Heterocyst differentiation and pattern formation in cyanobacteria: a chorus of signals

Heterocyst differentiation in filamentous cyanobacteria provides an excellent prokaryotic model for studying multicellular behaviour and pattern formation. In Anabaena sp. strain PCC 7120, for example, 5-10% of the cells along each filament are induced, when deprived of combined nitrogen, to differentiate into heterocysts. Heterocysts are specialized in the fixation of N(2) under oxic conditions and are semi-regularly spaced among vegetative cells. This developmental programme leads to spatial separation of oxygen-sensitive nitrogen fixation (by heterocysts) and oxygen-producing photosynthesis (by vegetative cells). The interdependence between these two cell types ensures filament growth under conditions of combined-nitrogen limitation. Multiple signals have recently been identified as necessary for the initiation of heterocyst differentiation, the formation of the heterocyst pattern and pattern maintenance. The Krebs cycle metabolite 2-oxoglutarate (2-OG) serves as a signal of nitrogen deprivation. Accumulation of a non-metabolizable analogue of 2-OG triggers the complex developmental process of heterocyst differentiation. Once heterocyst development has been initiated, interactions among the various components involved in heterocyst differentiation determine the developmental fate of each cell. The free calcium concentration is crucial to heterocyst differentiation. Lateral diffusion of the PatS peptide or a derivative of it from a developing cell may inhibit the differentiation of neighbouring cells. HetR, a protease showing DNA-binding activity, is crucial to heterocyst differentiation and appears to be the central processor of various early signals involved in the developmental process. How the various signalling pathways are integrated and used to control heterocyst differentiation processes is a challenging question that still remains to be elucidated.     

Phylogenetic and mutational analyses reveal key residues for UDP-glucuronic acid binding and activity of beta1,3-glucuronosyltransferase I (GlcAT-I)

The beta1,3-glucuronosyltransferases are responsible for the completion of the protein-glycosaminoglycan linkage region of proteoglycans and of the HNK1 epitope of glycoproteins and glycolipids by transferring glucuronic acid from UDP-alpha-D-glucuronic acid (UDP-GlcA) onto a terminal galactose residue. Here, we develop phylogenetic and mutational approaches to identify critical residues involved in UDP-GlcA binding and enzyme activity of the human beta1,3-glucuronosyltransferase I (GlcAT-I), which plays a key role in glycosaminoglycan biosynthesis. Phylogeny analysis identified 119 related beta1,3-glucuronosyltransferase sequences in vertebrates, invertebrates, and plants that contain eight conserved peptide motifs with 15 highly conserved amino acids. Sequence homology and structural information suggest that Y84, D113, R156, R161, and R310 residues belong to the UDP-GlcA binding site. The importance of these residues is assessed by site-directed mutagenesis, UDP affinity and kinetic analyses. Our data show that uridine binding is primarily governed by stacking interactions with the phenyl group of Y84 and also involves interactions with aspartate 113. Furthermore, we found that R156 is critical for enzyme activity but not for UDP binding, whereas R310 appears less important with regard to both activity and UDP interactions. These results clearly discriminate the function of these two active site residues that were predicted to interact with the pyrophosphate group of UDP-GlcA. Finally, mutation of R161 severely compromises GlcAT-I activity, emphasizing the major contribution of this invariant residue. Altogether, this phylogenetic approach sustained by biochemical analyses affords new insight into the organization of the beta1,3-glucuronosyltransferase family and distinguishes the respective importance of conserved residues in UDP-GlcA binding and activity of GlcAT-I.     

Analysis of Intraspecies Diversity in Wheat and Barley Genomes Identifies Breakpoints of Ancient Haplotypes and Provides Insight into the Structure of Diploid and Hexaploid Triticeae Gene Pools

A large number of wheat (Triticum aestivum) and barley (Hordeum vulgare) varieties have evolved in agricultural ecosystems since domestication. Because of the large, repetitive genomes of these Triticeae crops, sequence information is limited and molecular differences between modern varieties are poorly understood. To study intraspecies genomic diversity, we compared large genomic sequences at the Lr34 locus of the wheat varieties Chinese Spring, Renan, and Glenlea, and diploid wheat Aegilops tauschii. Additionally, we compared the barley loci Vrs1 and Rym4 of the varieties Morex, Cebada Capa, and Haruna Nijo. Molecular dating showed that the wheat D genome haplotypes diverged only a few thousand years ago, while some barley and Ae. tauschii haplotypes diverged more than 500,000 years ago. This suggests gene flow from wild barley relatives after domestication, whereas this was rare or absent in the D genome of hexaploid wheat. In some segments, the compared haplotypes were very similar to each other, but for two varieties each at the Rym4 and Lr34 loci, sequence conservation showed a breakpoint that separates a highly conserved from a less conserved segment. We interpret this as recombination breakpoints of two ancient haplotypes, indicating that the Triticeae genomes are a heterogeneous and variable mosaic of haplotype fragments. Analysis of insertions and deletions showed that large events caused by transposable element insertions, illegitimate recombination, or unequal crossing over were relatively rare. Most insertions and deletions were small and caused by template slippage in short homopolymers of only a few base pairs in size. Such frequent polymorphisms could be exploited for future molecular marker development.     

