Generation and epitope mapping of high-affinity scFv to eukaryotic elongation factor 1A by dual application of phage display.

To generate specific tools for, in particular, localization studies of the eukaryotic elongation factor 1A (eEF1A), we have applied phage display in various formats to affinity-improve and map epitopes of two previously isolated, low-affinity single-chain Fv (scFv) G3 and D1. The scFv differ in their reactivity toward the eEF1A isoforms, eEF1A-1 and eEF1A-2. By PCR-based randomization of six residues within the variable light chain CDR3 (LCDR3), and subsequent phage-based affinity-selection, two 'families' of affinity-improved scFv were obtained. The scFv of highest affinity, A8, has a Kd of 9 nM to eEF1A-1. Interestingly, two affinity-improved scFvs have abnormally short LCDR3 consisting of two and four residues compared to 11 in the parental scFv. Hence, the LCDR3 of the parental clones may play a modulating rather than a direct role in antigen-binding. Despite different preferences for the eEF1A isoforms, both families of scFv recognize antigenic determinant(s), which was mapped to residues 413-450 of eEF1A-1/2 by Western blot analysis of recombinant human eEF1A (hEF1A) fragments. Prior to the Western blotting analysis, the epitope location had been suggested using a novel approach where phage-antibody repertoire derived scFv were used to select phage-displayed peptides. Hereby, peptides containing a SFXD motif, matching the SFSD(414-418) sequence found in hEF1A-1 were isolated. The structure of eukaryotic EF1A from yeast indicates a discontinuous nature of the epitope with distal functional elements juxtaposed by the protein fold. Finally, the scFv A8 was applied for immunofluorescence studies of transformed human amnion cells and MCF-7 fibroblasts. In both cases a perinuclear localization of hEF1A was observed. No evidence for the reported nuclear localization of hEF1A was obtained.     

Water level fluctuations may decrease phosphate adsorption capacity of the sediment in oligotrophic high mountain lakes

The effect of sediment desiccation and re-wetting on phosphate adsorption and desorption properties was examined in two oligotrophic high mountain lakes (La Caldera and Río Seco, altitude ~3000 m) in the Sierra Nevada National Park, Spain. Decrease in phosphate sorption properties was observed in transects from the littoral zone to dry land (up to the high water mark) in both lakes concomitantly with loss of amorphous oxides of iron and aluminum as revealed by oxalate and dithionite extractions. X-ray diffraction did not indicate increased amounts of crystalline metal oxides, but there was a loss of fine particles (<20 μm) with distance from the shore, probably due to wind erosion. Likely this explains the loss of amorphous metal oxides as they are often enriched in the fine sediment fraction. Changes in P-speciation toward less available pools were also observed as a result of desiccation. When re-wetted under oxic conditions, the sediments, especially those closest to the shore, released phosphate to the overlying water. The loss of adsorption capacity for phosphate upon re-wetting was associated with loss of amorphous iron oxides, and the changes appear to be non-reversible. Thus, both desiccation and re-wetting will lead to a decrease in sediment phosphate sorption capacity and increased water level fluctuations, a possible scenario in climate change, can therefore increase P availability in the water column of these oligotrophic systems.     

The same regulatory point mutation changed seed-dispersal structures in evolution and domestication

It is unclear whether gene regulatory changes that drive evolution at the population and species levels [1-3] can be extrapolated to higher taxonomic levels. Here, we investigated the role of cis-regulatory changes in fruit evolution within the Brassicaceae family. REPLUMLESS (RPL, At5g02030) controls development of the replum, a structure with an important role in fruit opening and seed dispersal [6]. We show that reduced repla resembling the Arabidopsis rpl mutant correlated across the Brassicaceae with a point mutation in a conserved cis-element of RPL. When introduced in Arabidopsis, this nucleotide change specifically reduced RPL expression and function in the fruit. Conversely, Brassica RPL containing the Arabidopsis version of the cis-element was sufficient to convert the Brassica replum to an Arabidopsis-like morphology. A mutation in the same nucleotide position of the same cis-element in a RPL ortholog has been independently selected to reduce seed dispersal during domestication of rice, in spite of its very different fruit anatomy. Thus, single-nucleotide regulatory mutations at the same position explain developmental variation in seed-dispersal structures at the population and family levels and suggest that the same genetic toolkit is relevant to domestication and natural evolution in widely diverged species.     

Genetic variation in cultivars of diploid ryegrass,Lolium perenne andL. multiflorum,at five enzyme systems

Summary Samples of approximately 100 plants from each of 22 populations ofLolium perenne representing 15 cultivars, and from 13 populations ofLolium multiflorum representing six cultivars were scored for iso-zyme variants in five enzyme systems, PGI, GOT, ACP, PGM and 6-PGD. From the individual banding patterns a genetic interpretation of the variation was formulated and population studies of the resulting six polymorphic enzyme loci were performed. No strong indications of partial selfing was found since at four of the six loci,Pgi 2, Got 3, Pgm 1 andPgd 1, the genotypic proportions were in correspondence with the Hardy-Weinberg expectations. This indicated, further, that the genetical interpretations of the banding patterns might be correct. Deviations from Hardy-Weinberg proportions forAcp 1 andGot 2 indicated presumably selection working on the linkage group including these loci. Gametic phase disequilibrium was observed betweenPgi 2 andPgd 1 for populations of one cultivar. These results were discussed in relation to the variation expected within a cultivar.     

