Inhibitory properties of nucleotides with difluoromethylenephosphonic acid as a phosphate mimic versus calf spleen purine nucleoside phosphorylase and effect of these analogues on the viability of human blood lymphocytes

Several cyclic and acyclic 6-keto purine nucleotides with difluoromethylenephosphonic acid as phosphate mimic are proved to be potent inhibitors of mammalian purine nucleoside phosphorylase (PNP). Antiproliferative activity of these analogues on the growth of human blood lymphocytes was tested by MTT assay. Compared to inhibitory effects on the growth of human blood T-lymphocytes isolated from healthy donors, all analogues significantly slow down proliferation of T-lymphocytes isolated from patients with autoimmune thyroid disease--Hashimoto's thyroiditis.     

Identification of the flagellar chaperone FlgN in the phytopathogen <Emphasis Type="Italic">Xanthomonas axonopodis</Emphasis> pathovar <Emphasis Type="Italic">citri</Emphasis> by its interaction with hook-associated FlgK

Genome annotation of the plant pathogen Xanthomonas axonopodis pv. citri (Xac), identified flagellar genes in a 15.7 kb gene cluster. However, FlgN, a secretion chaperone for hook-associated proteins FlgK and FlgL, was not identified. We performed extensive screening of the X. axonopodis pv. citri genome with the yeast two-hybrid system to identify a protein with the characteristics of the flagellar chaperone FlgN. We found a candidate (XAC1990) encoded by an operon for components of the flagellum apparatus that interacted with FlgK. In order to further support this finding, Xac FlgK and XAC1990 were cloned, expressed, and purified. The recombinant proteins were characterized by spectroscopic methods and their interaction in vitro confirmed by pull-down assays. We, therefore, conclude that XAC1990 and its homologs in other Xanthomonas species are, in fact, FlgN proteins. These observations extend the sequence diversity covered by this family of proteins.     

The cellular stress response of the scleractinian coral <Emphasis Type="Italic">Goniopora columna</Emphasis> during the progression of the black band disease

Black band disease (BBD) is a widespread coral pathology caused by a microbial consortium dominated by cyanobacteria, which is significantly contributing to the loss of coral cover and diversity worldwide. Since the effects of the BBD pathogens on the physiology and cellular stress response of coral polyps appear almost unknown, the expression of some molecular biomarkers, such as Hsp70, Hsp60, HO-1, and MnSOD, was analyzed in the apparently healthy tissues of Goniopora columna located at different distances from the infection and during two disease development stages. All the biomarkers displayed different levels of expression between healthy and diseased colonies. In the healthy corals, low basal levels were found stable over time in different parts of the same colony. On the contrary, in the diseased colonies, a strong up-regulation of all the biomarkers was observed in all the tissues surrounding the infection, which suffered an oxidative stress probably generated by the alternation, at the progression front of the disease, of conditions of oxygen supersaturation and hypoxia/anoxia, and by the production of the cyanotoxin microcystin by the BBD cyanobacteria. Furthermore, in the infected colonies, the expression of all the biomarkers appeared significantly affected by the development stage of the disease. In conclusion, our approach may constitute a useful diagnostic tool, since the cellular stress response of corals is activated before the pathogens colonize the tissues, and expands the current knowledge of the mechanisms controlling the host responses to infection in corals.     

An agonistic mAb directed to the TrkC receptor juxtamembrane region defines a trophic hot spot and interactions with p75 coreceptors

The D5 domain of TrkC receptors is a docking site for Neurotrophin‐3 (NT‐3), but other domains may be relevant for function or harmonizing signals with p75^NTR coreceptors. We report a monoclonal antibody (mAb) 2B7 targeting the juxtamembrane domain of TrkC. mAb 2B7 binds to murine and human TrkC receptors and is a functional agonist that affords activation of TrkC, AKT, and MAPK. These signals result in cell survival but not in cellular differentiation. Monomeric 2B7 Fabs also affords cell survival. Binding of 2B7 mAb and 2B7 Fabs to TrkC are blocked by NT‐3 in a dose‐dependent manner but not by pro‐NT‐3. Expression of p75^NTR coreceptors on the cell surface block the binding and function of mAb 2B7, whereas NT‐3 binding and function are enhanced. mAb 2B7 defines a previously unknown neurotrophin receptor functional hot spot; that exclusively generates survival signals; that can be activated by non‐dimeric ligands; and potentially unmasks a site for p75‐TrkC interactions. © 2009 Wiley Periodicals, Inc. Develop Neurobiol, 2010.     

