The reticuloplasmin calreticulun is released into the medium by carrot cell cultures

We report here the presence of a 58-kDa protein in the cells of Daucus carota L. cultivated in vitro. Two lines of carrot cells are used: wild-type line (wt) and mutant line (ts11). We describe here also presence of this protein in the media of cultured cells. Strong reaction of this intracellular and extracellular protein with an anti-calreticulin antiserum indicates that it is a major high capacity, low affinity Ca²⁺-binding reticuloplasmin–calreticulin. No differences in biochemical characterization is found between calreticulin purified from the wild-type line and the mutant line. Moreover molecular mass, type of glycosylation and the ability of extracellular protein to bind calcium is found to be indistinguishable from those of the purified intracellular calreticulin. Calreticulin release is attributed to some stress imposed on cultured cells by growth conditions. It is shown that this process can be also induced in CR-non-releasing systems such as carrot somatic embryos by applying a high-cell-density stress.     

Elongation factor 1A is the target of growth inhibition in yeast caused by Legionella pneumophila glucosyltransferase Lgt1

Legionella is a pathogenic Gram-negative bacterium that can multiply inside of eukaryotic cells. It translocates numerous bacterial effector proteins into target cells to transform host phagocytes into a niche for replication. One effector of Legionella pneumophila is the glucosyltransferase Lgt1, which modifies serine 53 in mammalian elongation factor 1A (eEF1A), resulting in inhibition of protein synthesis and cell death. Here, we demonstrate that similar to mammalian cells, Lgt1 was severely toxic when produced in yeast and effectively inhibited in vitro protein synthesis. Saccharomyces cerevisiae strains, which were deleted of endogenous eEF1A but harbored a mutant eEF1A not glucosylated by Lgt1, were resistant toward the bacterial effector. In contrast, deletion of Hbs1, which is also an in vitro substrate of the glucosyltransferase, did not influence the toxic effects of Lgt1. Serial mutagenesis in yeast showed that Phe(54), Tyr(56) and Trp(58), located immediately downstream of serine 53 of eEF1A, are essential for the function of the elongation factor. Replacement of serine 53 by glutamic acid, mimicking phosphorylation, produced a non-functional eEF1A, which failed to support growth of S. cerevisiae. Our data indicate that Lgt1-induced lethal effect in yeast depends solely on eEF1A. The region of eEF1A encompassing serine 53 plays a critical role in functioning of the elongation factor.     

Rheological behaviour of the culture medium during growth of the microalga Botryococcus braunii

A non-axenic strain of the microalga Botryococcus braunii Kützing, isolated from a small lake in Portugal, when cultured at 25°C in mineral medium and under continuous illumination, showed a poor production of hydrocarbons (5% of the dry biomass) but excreted remarkably high quantities of an exopolysaccharide (4–4·5 g litre⁻¹) into the medium. The production of the soluble polysaccharide, which contains galactose, fucose and uronic acid residues, occurs mainly after the exponential phase of growth.The rheological properties of broth during growth were studied. The increase of polysaccharide concentration as a consequence of its continuous biosynthesis, changes the medium behaviour from Newtonian to non-Newtonian with a flow characterized by a power-law equation. This behaviour becomes Newtonian again, when the culture is maintained for a longer period of time.     

Repression of Rx gene on the left side of the sensory vesicle by Nodal signaling is crucial for right-sided formation of the ocellus photoreceptor in the development of Ciona intestinalis

Nodal signaling plays an essential role in the establishment of left–right asymmetry in various animals. However, it is largely unknown how Nodal signaling is involved in the establishment of the left–right asymmetric morphology. In this study, the role of Nodal signaling in the left–right asymmetric ocellus formation in the ascidian, Ciona intestinalis was dealt with. During the development of C. intestinalis, the ocellus pigment cell forms on the midline and moves to the right side of the midline. Then, the photoreceptor cells form on the right side of the sensory vesicle (SV). Ci-Nodal is expressed on the left side of the SV in the developing tail bud embryo. When Nodal signaling is inhibited, the ocellus pigment cell form but remain on the midline, and expression of marker genes of the ocellus photoreceptor cells is ectopically detected on the left side as well as on the right side of the SV in the larva. Furthermore, Ci-Rx, which is essential for the ocellus differentiation, turns out to be negatively regulated by the Nodal signaling on the left side of the SV, even though it is required for the right-sided photoreceptor formation. These results indicate that Nodal signaling controls the left–right asymmetric ocellus formation in the development of C. intestinalis.     

