Aloe spp. – plants with vertebrate-like telomeric sequences

Chromosome termini of most eukaryotes end in tracks of short tandemly repeated GC-rich sequences, the composition of which varies among different groups of organisms. Plant species predominantly contain (TTTAGGG)_n repeats at their telomeres. However, a few plant species, including members of Alliaceae and Aloe spp. (Asphodelaceae) were found to lack such Arabidopsis-type (T₃AG₃)_n telomeric repeats. Recently, it has been proposed that the lack of T₃AG₃ telomeric repeat sequences extends to all species forming the Asparagales clade. Here, we analysed the composition of Aloe telomeres by single-primer PCR and fluorescence in-situ hybridization (FISH) with directly labelled Arabidopsis-type (TTTAGGG)_28–43 DNA probe, and with vertebrate-type (TTAGGG)_33–50 DNA and a (C₃TA₂)₃ peptide nucleic acid (PNA) probe. It was found that Nicotiana tabacum contained Arabidopsis-type telomeric repeats, while Aloe telomeres lacked the corresponding FISH signals. Surprisingly, FISH with the highly specific vertebrate-type (C₃TA₂)₃ PNA probe resulted in strong T₂AG₃-specific FISH signals at the ends of chromosomes of both Aloe and Nicotiana tabacum, suggesting the presence of T₂AG₃ telomeric repeats in these species. FISH with a long (TTAGGG)_33–50 DNA probe also highlighted Aloe chromosome ends, while this probe failed to reveal FISH signals on tobacco chromosomes. These results indicate the presence of vertebrate-like telomeric sequences at the telomeres of Aloe spp. chromosomes. However, single-primer PCR with (T₂AG₃)₅ primers failed to amplify such sequences in Aloe, which could indicate a low copy number of T₂AG₃ repeats at the chromosome ends and/or their co-orientation and interspersion with other repeat types. Our results suggest that telomeres of plant species, which were thought to lack GC-rich repeats, may in fact contain variant repeat types. This revised version was published online in July 2006 with corrections to the Cover Date.     

Macroporous Silicon Electrical Sensor for DNA Hybridization Detection

Macroporous silicon (pore diameter 1-2 microm) was used in an electrical sensor for real time, label free detection of DNA hybridization. Electrical contacts were made exclusively on the back side of the substrate, which allowed complete exposure of the porous layer to DNA. Hybridization of a DNA probe with its complementary sequence produced a reduction in the impedance and a shift in the phase angle resulting from a change in dielectric constant inside the porous matrix and a modification of the depletion layer width in the crystalline silicon structure. The effect of the DNA charge on the response was corroborated using peptide nucleic acid (PNA), an uncharged analog of DNA. The sensitivity and selectivity of the device were characterized and the sensing properties of the porous layer alone were investigated using self-supporting macroporous silicon membranes.     

Difference in Ulex europaeus agglutinin I-binding activity of decay-accelerating factor detected in the stools of patients with colorectal cancer and ulcerative colitis

Expression of decay-accelerating factor (DAF, CD55), a complement-regulatory glycoprotein, is enhanced in colorectal-cancer (CC) cells and colonic epithelium in ulcerative colitis (UC), and stools from these patients contain increased amounts of DAF. Carbohydrate chains of glycoproteins are often altered during malignant transformation or inflammation. In this study, we investigated whether DAF molecules in patients with CC and those with UC differ with respect to oligosaccharide side chains. We analyzed DAF in stools and homogenates of colonic-tissue specimens obtained from patients with CC or UC using solid-phase enzyme-linked assay and Western blotting for reactivity with the lectins Ulex europaeus agglutinin I (UEA-I), wheat-germ agglutinin, peanut agglutinin, and concanavalin A. UEA-I bound to DAF in stools from patients with UC but not in that from the stools of CC patients, as demonstrated on the solid-phase enzyme-linked assay (P <.05, Mann-Whitney U test) and Western blotting. Binding of UEA-I was specifically inhibited by the addition of fucose. The difference in UEA-I reactivity with DAF was observed also in colonic-tissue homogenates from patients with UC and those with CC. DAF expressed in the mucosa and excreted into the stools of UC patients is different from that expressed in CC with regard to UEA-I reactivity. Future studies should be directed toward determining whether a qualitatively unique isoform of DAF is present, of which sugar chains are specific to CC in UC patients.     

肽核酸单体骨架修饰的研究进展

肽核酸(PNA)是DNA模拟物,能与DNA和RNA以序列特异性方式结合,在基因研究和基因治疗方面有广泛的应用前景。为了优化肽核酸的性质(如水溶性、对细胞的透膜能力、杂交专一性),许多骨架修饰的PNA被合成出来。该文综述了近年来肽核酸单体骨架修饰的合成研究进展。指出设计新型的PNA结构是改良PNA性能、拓宽DNA应用的主要途径。     

Identification of peanut agglutinin binding glycoproteins restricted to Hodgkin's disease-derived cell lines

