Changes in the LHCI aggregation state during iron repletion in the unicellular red alga Rhodella violacea

Red algae are well suited to study the effects of iron deficiency on light-harvesting complex for photosystem I (LHCI), since they are totally devoid of light-harvesting complex for photosystem II (LHCII). Iron starvation results in a reduction of the pigment content, an increase of the fluorescence yield and a new emission band at 705 nm in the 77 K fluorescence emission spectra. These changes reflect the accumulation of uncoupled, aggregated LHCI in iron-depleted cells. Reconnection of LHCI to de novo synthesized reaction center I (RCI) is the first event, which takes place after iron addition. The changes in the aggregation state of LHCI are likely to occur also in brown and green algae.     

Heterochronic expression of sexual reproductive programs during apomictic development in Tripsacum

Some angiosperms reproduce by apomixis, a natural way of cloning through seeds. Apomictic plants bypass both meiosis and egg cell fertilization, producing progeny that are genetic replicas of the mother plant. In this report, we analyze reproductive development in Tripsacum dactyloides, an apomictic relative of maize, and in experimental apomictic hybrids between maize and Tripsacum. We show that apomictic reproduction is characterized by an alteration of developmental timing of both sporogenesis and early embryo development. The absence of female meiosis in apomictic Tripsacum results from an early termination of female meiosis. Similarly, parthenogenetic development of a maternal embryo in apomicts results from precocious induction of early embryogenesis events. We also show that male meiosis in apomicts is characterized by comparable asynchronous expression of developmental stages. Apomixis thus results in an array of possible phenotypes, including wild-type sexual development. Overall, our observations suggest that apomixis in Tripsacum is a heterochronic phenotype; i.e., it relies on a deregulation of the timing of reproductive events, rather than on the alteration of a specific component of the reproductive pathway.     

Nitric oxide binding properties of neuroglobin. A characterization by EPR and flash photolysis

Neuroglobin is a recently discovered member of the globin superfamily. Combined electron paramagnetic resonance and optical measurements show that, in Escherichia coli cell cultures with low O(2) concentration overexpressing wild-type mouse recombinant neuroglobin, the heme protein is mainly in a hexacoordinated deoxy ferrous form (F8His-Fe(2+)-E7His), whereby for a small fraction of the protein the endogenous protein ligand is replaced by NO. Analogous studies for mutated neuroglobin (mutation of E7-His to Leu, Val, or Gln) reveal the predominant presence of the nitrosyl ferrous form. After sonication of the cells wild-type neuroglobin oxidizes rapidly to the hexacoordinated ferric form, whereas NO ligation initially protects the mutants from oxidation. Flash photolysis studies of wild-type neuroglobin and its E7 mutants show high recombination rates (k(on)) and low dissociation rates (k(off)) for NO, indicating a high intrinsic affinity for this ligand similar to that of other hemoglobins. Since the rate-limiting step in ligand combination with the deoxy-hexacoordinated wild-type form involves the dissociation of the protein ligand, NO binding is slower than for the related mutants. Structural and kinetic characteristics of neuroglobin and its mutants are analyzed. NO production in rapidly growing E. coli cell cultures is discussed.     

Phosphorylation-dependent interaction between the splicing factors SAP155 and NIPP1

NIPP1 is a ubiquitously expressed nuclear protein that functions both as a regulator of protein Ser/Thr phosphatase-1 and as a splicing factor. The N-terminal part of NIPP1 consists of a phosphothreonine-interacting Forkhead-associated (FHA) domain. We show here that the FHA domain of NIPP1 interacts in vitro and in vivo with a TP dipeptide-rich fragment of the splicing factor SAP155/SF3b(155), a component of the U2 small nuclear ribonucleoprotein particle. The NIPP1-SAP155 interaction was entirely dependent on the phosphorylation of specific TP motifs in SAP155. Mutagenesis and competition studies revealed that various phosphorylated TP motifs competed for binding to the same site in the FHA domain. The SAP155 kinases in cell lysates were blocked by the Ca(2+) chelator EGTA and by the cyclin-dependent protein kinase inhibitor roscovitine. The phosphorylation level of SAP155 was dramatically increased during mitosis, and accordingly the activity of SAP155 kinases was augmented in mitotic lysates. We discuss how the interaction between NIPP1 and SAP155 could contribute to spliceosome (dis)assembly and the catalytic steps of splicing.     

Dermal nevus cells from congenital nevi cannot penetrate the dermis in skin reconstructs

Congenital nevi are composed of pigment cells bearing common features with melanocytes but showing altered differentiation which leads to nesting and dermal involvement. Using a dead de-epidermized dermis seeded with a combination of keratinocytes and various sources of pigment cells (normal melanocytes, dermal nevus cells from congenital nevi, Bowes melanoma cells), we have studied the formation of nests and the dermal migration of pigment cells together with their secretion profiles of matrix metalloproteinases (MMP). Dermal fibroblasts were also used as control cells in epidermal reconstructs. Besides their morphologic features, the absence of pigment donation to keratinocytes was the major characteristic of dermal nevus cells. A positive correlation was established between the increasing percentage of seeded nevus cells and the patchy pigmentation of reconstructs, as well as the clustering of cells in junctional nests. However, the presence of nevus cells in the dermis of reconstructs was never detected, whereas melanoma cells and dermal fibroblasts could invade the dermis during the time span of the experiments. MMP9 was never expressed in congenital dermal nevus cells but pro-MMP2 was constitutively expressed by all strains of congenital nevus cells and dermal fibroblasts. Melanocytes produced comparable amounts of both pro-MMP2 and pro-MMP9, and Bowes melanoma cells secreted a marginal level of pro-MMP2. In view of their three-dimensional behaviour and secretion of MMPs, we propose that dermal congenital nevus cells correspond to an intermediate status of differentiation between normal melanocytes and melanoma cells. Activation of MMPs by a cofactor or the activation of another signalling pathway seems necessary to induce the dermal passage of nevus cells.     

