Glycosylation sites and site-specific glycosylation in human Tamm- Horsfall glycoprotein

The N-glycosylation sites of human Tamm-Horsfall glycoprotein from one healthy male donor have been characterized, based on an approach using endoproteinase Glu-C (V-8 protease, Staphylococcus aureus ) digestion and a combination of chromatographic techniques, automated Edman sequencing, and fast atom bombardment mass spectrometry. Seven out of the eight potential N-glycosylation sites, namely, Asn52, Asn56, Asn208, Asn251, Asn298, Asn372, and Asn489, turned out to be glycosylated, and the potential glycosylation site at Asn14, being close to the N-terminus, is not used. The carbohydrate microheterogeneity on three of the glycosylation sites was studied in more detail by high-pH anion-exchange chromatographic profiling and 500 MHz1H-NMR spectroscopy. Glycosylation site Asn489 contains mainly di- and tri-charged oligosaccharides which comprise, among others, the GalNAc4 S (beta1-4)GlcNAc terminal sequence. Only glycosylation site Asn251 bears oligomannose-type carbohydrate chains ranging from Man5GlcNAc2to Man8GlcNAc2, in addition to a small amount of complex- type structures. Profiling of the carbohydrate moieties of Asn208 indicates a large heterogeneity, similar to that established for native human Tamm-Horsfall glycoprotein, namely, multiply charged complex-type carbohydrate structures, terminated by sulfate groups, sialic acid residues, and/or the Sda-determinant.      

Cytokinin-induced <Emphasis Type="Italic">VvTFL1A</Emphasis> expression may be involved in the control of grapevine fruitfulness

Grapevine bud fruitfulness is determined by the differentiation of uncommitted meristem (UCM) into either tendril or inflorescence. Since tendril and inflorescence differentiation have long been considered sequential steps in inflorescence development, factors that control the progression of floral meristem development may regulate the final outcome of UCM differentiation, and thus affect fruitfulness. A comparison of the expression profiles of the master regulators of floral meristem identity (FMI) during development of fruitful and non-fruitful buds along the same cane allowed associating the expression of a homolog of terminal flower 1 (TFL1, a negative regulator of FMI) to fruitful buds, and the expression of positive FMI regulators to non-fruitful buds. Combined with (a) cytokinin-induced upregulation of VvTFL1A expression in cultured tendrils, which accompanied cytokinin-derived tendril transformation into branched, inflorescence-like structures, (b) positive regulation of VvTFL1A expression by cytokinin, which was demonstrated in transgenic embryonic culture expressing GUS reporter under the control of VvTFL1A promoter, and (c) a significantly higher level of active cytokinins in fruitful positions, the data may support the assumption of cytokinin-regulated VvTFL1A activity’s involvement in the control of inflorescence development. Such activity may delay acquisition of FMI and allow an extended branching period for the UCM, resulting in the differentiation of inflorescence primordia.     

Phosphatase and ATPase activities in isonuclear lines of cytoplasmic male-sterile and male-fertile petunia

Summary Soluble and membrane-bound fractions of plant leaves, cell suspension cultures and seedlings of petunia were examined for phosphohydrolase activity on p-nitrophenyl phosphate (pNPPase) and adenosine triphosphate (ATPase). One cytoplasmic male-sterile (CMS) and one fertile (F) line was examined for each tissue. Both pNPPase and ATPase exhibited a broad optimal activity between pH 5.5–7.0 for the membrane-bound fraction and between 4.5–7.0 for the soluble fractions. The activity of both were inhibited by divalent ions including Mg²⁺. At pH 7.2, the activities on various triphosphonucleotides were similar and they were hydrolyzed by a rate of 20–50% of that of ATP. Significant differences between CMS and F extracts were: (a) higher activities in CMS membranes; (b) lower Ea (energy of activation) values for activities in CMS membrane functions; (c) seedling and cell-culture CMS extracts exhibited a higher sensitivity to high temperature denaturation; (d) the hydrolase activity on monoand triphospho-cytosine compounds was significantly higher in CMS than in F membranes.Contribution from the Agricultural Research Organization, The Volcani Center, Bet-Dagan, Israel No. 355-E, 1992 series     

Tentoxin sensitivity of citrus: cotyledon-dependency of growth inhibition and reversibility of chlorosis

Tentoxin is a cyclic tetrapeptide, produced by the fungus Alternariaalternata, that induces chiorosis in germinating seedlings ofsome angiosperms. Since the most pronounced chiorotic effectof tentoxin is at the initial stages of germination most studieshave evaluated the effects of tentoxin on cotyledons. In thispreliminary work a unique biological system was establishedfor the study of the mechanism of tentoxin induced chiorosisin developing citrus seedlings. This system was used to comparethe effects of tentoxin on the in vitro germination of intactversus decotyle donized embryos. It is demonstrated here thatthe chlorotic effect of tentoxin is reversible and that ten-toxin blocks the ability of decotyledonized embryos to utilizenutrients from the growth medium and, there fore, to compensatefor the lack of cotyledons. The citrus system offers a uniqueway to study the relation between the effect of tentoxin onthe activity of choloplast ATPase and the induction of chlorosis. Key words: Tentoxin, citrus, chiorosis     

Cysteine is the presumed catalytic residue of Citrus sinensis phospholipid hydroperoxide glutathione peroxidase over‐expressed under salt stress