Calcium-dependent proteolytic activity of a cysteine protease caldonopain is detected during Leishmania infection

A calcium-activated protease caldonopain in the cytosolic fraction of Leishmania donovani has been found to digest different endogenous proteins when subjected to SDS-PAGE. Gelatin-embedded gel electrophoresis confirms presence of calcium-dependent protease activity. Ca²⁺ affects proteolytic activity after 10 h. When host–parasite interaction was conducted in vitro, caldonopain was found to be active after 10 h of incubation with calcium. A 67-kDa protein is specifically digested during this time and two new proteins of 45 and 36 kDa appeared in SDS-PAGE electrophoregram. This belated action of calcium towards protease activity may be pre-requisite to facilitate invasion of host tissues and thereby mediate protein metabolism during survival of this pathogen both independently and intracellularly. It is likely that calcium metabolism in promastigotes and amastigotes does not propagate in the same manner. Involvement of calcium to initiate caldonopain activity may be critically associated with signal transduction pathways which may be responsible for the pathobiological action of this parasite. We propose that caldonopain could be a potential target to develop new chemotherapeutic approach against leishmaniasis.     

Structure and the evolutionary implication of the triplicated complement factor B genes of a urochordate ascidian, Ciona intestinalis

To elucidate the evolution of the complement system and MHC class III region, we analyzed the complement factor B (Bf) genes of a urochordate ascidian, Ciona intestinalis. Three different cDNA species, termed CiBf-1, CiBf-2 and CiBf-3, were identified. The deduced amino-acid sequences all contained the usual domains of vertebrate Bf and, in addition, three extra domains at the N-terminus. Furthermore, the serine protease domain of these CiBfs shared unique features with vertebrate complement components C1r/s and mannose-binding lectin-associated serine protease (MASP)-2/3, the absence of the disulfide bond designated histidine loop, and the usage of the AGY codon for the catalytic serine residue. These results indicate that complement genes have evolved through extensive exon shuffling events in the early stage of chordate evolution. Overall deduced amino-acid identity between CiBf-1 and -2 was 88%, whereas CiBf-3 showed 49% identity to both CiBf-1 and CiBf-2. These three CiBf genes were located within an approximately 50-kb genomic region, and exons 3 and 5 of all the three Bf genes showed an extremely high degree of nucleotide identity, indicating that the CiBf genes experienced extensive reorganization, such as duplication and gene conversion, since its divergence from the vertebrate Bf/C2 gene. Fluorescent in situ hybridization (FISH) to the chromosomes showed that genetic loci for the CiBfs, CiC3-1 and CiC3-2 genes are present on three different chromosomes, suggesting the possibility that the linkage among the MHC class III complement genes was established in the vertebrate lineage after its divergence from urochordates.The sequences reported in this paper have been deposited in the DDBJ database (accession nos. AB180044–AB180051).     

Vascular endothelial growth factor is involved in neoangiogenesis in Hirudo medicinalis (Annelida,Hirudinea)

Vascular endothelial growth factor (VEGF) is fundamental in vertebrates for correct development of blood vessels. However, there are only few data about the presence of VEGF in invertebrates. In this study the role of VEGF in neovessel formation is investigated in Hirudo medicinalis. The leech is able to respond to administration of human VEGF by formation of new vessels. The response of H. medicinalis to this growth factor is explained by the presence of two specific VEGF-like receptors (Flt-1/VEGFR-1 and Flk-1/VEGFR-2) as demonstrated by immunohistochemistry and biochemical analysis. The VEGF-like produced by this annelid following surgical stimulation determines not only blood vessel formation, proliferation of vascular endothelial cells but also an increase of cytoplasmic calcium levels. The administration of specific VEGF receptor antibodies can inhibit angiogenesis in leeches previously stimulated with VEGF.     

