Characterization of Arabidopsis thaliana mutant ror-1 (roscovitine-resistant) and its utilization in understanding of the role of cytokinin N-glucosylation pathway in plants

Cytokinin analogue roscovitine exhibits a strong inhibitory effect on cytokinin N-glucosylation, one of the most important pathways of cytokinin inactivation in plants. Roscovitine-resistant mutant. (ror-1) was isolated using T-DNA tagged lines of Arabidopsis thaliana (L.) Heynh in order to find a gene putatively involved in cytokinin N-glucosylation. The amount of cytokinin N-glucosides of trans-zeatin- and isopentenyladenine-type was elevated by 20% in ror-1 mutant compared to WT. The cytokinin oxidase/dehydrogenase activity exhibited a mild elevation in ror-1 compared to WT in basal media. Additionally, ror-1 plants showed slightly enhanced resistance to exogenously supplied aromatic cytokinins (benzyladenine). Incubation with exogenous cytokinin (5 μM BA for 24 h) resulted in significant up-regulation of ROR-1 gene expression in ror-1 mutant. In silico analysis showed that ROR-1 gene encoded for a protein consisting of GRAM (Glycosyltransferases Rab-like GTPase activators and Myotubularins) and C2 domains. Here, we report on the role of ROR-1 gene in metabolism of bioactive cytokinins in the plants.     

Molecular Phylogeny Reveals High Diversity,Geographic Structure and Limited Ranges in Neotenic Net-Winged Beetles Platerodrilus (Coleoptera: Lycidae)

The neotenic Platerodrilus net-winged beetles have strongly modified development where females do not pupate and retain larval morphology when sexually mature. As a result, dispersal propensity of females is extremely low and the lineage can be used for reconstruction of ancient dispersal and vicariance patterns and identification of centres of diversity. We identified three deep lineages in Platerodrilus occurring predominantly in (1) Borneo and the Philippines, (2) continental Asia, and (3) Sumatra, the Malay Peninsula and Java. We document limited ranges of all species of Platerodrilus and complete species level turnover between the Sunda Islands and even between individual mountain regions in Sumatra. Few dispersal events were recovered among the major geographical regions despite long evolutionary history of occurrence; all of them were dated at the early phase of Platerodrilus diversification up to the end of Miocene and no exchange of island faunas was identified during the Pliocene and Pleistocene despite the frequently exposed Sunda Shelf as sea levels fluctuated with each glacial cycle. We observed high diversity in the regions with persisting humid tropical forests during cool periods. The origins of multiple species were inferred in Sumatra soon after the island emerged and the mountain range uplifted 15 million years ago with the speciation rate lower since then. We suppose that the extremely low dispersal propensity makes Platerodrilus a valuable indicator of uninterrupted persistence of rainforests over a long time span. Additionally, if the diversity of these neotenic lineages is to be protected, a high dense system of protected areas would be necessary.     

New oligo-β-(1,3)-glucan derivatives as immunostimulating agents

Oligo-β-(1,3)-glucans were chemically modified in order to introduce a structural variation specifically on the reducing end of the oligomers. The impact of well defined structural modulations was further studied on cancer cells and murin models to evaluate their cytotoxicity and immunostimulating potential.     

Monoclonal antibodies specific for the empty conformation of HLA-DR1 reveal aspects of the conformational change associated with peptide binding

Class II major histocompatibility complex (MHC) proteins bind peptides and present them at the cell surface for interaction with CD4+ T cells as part of the system by which the immune system surveys the body for signs of infection. Peptide binding is known to induce conformational changes in class II MHC proteins on the basis of a variety of hydrodynamic and spectroscopic approaches, but the changes have not been clearly localized within the overall class II MHC structure. To map the peptide-induced conformational change for HLA-DR1, a common human class II MHC variant, we generated a series of monoclonal antibodies recognizing the beta subunit that are specific for the empty conformation. Each antibody reacted with the empty but not the peptide-loaded form, for both soluble recombinant protein and native protein expressed at the cell surface. Antibody binding epitopes were characterized using overlapping peptides and alanine scanning substitutions and were localized to two distinct regions of the protein. The pattern of key residues within the epitopes suggested that the two epitope regions undergo substantial conformational alteration during peptide binding. These results illuminate aspects of the structure of the empty forms and the nature of the peptide-induced conformational change.     

Expression of class III beta-tubulin in neuroendocrine tumours of gastrointestinal tract

Class III b-tubulin is presented as a specific marker for the cells of neuronal origin as well as for the tumours originating from these cells. Its expression is considered one of the earliest events that appear in the cells revealing neuronal differentiation. Using monoclonal antibody TU-20 in an immunohistochemical analysis, we studied the expression of class III b-tubulin in gastrointestinal carcinoid tumours. Paraffin-embedded, formalin-fixed tissue sections from 49 tumour samples obtained from following locations: stomach (4 cases), small intestine (8 cases), appendix (18 cases), rectum (3 cases), pancreas (5 cases), liver metastases (7 cases) and lymph node metastases (4 cases) were used in the study. In 41 of the 49 tumour samples (83.7%), positive staining for class III b-tubulin was detected, while 8 tumour samples (16.3%) were negative. Expression of class III b-tubulin was prominent in all three rectal carcinoids and in three atypical carcinoids located in small intestine. Pancreatic neuroendocrine tumours revealed either weak immunostaining (2 cases), or were negative for this marker (3 cases). The intensity of class III b-tubulin immunolabelling was not related to the degree of tumour differentiation. The results of this study suggest that class III b-tubulin could be a perspective marker for gastrointestinal neuroendocrine tumours. Moreover, the differences in its expression could also elucidate some aspects of histogenetic relationships of neuroendocrine tumours of gastrointestinal tract.     