Peanut agglutinin (PNA) binding glycoproteins from four Hodgkin's disease (HD)-derived cell lines and a variety of cell lines/peripheral blood cells representative of the lymphoid and myeloid lineages were identified by probing nitrocellulose membranes of SDS-PAGE separated NP40 solubilized cellular glycoproteins with [125I]-labelled PNA. The two Hodgkin's cell lines Ho and L428 demonstrated the most heterogeneous glycoprotein profiles each expressing 15 PNA binding glycoproteins, respectively. The two remaining Hodgkin's lines Co and L591 expressed only four glycoproteins each and these were all also commonly expressed by Ho and L428. Comparative analysis with all other cell types studied revealed the expression of five glycoproteins restricted to Ho (gp42, gp40, gp38, gp24 and gp22) and six restricted to L428 (gp180, gp75, gp40, gp38, gp24 and gp22). Four of these, gp40, gp38, gp24 and gp22 were commonly expressed by both Ho and L428. Of cell lines of myeloid lineage studied only the erythroleukemia cell line K562 expressed detectable glycoproteins also expressed by some of the Hodgkin's cell lines (gp110, gp96, gp50 and gp45). Only one glycoprotein, gp20 expressed by Ho was also commonly expressed by normal peripheral blood granulocytes. This limited study has thus succeeded in demonstrating for the range of cell types studied, that some glycoproteins with terminal D-galactose beta (1----3) N-acetyl galactosamine oligosaccharide sequences are apparently restricted to two of the HD cell lines. Moreover, the heterogeneous glycoprotein profiles obtained for the HD cell lines Ho and L428 suggests that galactosylation processes in these two cell lines is aberrant.     

Differential lectin-bindings in normal and precancerous epithelium and squamous cell carcinoma of the oral mucosa

In order to establish a useful and objective marker of malignancy of oral mucosa, the binding sites for Ulex europaeus agglutinin I (UEA-I). Bandeiraea simplicifolia agglutinin I (BSA-I) and peanut agglutinin (PNA) were comparatively examined in the surgical materials from the normal, dysplastic and cancerous epithelium of the oral mucosa by a novel lectin-antilectin immunoperoxidase method. Based on the staining patterns of the normal keratinized epithelium, UEA-I was regarded as the marker for the prickle cells, BSA-I for the cells in the upper prickle to the horny layers, and PNA for those in the basal layer. As the degree of dysplasia advanced, all layers of epithelium came to react with UEA-I and PNA, whereas the BSA-I binding was negative. Positive reactions for UEA-I and PNA were seen in most carcinoma cells other than the keratinizing foci stained by BSA-I. The results indicate that a UEA-I-positive reaction in the basal cells, a PNA-positive in the prickle cells and loss of receptor for BSA-I occur in the course of malignant transformation of oral mucosa, and that these lectins may be regarded as useful markers of oral epithelial cytoplasmic differentiation.     

Binding of FITC-labelled lectins to the gastrointestinal epithelium of the rat

Biotechnology uses lectin genes to transfect into crop plants for protection against insects and nematodes. On the other hand, the information is limited on lectin-binding properties of cells in the gastrointestinal tract. Therefore, binding of a panel of FITC-labelled plant lectins to gastrointestinal cells of the rat was studied. In the stomach, cytoplasmic staining of parietal cells by PHA appeared to be due to glycoproteins attached to the tubulovesicles. PNA also stained the parietal cells, but only in the isthmus and neck regions, reacting with desialylated glycoproteins. WGA bound to the mucous neck cells with higher affinity than to the surface and foveolar mucous cells. The mucous cells were also stained by SNA-I, UEA-I and, less intensively, by LCA. Chief cells did not show detectable reaction with any of the applied lectins. Binding of PHA to gastric cells showed differences when compared with the results of in vivostudies. Small intestinal brush border was stained with UEA-I and SNA-I, the latter lectin also strongly stained the surface of small intestinal crypts. Both lectins reacted with the mucus of goblet cells. In the large intestine UEA-I and SNA-I stained the goblet cells at the base and upper part of the crypts, respectively. Accordingly, we provided evidences for the unique lectin-binding phenotype of the various segments of the gastrointestinal tract.     

Seven years of clinical experience with the Yeast Traffic Light PNA FISH: Assay performance and possible implications on antifungal therapy

We evaluated the performance of Yeast Traffic Light PNA FISH (YTL PNA FISH) in identification of Candida spp. from blood cultures. A total of 200 new episodes of candidaemia were analysed prospectively. The YTL PNA FISH results were reported to the clinicians and data on antifungal therapy were documented. In total, there were 164/200 (82%) positive blood culture bottles with monomicrobial growth. Coverage of monomicrobial yeast was 150/164 (91.5%). YTL PNA FISH could identify 23/24 (95.8%) Candida spp. in bottles with concomitant growth of bacteria and one yeast. Growth of two or more different yeast was observed in 12/200 (6%) blood culture bottles and the method could identify all yeast in 8/12 (66.7%). Data on antifungal treatment were available for 181/200 patients (90.5%). In 132/137 (96.4%) samples from patients without antifungal treatment, YTL PNA FISH could identify the Candida spp. or gave a negative result for yeast not included in panel, and based on the result guide appropriate antifungal therapy the same day when the blood culture bottle signalled positive. This study shows that YTL PNA FISH is a rapid, reliable diagnostic method which significantly reduces time delay for choice of appropriate antifungal therapy for critically ill patients.     

Lectin binding to glycoproteins in the surface membrane of Schistosoma mansoni

A number of lectins were assessed for their ability to bind to glycoproteins in the surface membrane of Schistosoma mansoni. The membrane polypeptides were separated by SDS-PAGE and the glycoproteins visualised by incubating the gel with radio-iodinated lectin followed by autoradiography. Most of the individual lectins bound to a variety of glycoproteins but peanut agglutinin and Dolichos biflorus agglutinin bound preferentially to a single glycoprotein of apparent molecular weight 170 000. This glycoprotein was subsequently shown to be exposed at the surface of the parasite and localised at the tubercles.