Cmk2, a novel serine/threonine kinase in fission yeast

The cmk2 gene of Schizosaccharomyces pombe encodes a 504 amino acid protein kinase with sequence homology with the calmodulin-dependent protein kinase family. The cmk2(+) gene is not essential for cell viability but overexpression of cmk2(+) blocks the cell cycle at G2 phase and this inhibition is cdc2-dependent. The Cmk2 is a cytoplasmic protein expressed in a cell cycle-dependent manner, peaking at the G1/S boundary. Overexpression of Cmk2 suppresses fission yeast DNA replication checkpoint defects but not DNA damage checkpoint defects, suggesting that the G2 cell cycle arrest mediated by high levels of Cmk2 provides sufficient time to correct DNA replication alterations.     

Seasonal and interannual variability of size-fractionated phytoplankton biomass and community structure at station Kerfix, off the Kerguelen Islands, Antarctica

Time series of phytoplankton biomass and taxonomic compositionhave been obtained for the 3 years 1992, 1993 and 1994 in thenorthern part of the Southern Ocean (station Kerfix, 5040'S,6825;E) Autotrophic biomass was low throughout the year (<0.2mg m^–3 except during a short period in summer when a maximumof 1.2 mg chlorophyll (Chl) a m– was reached. During winter,the integrated biomass was low (<10 mg m^–2) and associatedwith deeply mixed water, whereas the high summer biomass (>20mg m^–2) was associated with increased water column stability.During summer blooms, the >10 µ;m size fraction contributed60% to total integrated biomass. Large autotrophic dinoflagellates,mainly Prorocentrum spp., were associated with the summer phytoplankton maxima and accounted for >80% of the total autotrophcarbon biomass. In November and December, the presence of thelarge heterotrophic dinoflagellates Protoperidinium spp. andGyro dinium spp. contributed a high proportion of total carbonbiomass. During winter, the <10 µm size fraction contributed80% of total Chi a biomass with domination of the picoplanktonsize fraction. The natural assemblage included mainly nakedflagellates such as species of the Prasinophyceae, Cryptophyceaeand Prymnesiophyceae. During spring, picocyanobacteria occurredin sub-surface water with a maximum abundance in September of106 cells 1^–1     

A proposed bioactive form of peptide T and the design of peptidomimetics

Peptide T is a non-natural octapeptide of sequence Ala-Ser-Thr-Thr-Thr-Asn-Tyr-Thr, taken from the sequence of the protein gp120 of HIV. The peptide has been shown to bind competitively to the CD4 receptors of the helper/inducer lymphocytes T. The peptide is presently used for the treatment of AIDS-associated dementia and has been proven useful for the treatment of psoriasis. Using molecular modeling procedures, we studied the conformational profile of this peptide as well as those of several active and inactive analogs. The analysis of these results gave rise to the proposal of a bioactive conformation of the peptide, which can be described as a pseudo -turn structure, involving the last four residues at the C-terminus of the peptide. The secondary structure is stabilized by a hydrogen bond between the hydroxyl hydrogen of the side chain of Thr⁵ and the carbonyl oxygen of Tyr⁷. From the bioactive form and different structure–activity relationship studies, a pharmacophore was proposed. This hypothesis was used to search on several 3D data bases. One of the hits obtained was the natural compound amigdalin, which was tested and exhibited moderate activity.     

A new genetic model proposing that the Se gene is a structural gene closely linked to the H gene.

The Se gene is classically considered as a regulatory gene controlling the expression of the structural gene H in external secretions. Under this hypothesis, Bombay (h/h) individuals should not be able to express the Se gene. Statistical analysis of the 44 published Bombay pedigrees suggests on the contrary that there is no suppression of Se in Bombay individuals, and that both Se and H loci can be fully expressed at the phenotypic level. Based on a lod score of 12.9 at 1% recombination units and the existence of two different acceptors for the biosynthesis of the H antigen, a new genetic model is proposed in which H and Se would be two closely linked structural genes coding for two different 2-alpha-L-fucosyltransferases.     