Giardia lamblia EB1 is a functional homolog of yeast Bim1p that binds to microtubules

Giardia lamblia, with two nuclei and a distinct polarized morphology, is an interesting organism for investigating how distribution of its microtubule (MT) is controlled during its cell cycle. In this study, we identified the end-binding protein 1 (EB1) of G. lamblia, a well-known microtubule-associated protein that organizes MTs in eukaryotes. Immunofluorescence assays using recombinant EB1 (rEB1)-specific antibodies demonstrated EB1 localization in nuclear membrane as well as in some cytoskeletal structures such as axomenes and median bodies of trophozoites of G. lamblia. Complementation experiments using the BIM1 knock-out mutant of yeast, the yeast homolog of mammalian EB1, showed that giardial EB1 was able to carry out a homologous function in controlling MT dynamics. In addition, rEB1 of G. lamblia co-precipitated with MTs by an in vitro binding assay, thereby demonstrating that G. lamblia EB1 is a MT-associated protein. These results, taken together, suggest that G. lamblia EB1 is a functional homolog of eukaryotic EB1 and is likely to be a determinant for MT distribution.     

Preliminary characterization of the material released to the culture medium byCandida albicans yeast and mycelial cells

Culture filtrate concentrates were obtained fromCandida albicans yeast and mycelial cells grown in the presence of¹⁴C-protein hydrolysate for radioactive labeling of cellular polypeptides. Both growth forms released to the medium minor but significant amounts of proteinaceous materials. The analysis of culture filtrate concentrates by means of SDS-polyacrylamide gel electrophoresis and fluorography revealed a similar and complex electrophoretic pattern, though some qualitative and quantitative differences between samples obtained from yeast and mycelial cells were observed. Materials released, mostly composed of mannoproteins as shown by their affinity towards concanavalin A, presented (i) cross-reactivity (by Western immunoblotting and ELISA) against polyclonal antisera to genuine cell wall components (among them the 58-kilodalton fibrinogen-binding mannoprotein) and (ii) high affinity for polystyrene-latex microbeads. Results presented suggest a possible common identity for the molecules shed to the medium and the cell wall protein and mannoprotein constituents.Abbreviations SDS-PAGE sodium dodecyl sulphate-polyacrylamide gel electroporesis - PAS periodic acid/Schiff method     

Influence of culture temperature on the growth,biochemical composition and fatty acid profiles of six Antarctic microalgae

The growth, biochemical composition and fatty acid profiles of six Antarctic microalgae cultured at different temperatures, ranging from 4, 6, 9, 14, 20 to 30 ^∘C, were compared. The algae were isolated from seawater, freshwater, soil and snow samples collected during our recent expeditions to Casey, Antarctica, and are currently deposited in the University of Malaya Algae Culture Collection (UMACC). The algae chosen for the study were Chlamydomonas UMACC 229, Chlorella UMACC 234, Chlorella UMACC 237, Klebsormidium UMACC 227, Navicula UMACC 231 and Stichococcus UMACC 238. All the isolates could grow at temperatures up to 20 ^∘C; three isolates, namely Navicula UMACC 231 and the two Chlorella isolates (UMACC 234 and UMACC 237) grew even at 30 ^∘C. Both Chlorella UMACC 234 and Stichococcus UMACC 238 had broad optimal temperatures for growth, ranging from 6 to 20 ^∘C (μ = 0.19 – 0.22 day^–1) and 4 to 14 ^∘C (μ = 0.13 – 0.16 day^–1), respectively. In contrast, optimal growth temperatures for NaviculaUMACC 231 and Chlamydomonas UMACC 229 were 4 ^∘C (μ = 0.34 day^–1) and 6–9 ^∘C (μ = 0.39 – 0.40 day^–1), respectively. The protein content of the Antarctic algae was markedly affected by culture temperature. All except Navicula UMACC 231 and Stichococcus UMACC 238 contained higher amount of proteins when grown at low temperatures (6–9 ^∘C). The percentage of PUFA, especially 20:5 in Navicula UMACC 231 decreased with increasing culture temperature. However, the percentages of unsaturated fatty acids did not show consistent trend with culture temperature for the other algae studied.     

