The S1 pocket of GCPII could be accurately defined and analyzed for the first time, and the data indicate the importance of Asn519, Arg463, Arg534, and Arg536 for recognition of the penultimate (i.e., P1) substrate residues. Direct interactions between the positively charged guanidinium groups of Arg534 and Arg536 and a P1 moiety of a substrate/inhibitor provide mechanistic explanation of GCPII preference for acidic dipeptides. Additionally, observed conformational flexibility of the Arg463 and Arg536 side chains likely regulates GCPII affinity toward different inhibitors and modulates GCPII substrate specificity. The biochemical experiments assessing the hydrolysis of several GCPII substrate derivatives modified at the P1 position, also included in this report, further complement and extend conclusions derived from the structural analysis. The data described here form an a solid foundation for the structurally aided design of novel low-molecular-weight GCPII inhibitors and imaging agents.