A citrus salt‐stress associated protein (Cit‐SAP), partially purified from citrus cultured cells, was previously identified as the first plant phospholipid hydroperoxide glutathione peroxidase (PHGPx). The nucleotide sequence of its isolated gene ( csa ) revealed that a TGT, known as codon for Cys, encodes the presumed catalytic residue 41 in the polypeptide chain of Cit‐SAP. In animals, a TGA encodes the rare amino acid selenocysteine (Sec) as the catalytic residue of the analogous enzyme. It is of interest to establish whether the TGT codon for this catalytic residue in the plant enzyme is indeed translated to Cys and not to Sec, and to demonstrate the effect of such a change, if it exists, on the nature of the enzymatic activity of the plant enzyme as compared to that of the animal. In the present study, we have purified for the first time, by affinity chromatography, enzymatically active citrus PHGPx from recombinant Escherichia coli bearing the csa gene. Tryptic digestion of the purified enzyme followed by HPLC afforded the isolation of a peptide which contains residue 41, and its sequence analysis revealed that this residue is indeed a Cys, and not Sec. The enzymatic activity and specificity of the recombinant Cit‐SAP was found to be similar to that observed before for the partially purified plant enzyme. However, the rate of this activity was much lower towards phospholipid hydroperoxides, and none towards hydrogen peroxide, as compared to that of the animal analogue. It is therefore suggested that the presence of a Cys, and not Sec, as the catalytic residue in the plant enzyme, affects its enzymatic activity and may determine a different biological role for the plant PHGPx from that of the animal.     

Studies of genotoxicity and mutagenicity of nitroimidazoles: demystifying this critical relationship with the nitro group

Nitroimidazoles exhibit high microbicidal activity, but mutagenic, genotoxic and cytotoxic properties have been attributed to the presence of the nitro group. However, we synthesised nitroimidazoles with activity against the trypomastigotes of Trypanosoma cruzi, but that were not genotoxic. Herein, nitroimidazoles (11-19) bearing different substituent groups were investigated for their potential induction of genotoxicity (comet assay) and mutagenicity (Salmonella/Microsome assay) and the correlations of these effects with their trypanocidal effect and with megazol were investigated. The compounds were designed to analyse the role played by the position of the nitro group in the imidazole nucleus (C-4 or C-5) and the presence of oxidisable groups at N-1 as an anion receptor group and the role of a methyl group at C-2. Nitroimidazoles bearing NO2 at C-4 and CH3 at C-2 were not genotoxic compared to those bearing NO₂ at C-5. However, when there was a CH3 at C-2, the position of the NO2 group had no influence on the genotoxic activity. Fluorinated compounds exhibited higher genotoxicity regardless of the presence of CH3 at C-2 or NO2 at C-4 or C-5. However, in compounds 11 (2-CH3; 4-NO2; N-CH2OHCH2Cl) and 12 (2-CH3; 4-NO2; N-CH2OHCH2F), the fluorine atom had no influence on genotoxicity. This study contributes to the future search for new and safer prototypes and provide.     

WASp Family Verprolin-homologous Protein-2 (WAVE2) and Wiskott-Aldrich Syndrome Protein (WASp) Engage in Distinct Downstream Signaling Interactions at the T Cell Antigen Receptor Site

T cell antigen receptor (TCR) engagement has been shown to activate pathways leading to actin cytoskeletal polymerization and reorganization, which are essential for lymphocyte activation and function. Several actin regulatory proteins were implicated in regulating the actin machinery, such as members of the Wiskott-Aldrich syndrome protein (WASp) family. These include WASp and the WASp family verprolin-homologous protein-2 (WAVE2). Although WASp and WAVE2 share several structural features, the precise regulatory mechanisms and potential redundancy between them have not been fully characterized. Specifically, unlike WASp, the dynamic molecular interactions that regulate WAVE2 recruitment to the cell membrane and specifically to the TCR signaling complex are largely unknown. Here, we identify the molecular mechanism that controls the recruitment of WAVE2 in comparison with WASp. Using fluorescence resonance energy transfer (FRET) and novel triple-color FRET (3FRET) technology, we demonstrate how WAVE2 signaling complexes are dynamically regulated during lymphocyte activation in vivo. We show that, similar to WASp, WAVE2 recruitment to the TCR site depends on protein-tyrosine kinase, ZAP-70, and the adaptors LAT, SLP-76, and Nck. However, in contrast to WASp, WAVE2 leaves this signaling complex and migrates peripherally together with vinculin to the membrane leading edge. Our experiments demonstrate that WASp and WAVE2 differ in their dynamics and their associated proteins. Thus, this study reveals the differential mechanisms regulating the function of these cytoskeletal proteins.     

The effect of temperature and relative humidity on the formation of Metarhizium anisopliae chlamydospores in tick eggs

The influence of ambient conditions on the development of Metarhizium anisopliae chlamydospores in tick eggs is reported for the first time. The infection of tick eggs by M. anisopliae involves common events, such as adhesion, conidial germination, appressoria formation, invasion, and development within the eggs. However, the final stage of fungal development differs according to the environmental conditions. At high humidity (close to 100%) and moderate temperature (25°C) the fungus emerged from the eggs and formed conidiophores and conidia externally on the dead eggs. Elevating the temperature to 30°C or reducing humidity to 55-75% induced the production of chlamydospores inside the eggs, without conidiogenesis. When eggs with mature chlamydospores were returned to the appropriate conditions (25°C and 100% RH), conidiogenesis was recovered. Formation of chlamydospores, observed by means of histology and TEM, began with the thickening and septation of hyphae. As the chlamydospore wall thickened a new external undulated wall layer appeared. The mature chlamydospore in eggs has an oval shape (5.3 ± 0.9 microm long, 2.5 ± 0.2 microm wide); its wall comprises three distinct layers. The ability of M. anisopliae to produce chlamydospores under harsh conditions is advantageous and should be considered in application.