RIP4 (DIK/PKK), a novel member of the RIP kinase family,activates NF-kappa B and is processed during apoptosis

RIP1 and its homologs, RIP2 and RIP3, form part of a family of Ser/Thr kinases that regulate signal transduction processes leading to NF-κB activation. Here, we identify RIP4 (DIK/PKK) as a novel member of the RIP kinase family. RIP4 contains an N-terminal RIP-like kinase domain and a C-terminal region characterized by the presence of 11 ankyrin repeats. Overexpression of RIP4 leads to activation of NF-κB and JNK. Kinase inactive RIP4 or a truncated version containing the ankyrin repeats have a dominant negative (DN) effect on NF-κB induction by multiple stimuli. RIP4 binds to several members of the TRAF protein family, and DN versions of TRAF1, TRAF3 and TRAF6 inhibit RIP4-induced NF-κB activation. Moreover, RIP4 is cleaved after Asp340 and Asp378 during Fas-induced apoptosis. These data suggest that RIP4 is involved in NF-κB and JNK signaling and that caspase-dependent processing of RIP4 may negatively regulate NF-κB-dependent pro-survival or pro-inflammatory signals.     

Protein mutagenesis with monodispersity-based quality probing: selective inactivation of p53 degradation and DNA-binding properties of HPV E6 oncoprotein

Interpretation of protein mutagenesis experiments requires the ability to distinguish functionally relevant mutations from mutations affecting the structure. When a protein is expressed soluble in bacteria, properly folded mutants are expected to remain soluble whereas misfolded mutants should form insoluble aggregates. However, this rule may fail for proteins fused to highly soluble carrier proteins. In a previous study, we analysed the biophysical status of HPV oncoprotein E6 fused to the C-terminus of maltose-binding protein (MBP) and found that misfolded E6 moieties fused to MBP formed soluble aggregates of high molecular weight. By contrast, preparations of properly folded E6 fused to MBP were monodisperse. Here, we have used this finding to evaluate the quality of 19 MBP-fused E6 site-directed mutants by using a light scattering assay performed in a fluorimeter. This assay guided us to rule out structurally defective mutants and to obtain functionally relevant E6 mutants selectively altered for two molecular activities: degradation of tumour suppressor p53 and DNA recognition.     

Presence and release of SR-17 (chromogranin B(586-602)) in the porcine splenic nerve and its enzymatic degradation by CD26/dipeptidyl peptidase IV

Using the pig splenic nerve as a model, we investigated the proteolytic processing of porcine chromogranin B (CgB) during its axonal transport. An ELISA was developed for SR-17 (CgB(586-602)), a novel CgB-derived peptide, originally found in the adrenal medulla. The results demonstrate that CgB is processed in an early stage during its axonal transport. Immunohistochemical data, based on a rabbit anti-SR-17 antiserum, show that the spleen CgB/SR-17 is exclusively present in the nerve endings. No SR-17 immunoreactivity (IR) was found in splenocytes. We also provide evidence that SR-17 is co-released with noradrenaline (NA) upon electrical stimulation of the splenic nerve. Its release is frequency-dependent and strongly enhanced in the presence of the alpha-blocking agent phentolamine. In addition, we show that the new CgB-peptide can serve as a substrate for the lymphocyte surface glycoprotein CD26, also known as dipeptidyl peptidase IV (DPP IV), generating a new peptide ER-15 (CgB(588-602)).     

Comparison of somatic embryogenesis-derived coffee (Coffea arabica L.) plantlets regenerated in vitro or ex vitro: morphological,mineral and water characteristics

Coffea arabica L. plantlets obtained ex vitro after sowing somatic embryos produced in a bioreactor in horticultural substrate were compared with those obtained in vitro from the same embryo population under conventional culturing conditions on semi-solid media. The intensity and quality of aerial and root system development were compared. Shoot emergence was more efficient in vitro but rooting frequencies were low. In contrast, all ex vitro-regenerated embryos rooted. The cotyledon area of mature embryos produced in a bioreactor positively affected plantlet development when regeneration was carried out ex vitro. Embryos with an intermediate cotyledon area (0.86 cm2) had the highest rates of plant conversion ex vitro (63%), and also resulted in vigorous plantlets. Mortality was higher in nursery conditions, but better plant development was obtained. The quality of plantlets produced under ex vitro conditions was reflected in better growth of the aerial and root systems, and also by similar morphological, mineral and water status characteristics to seedlings. Unlike roots formed on semi-solid media, those produced in soil were branched, fine (30-50% had a diameter of less than 0-5 mm) and they bore root hairs. Leaves of plantlets regenerated ex vitro had a histological structure similar to that of seedling leaves, and a lower stomatal density (100 vs. 233 mm-2). Moreover, they were more turgid, as indicated by higher pressure potential (psiP) (0.91 s. 0.30 MPa) and relative water content values (97 vs. 93%). Furthermore, under in vitro conditions, leaves had larger stomata which were abnormally round and raised. Direct sowing of germinated somatic embryos resulted in the rapid production of vigorous plantlets under ex vitro conditions, whilst removing the need for problematical and costly conventional acclimatization procedures.