Improving NaCl resistance of red-osier dogwood: role of CaCl2 and CaSO4

The influence of Ca²⁺ salts on the resistance of red-osier dogwood (Cornus sericea) seedlings to salinity was investigated. Red-osier dogwood seedlings were exposed to 5 and 10 mM of CaCl₂ or CaSO₄ in the presence or absence of 50 mM NaCl for 40 days in a controlled environment. Seedlings exposed to CaCl₂ and CaSO₄ recovered from NaCl-induced transpiration reduction after 20 days at a concentration of 10 mM and after 30 days at a concentration of 5 mM; while in absence of additional Ca²⁺, the seedlings recovered only after 40 days. Addition of 10 mM Ca²⁺ to NaCl treatment also limited the accumulation of proline in leaf tissues and caused an increase in leaf and lateral shoot K⁺ content. These results suggest that 10 mM Ca²⁺ could alleviate, at least in part, the osmotic effect of NaCl on red-osier dogwood via control of stomatal closure. On the other hand, ion analysis showed that Ca²⁺ addition was able to reduce the NaCl-induced Na⁺ concentration only in stem tissues suggesting that Ca²⁺ had only a limited effect on the ionic stress. The present study also showed an unexpected NaCl-induced increase in Ca²⁺ content of leaves, lateral shoots and stems that was not observed in our previous hydroponics experiments and seems to be more characteristic of plants growing on sandy soils.     

EXAFS structural study of platinum-based anticancer drugs degradation in presence of sulfur nucleophilic species

Three platinum complexes, cisplatin, carboplatin and oxaliplatin are currently used worldwide. Investigation of their main structural modifications in presence of sulfur nucleophiles is of particular interest because of the implication of thiol and thioether groups in biochemical mechanism of action, resistance mechanism and in vivo or in vitro detoxification. We present the main structural results we have obtained concerning the reaction of these drugs with diverse sulfur nucleophiles (cysteine, glutathione, methionine, thiosulfate and thiocyanate), monitored in solution or as precipitates by EXAFS spectroscopy. The reactivities of the carboxylate and amine ligands of both carboplatin and oxaliplatin are compared, on the basis of first-coordination sphere modeling. Among the new results of this EXAFS study, we present the first observation of oxaliplatin diaminocyclohexane ligand displacement by sulfur nucleophiles.     

Soluble HLA/peptide monomers cross-linked with co-stimulatory antibodies onto a streptavidin core molecule efficiently stimulate antigen-specific T cell responses

Soluble MHC–peptide complexes, commonly referred to as tetramers, have been shown to induce strong cross-linking of TCR and CD8, resulting in a vigorous activation followed by a rapid non-apoptotic CD8⁺ T cell death. This has limited tetramer use for antigen-specific T cells isolation and cloning, as sorted tetramer positive cells were shown to possess compromised functional integrity. Here we show that the cross-linking of a secondary co-stimulatory signal into oligomeric MHC:peptide complexes prevents such cell death, and in contrast strongly stimulates antigen-specific T cell responses. Such soluble antigen-presenting complexes (sAPCs) containing MHC:peptide complexes linked to either anti-CD27 or anti-CD28 antibodies were capable of priming and expanding HLA-A*0201 restricted CMV specific T cells and also of generating functional HLA-A*0301 restricted BCR/ABL-specific T cell responses. These sAPCs constitute an encouraging alternative method for generating antigen-specific T cells that could be applied to a variety of antigens. A. I. Dodi and P. J. Travers contributed equally to this work. This paper is an original contribution from the meeting which took place on 28 and 29 May 2008 in Nottingham, UK, celebrating the contribution of Prof. I. A. “Tony” Dodi (^†29.1.2008) to the EU project “Network for the identification and validation of antigens and biomarkers in cancer and their application in clinical tumour immunology (ENACT)”.     

The prevention and treatment of cytomegalovirus infection in haematopoietic stem cell transplantation

Allogeneic haematopoietic stem cell transplantation (HSCT) is an intensive medical treatment involving myeloablative chemo-radiotherapy followed by stem cell rescue using allogeneic haematopoietic stem cells harvested from HLA-matched donors, which is primarily used for the treatment of haematological malignancies. Cytomegalovirus (CMV) infection is one of the major causes of morbidity and death after HSCT. This focused research review highlights the advances made with research into CMV in the HSCT setting. It provides the reader with an overview of current CMV research into the prevention and management of CMV infection. This paper is a Focussed Research Review from the meeting which took place 28–29 May 2008 in Nottingham, UK, celebrating the contribution of Prof. I. A. “Tony” Dodi (+29.1.2008) to the EU project “Network for the identification and validation of antigens and biomarkers in cancer and their application in clinical tumour immunology (ENACT)”.     

Flavobacterial Community Structure in a Hardwater Rivulet and Adjacent Forest Soil,Harz Mountain,Germany

The great increase in the abundance and phylogenetic diversity of Flavobacterium spp. within a few hundred meters downstream of the discharge site of the Westerhöfer Bach, a hardwater rivulet, raised the question whether adjacent soil may serve as a reservoir of bacteria not detected in discharge water. To address this question, denaturing gradient gel electrophoresis (DGGE) analyses of the V3 region of Flavobacterium 16S rRNA genes were performed on DNA from nine soil samples and five rivulet sites. The resulting patterns were tested for the significance of differences between the sampling habitats using the nonparametric analysis of similarities and multidimensional scaling procedures. Even though both habitats were sampled in two consecutive years DGGE patterns of soil and downstream water samples showed significant overlap (R = 0.614). Sequencing of 57 DGGE bands resulted in 30 different sequences, which, on the basis of BLAST analyses, were between 96% and 100% similar to published clone, DGGE, and strain sequences from a wide range of different habitats. Forty-five percent of the highly similar sequences included those of isolates from the Westerhöfer Bach, while the other sequences were more closely related to clones and cultures from other habitats, especially agricultural soil. Based on these results we suggest that the increase in flavobacterial strain diversity and abundance in the rivulet may originate from soil microflora.