Bacterial secretion chaperones

Many Gram-negative bacteria are able to invade hosts by translocation of effectors directly into target cells in processes usually mediated by two very complex secretion systems (SSs), named type III (T3) and type IV (T4) SSs. These syringe-needle injection devices work with intervention of specialized secretion chaperones that, unlike traditional molecular chaperones, do not assist in protein folding and are not energized by ATP. Controversy still surrounds secretion chaperones primary role, but we can say that these chaperones act as: (i) bodyguards to prevent premature aggregation, or as (ii) pilots to direct substrate secretion through the correct secretion system. This family of chaperones does not share primary structure similarity but amazingly equal 3D folds. This mini review has the intent to present updated structural and functional data for several important secretion chaperones, either alone or in complex with their cognate substrates, as well to report on the common features and roles of T3, T4 and flagellar chaperones.     

Structural studies of phosphoinositide 3-kinase-dependent traffic to multivesicular bodies

Three large protein complexes known as ESCRT I, ESCRT II and ESCRT III drive the progression of ubiquitinated membrane cargo from early endosomes to lysosomes. Several steps in this process critically depend on PtdIns3P, the product of the class III phosphoinositide 3-kinase. Our work has provided insights into the architecture, membrane recruitment and functional interactions of the ESCRT machinery. The fan-shaped ESCRT I core and the trilobal ESCRT II core are essential to forming stable, rigid scaffolds that support additional, flexibly-linked domains, which serve as gripping tools for recognizing elements of the MVB (multivesicular body) pathway: cargo protein, membranes and other MVB proteins. With these additional (non-core) domains, ESCRT I grasps monoubiquitinated membrane proteins and the Vps36 subunit of the downstream ESCRT II complex. The GLUE (GRAM-like, ubiquitin-binding on Eap45) domain extending beyond the core of the ESCRT II complex recognizes PtdIns3P-containing membranes, monoubiquitinated cargo and ESCRT I. The structure of this GLUE domain demonstrates that it has a split PH (pleckstrin homology) domain fold, with a non-typical phosphoinositide-binding pocket. Mutations in the lipid-binding pocket of the ESCRT II GLUE domain cause a strong defect in vacuolar protein sorting in yeast.     

History of vegetation and land use from 3200 B.P. to the present in the north-west Burren,a karstic region of western Ireland

A radiocarbon-dated pollen profile provides the first evidence for the vegetation history of the north-west Burren, a karstic region in Co. Clare, western Ireland. The profile, which spans the period ca. 3200-0 B.P., shows that Corylus was the main woody species and that, apart from the later eighteenth and nineteenth centuries, when woody species were almost totally cleared, it constituted a more important element in the landscape than at present. The data indicate that small populations of Quercus, Fraxinus, Betula, Alnus and possibly Ulmus were also present until at least the mid seventeenth century. Exceptionally high values of Plantago lanceolata (mostly in excess of 20% total terrestrial pollen) suggest intensive pastoral farming over most of the period. Between A.D. 200–580, a lull in activity is recorded during which woody species and, in particular, hazel scrub regenerated. The separation of pollen of cereal origin from large cereal-type pollen produced by non-cultivated grasses proved particularly problematic. Reliance has therefore been placed on the weed pollen flora as a guide to arable activity. This flora indicates that arable farming was of some importance in the Burren from A.D. 1000 onwards. The implications of the various findings for the longer term history of the rarer elements of the Burren flora and the relationship between the pollen record and the archaeological and historical evidence for settlement and land use are discussed.