Alteration of collagenous protein profile in congestive heart failure secondary to myocardial infarction

The rat model of myocardial infarction is characterized by progressive cardiac hypertrophy and failure. Rats with infarcts greater than 30% of the left ventricle exhibited early and moderate, stages of heart failure 4 and 8 weeks after the occlusion of the left coronary artery, respectively. As heart failure is usually associated with remodeling of the extracellular matrix, a histological and biochemical study of cardiac collagenous proteins was carried out using failing hearts. Total collagen content in the right ventricle increased at 2, 4, and 8 weeks following occlusion of the left coronary artery whereas such a change in viable left ventricle was seen after 4 and 8 weeks. Total cardiac hydroxyproline concentration was increased in both right and left ventricular samples from the infarcted animals when compared to those of control; this increase was due to elevation of pepsin-insoluble collagen fraction. The myocardial noncollagenous/collagenous protein ratio was decreased in experimental right and left ventricular samples when compared to control samples. These findings suggest that an increase in cross-linking of cardiac collagen as well as disparate synthesis of collagenous and noncollagenous proteins occurs in this model of congestive heart, failure.     

Detailed analysis of pronucleus development in bovine zygotes in vivo: Ultrastructure and cell cycle chronology

The ultrastructural development of pronuclei and cytoplasm was studied in bovine zygotes developed in the oviducts. The timing of the morphological events was related to sonographically detected ovulation and to the progress of the cell cycle determined by double labelling (³H and ¹⁴C-thymidine) of newly synthesized DNA combined with autoradiographic detection. The onset of the S-phase occurred at 11–12 hr after the estimated time of ovulation (EO), and this phase of the cell cycle lasted for 7–9 hr. During the G1-phase, the pronuclei contained spheres of compact, electron-dense fibrillar material classified as nucleolus precursor bodies. Early in the S-phase (13 hr aver EO) spherical fibrillogranular bodies containing larger rounded electron-dense components were detected in the periphery of the pronuclei as well. At 15 hr, the latter bodies had become connected through electron-dense material with spherical multivacuolated fibrillar bodies of the same electron density as the nucleolus precursor bodies. At 17 hr, similar compact spherical bodies, now presenting a single large vacuole, were observed on some occasions, while in other zygotes the morphology remained unchanged throughout the rest of the S and G2-phases. © 1996 Wiley-Liss, Inc.     

Regulation of Src family kinases involved in T cell receptor signaling by protein-tyrosine phosphatase CD148

CD148 is a receptor-like protein-tyrosine phosphatase known to inhibit transduction of mitogenic signals in non-hematopoietic cells. Similarly, in the hematopoietic lineage, CD148 inhibited signal transduction downstream of T cell receptor. However, it also augmented immunoreceptor signaling in B cells and macrophages via dephosphorylating C-terminal tyrosine of Src family kinases (SFK). Accordingly, endogenous CD148 compensated for the loss of the main SFK activator CD45 in murine B cells and macrophages but not in T cells. Hypothetical explanations for the difference between T cells and other leukocyte lineages include the inability of CD148 to dephosphorylate a specific set of SFKs involved in T cell activation or the lack of CD148 expression during critical stages of T cell development. Here we describe striking differences in CD148 expression between human and murine thymocyte subsets, the only unifying feature being the absence of CD148 during the positive selection when the major developmental block occurs under CD45 deficiency. Moreover, we demonstrate that similar to CD45, CD148 has both activating and inhibitory effects on the SFKs involved in TCR signaling. However, in the absence of CD45, activating effects prevail, resulting in functional complementation of CD45 deficiency in human T cell lines. Importantly, this is independent of the tyrosines in the CD148 C-terminal tail, contradicting the recently proposed phosphotyrosine displacement model as a mechanism of SFK activation by CD148. Collectively, our data suggest that differential effects of CD148 in T cells and other leukocyte subsets cannot be explained by the CD148 inability to activate T cell SFKs but rather by its dual inhibitory/activatory function and specific expression pattern.     

Dysregulation of Src family kinases in mast cells from epilepsy-resistant ASK versus epilepsy-prone EL mice

EL mice have been used as a model of epilepsy, whereas ASK mice are an epilepsy-resistant variant originating from a colony of EL mice. Mast cell-dependent anaphylaxis is easily inducible by stimulation with IgE and Ag in ASK mice, whereas EL mice are resistant to such stimuli. In this study we have characterized mast cells derived from these two strains. ASK mast cells proliferated more vigorously than EL cells in response to IL-3 and stem cell factor. Although ASK mast cells degranulated less vigorously than EL mast cells upon stimulation with IgE and Ag, ASK cells produced and secreted several-fold more TNF-alpha and IL-2 than EL cells. Consistent with the similarities of these ASK and EL mast cell responses with phenotypes of lyn(-/-) and wild-type mast cells, respectively, Lyn activity was reduced in ASK cells. In addition to the impaired Lyn activity, ASK cells just like lyn(-/-) cells exhibited reduced Syk activity, prolonged activation of ERK and JNK, and enhanced activation of Akt. Furthermore, the lipid raft-resident transmembrane adaptor protein Cbp/PAG that associates with Lyn was hypophosphorylated in ASK cells. Importantly, similar to lyn(-/-) cells, Fyn was hyperactivated in ASK cells. Therefore, these results are consistent with the notion that Lyn-dependent phosphorylation of Cbp/PAG negatively regulates Src family kinases. This study also suggests that reduced activity of Lyn, a negative regulator of mast cell activation, underlies the susceptibility of ASK mice to anaphylaxis and implies that dysregulation of Lyn and other Src family kinases contributes to epileptogenesis.