21-kDa polypeptide,a low-molecular-weight cyclophilin,is released from pollen of higher plants into the extracellular medium in vitro

Calcium ions play a key role in the elongation and orientation of pollen tubes. We found that significant amounts of 21-kDa polypeptide were specifically released into the extracellular medium when pollen grains of lily, Lilium longiflorum Thunb., were incubated in the presence of EGTA or at low concentrations of Ca²⁺. This phenomenon was also dependent on pH and on the concentrations of MgCl₂ in the medium; the release of 21-kDa polypeptide from pollen was suppressed by increasing the MgCl₂ concentration and by lowering pH. Germination of pollen grains was inhibited in the medium into which the 21-kDa polypeptide had been released. This inhibition was irreversible; germination did not occur on transfer of the pollen grains into basal culture medium. Immuno-electron microscopy using an antibody against 21-kDa polypeptide showed that this polypeptide was present in the cytoplasm, vegetative nucleus and generative cell. When the pollen was treated with a medium containing EGTA, the density of 21-kDa polypeptide in the cytoplasm significantly decreased, but its density in vegetative nuclei and the generative cell did not, suggesting that only cytoplasmic 21-kDa polypeptide was released into the extracellular medium. The 21-kDa polypeptide was also present in the pollen of other higher-plant species, such as Tradescantia virginiana L., Nicotiana tabacum L. (angiosperms), and Cryptomeria japonica D. Don. (gymnosperm), and was also released into the medium in the presence of EGTA. In the case of C. japonica, however, it was released from pollen at alkaline pH above 8.5. The expression of 21-kDa polypeptide was not pollen-specific, because 21-kDa components immunoreactive with the anti-21-kDa polypeptide serum also existed in vegetative organs and cells of lily or tobacco. However, the 21-kDa polypeptide was not released into the extracellular medium from cultured tobacco BY-2 cells, even in the presence of EGTA. Amino acid sequences of two peptide fragments derived from 21-kDa polypeptide matched well those of low-molecular-weight cyclophilin (CyP). The antiserum against 21-kDa polypeptide recognized the CyP A from calf thymus and that in A431 carcinoma cells. The 21-kDa polypeptide fraction purified from lily pollen possessed peptidyl-prolyl cis-trans isomerase activity, which was suppressed by cyclosporin A (CsA), an inhibitor of enzyme activities of CyPs. From these results, we concluded that the 21-kDa polypeptide is a low-molecular-weight CyP. The present study showed that CyP in the pollen of higher plants is released into the extracellular matrix under unfavorable conditions.Abbreviations CaM Calmodulin - CBB Coomassie-brilliant-blue - CsA Cyclosporin A - CyP Cyclophilin     

Lrrn1 is required for formation of the midbrain-hindbrain boundary and organiser through regulation of affinity differences between midbrain and hindbrain cells in chick

The midbrain–hindbrain boundary (MHB) acts as an organiser/signalling centre to pattern tectal and cerebellar compartments. Cells in adjacent compartments must be distinct from each other for boundary formation to occur at the interface. Here we have identified the leucine-rich repeat (LRR) neuronal 1 (Lrrn1) protein as a key regulator of this process in chick. The Lrrn family is orthologous to the Drosophila tartan/capricious (trn/caps) family. Differential expression of trn/caps promotes an affinity difference and boundary formation between adjacent compartments in a number of contexts; for example, in the wing, leg and eye imaginal discs. Here we show that Lrrn1 is expressed in midbrain cells but not in anterior hindbrain cells. Lrrn1 is down-regulated in the anterior hindbrain by the organiser signalling molecule FGF8, thereby creating a differential affinity between these two compartments. Lrrn1 is required for the formation of MHB — loss of function leads to a loss of the morphological constriction and loss of Fgf8. Cells overexpressing Lrrn1 violate the boundary and result in a loss of cell restriction between midbrain and hindbrain compartments. Lrrn1 also regulates the glycosyltransferase Lunatic Fringe, a modulator of Notch signalling, maintaining its expression in midbrain cells which is instrumental in MHB boundary formation. Thus, Lrrn1 provides a link between cell affinity/compartment segregation, and cell signalling to specify boundary cell fate.