Proteomic strategies to reveal tumor heterogeneity among urothelial papillomas

Proteomics and immunohistochemistry were used to reveal tumor heterogeneity among urothelial papillomas (UPs) with the long term goal of predicting their biological potential in terms of outcome. First, we identified proteins that were deregulated in invasive fresh lesions as compared with normal urothelium, and thereafter we immunostained UPs with a panel of antibodies against some of the markers. Twenty-two major proteins showing variations of 2-fold or more in at least one-third of the invasive lesions were selected. Specific antibodies against several of the proteins were obtained, but only a few reacted positively in immunostaining. A panel consisting of antibodies against keratinocytes (CKs) 5, 13, 18, and 20 and markers of squamous metaplasia (CKs 7, 8, and 14) was used to probe normal urothelium and 30 UPs collected during a period of five years. Four UPs showed a normal phenotype, whereas the rest could be grouped in five major types that shared aberrant staining with the CK20 antibody. Type 1 heterogeneity (n = 4) showed preferred staining of the umbrella cells with the CK8 antibody. Type 2 (n = 11) was typified by the staining of the basal and intermediate layers with the CK20 antibody. Type 3 (n = 7) was characterized by the predominant staining of the basal cell layer with the CK5 antibody. Type 4 (n = 1) showed areas of CK7 negative cells, whereas type 5 (n = 3) showed loss of staining of the basal cells with the CK20. 29% of the patients experienced recurrences, but none progressed to invasive disease. Patients harboring phenotypic alterations in the basal cell compartment (types 3 and 5) showed the highest number of recurrences (4/7 and 2/3, respectively), and all type 3 lesions progressed to a higher degree of dedifferentiation. Even though a long term prospective study involving a larger sample size is required to assess the biological potential of these lesions, we believe that this approach will prove instrumental for revealing early phenotypic changes in different types of cancer.     

The effect of prolonged stress-inducing electrical stimulation on cell proliferation in rat palatal epithelium

Abstract. This study examines whether blocking of cell division by stress-inducing electrical stimulation can be maintained for a prolonged period of time and whether this results in the accumulation of a large number of cells ready to divide when the block is released.
A total of 96 rats was subjected to electrical stimulation at intervals of 30 min for periods of 2,4,6 or 12 hr. Groups of animals were killed every second hour during these stimulation periods. After 2, 4 and 6 hr groups of animals were exempted from further stimulation and killed 2 or 4 hr later. After termination of the 12 hr stimulation period, groups of animals were killed every second hour during the succeeding 24 hr. Forty-eight animals were left undisturbed and served as controls. Two and a half hours prior to death, each of the experimental and control animals was injected intraperitoneally (i.p.) with colchicine. Histological sections of the palatal mucosa were prepared and the numbers of arrested metaphases were counted.
The electrical stimulation for 2, 4, 6 and 12 hr consistently resulted in a reduced number of cells entering mitosis, indicating a blockade at the G₂/M transition. However, with an increase in the length of electrical stimulation, an increasing number of cells passed the block, while a decreasing number entered mitosis during the first 4 hr following cessation of the stimulation. The sum of metaphases accumulated during the various stress periods and the following 4 hr was always similar and consistently lower than the number of metaphases accumulated in the control animals within the same time periods. This observation indicates that, during stress-inducing electrical stimulation and the succeeding 4 hr, only a defined number of cells can enter the mitotic phase. Thus, in addition to the block at the G₂/M transition, there appears to be another block of cell division at an earlier stage in the cell cycle.     

VsENOD5, VsENOD12 and VsENOD40 expression during Rhizobium-induced nodule formation on Vicia sativa roots

We isolated ENOD5, ENOD12 and ENOD40 homologues from Vicia sativa and studied their expression pattern during Rhizobium-induced nodule formation. Comparison of the VsENOD40 nucleotide sequence with the pea, soybean and alfalfa ENOD40 sequences showed that the sequences contain two conserved regions, called region I and region II. Comparison of all the potential open reading frames (ORFs) showed that all the five different ENOD40 clones encode a highly conserved small polypeptide of 12 or 13 amino acids encoded by an ORF located in region I. Furthermore we studied with in situ hybridization the expression pattern of VsENOD5, VsENOD12 and VsENOD40 during Rhizobium-induced nodule formation. Although the expression of these genes is largely similar to that of the pea counterparts, differences where found for the expression of VsENOD12 and VsENOD40 in Vicia. VsENOD12 is expressed in the whole prefixation zone II, whereas in pea ENOD12 is only expressed in the distal part of this zone. VsENOD40 is expressed in the uninfected cells of interzone II–III; while in pea ENOD40 is expressed in both the uninfected and infected cells of this zone.