Acceptor specificity and tissue distribution of three human alpha-3-fucosyltransferases

Based on the capacity to transfer alpha-L-fucose onto type-1 and type-2 synthetic blood group H and sialylated acceptors, a comparison of the alpha-3-fucosyltransferase activities of different human tissues is shown. Three distinct acceptor specificity patterns are described: (I) myeloid alpha-3-fucosyltransferase pattern, in which leukocytes and brain enzymes transfer fucose actively onto H type-2 acceptor and poorly onto sialylated N-acetyllactosamine: (II) plasma alpha-3-fucosyltransferase (EC 2.4.1.152), in which plasma and hepatocyte enzymes transfer, in addition, onto the sialylated N-acetyllactosamine; (III) Lewis alpha-3 4-fucosyltransferase (EC 2.4.1.65), in which gall-bladder kidney and milk enzymes transfer, in addition, onto type-1 acceptors. The small amount (less than 10%) of alpha-3-fucosyltransferase activity found in the plasma of an alpha-3-fucosyltransferase-deficient individual had a myeloid-type acceptor pattern, suggesting that this small proportion of the plasma enzyme is derived from leukocytes. In addition to the three acceptor specificity patterns, these enzyme activities can be differentiated by their optimum pH: 8.0-8.7 for the enzymes from myeloid cells and brain. 7.2-8.0 for liver enzymes and 6.0-7.2 for gallbladder enzymes. Milk samples had two alpha-3-fucosyltransferase activities, the Lewis or alpha-3/4-fucosyltransferase under control of the Lewis gene and an alpha-3-fucosyltransferase with plasma acceptor pattern which was independent of the control of the Lewis gene. The apparent affinity for GDP-fucose of the myeloid-like enzyme was weaker than those of the plasma and Lewis-like enzymes. The apparent affinities for H type 2 and sialylated N-acetyllactosamine were stronger for exocrine secretions as compared to the plasma and myeloid enzymes. The plasma type of alpha-3-fucosyltransferase activity was more sensitive to N-ethylmaleimide and heat inactivation than the samples with myeloid-like alpha-3-fucosyltransferase activity.     

H-deficient blood groups ( Bombay) of Reunion Island.

Forty-two H-deficient individuals (lacking H antigen on erythrocytes) with anti-H in their sera were found on Reunion Island. A, B, and AB Bombay subjects had small but detectable amounts of A and/or B antigens on erythrocytes. All the H-deficient phenotypes tested were nonsecretors of ABH in their saliva, and one-third were Lewis negative. Fifty-three of the 108 (49%) unaffected members in the 14 Bombay pedigrees analyzed were se/se, showing that the families were selected for the nonsecretor trait, and suggesting that the Bombay probands used to select the families have se/se genotype. In accordance with this concept, all the children from Bombay nonsecretor x unaffected nonsecretor matings were se/se. Segregation of H and Se is compatible with the genetic model proposing that Se and H are closely linked structural genes, and the analysis of the present and previously published Bombay pedigrees strongly supports this model.     

Forssman penta- and tetraglycosylceramide are xenoantigens of ostrich kidney and liver.

The heterophile antigens Galalpha1-->3Gal and N-glycolylneuraminic acid are the major obstacle to grafting mammal organs, especially from pig, to man. Lack of expression of these common xenoantigens by birds has raised interest in ostrich as a potential organ donor for xenotransplantation. Glycosphingolipids of ostrich liver and kidney were investigated for their carbohydrate determinants. Both organs were found similar in their glycolipid composition with three major species, mono-, di-, and pentaglycosylceramide. The pentaglycosylceramide was characterized as the Forssman antigen. In both organs, the ceramide portion was highly hydroxylated with prevalence of alpha-hydroxylated fatty acids, C18 phytosphingosine in kidney and C18 sphingosine in liver Forssman glycolipid. These data indicate that hydroxylation of kidney glycosphingolipids, which is found in mammals, has been maintained since the divergence of birds from other vertebrates. Characterization of a minor glycolipid as a Forssman tetraglycosylceramide built on the galabiosylceramide core indicates that the Forssman tetraglycosylceramide also exists in vivo. Its precursors, galactosyl- and galabiosylceramide, were characterized in kidney and liver. The Forssman antigen is the third heterophile antigen against which man raises natural antibodies. Its localization in the vascular endothelium and connective tissue makes ostrich an unpromising organ or cell donor for xenotransplantation to man.     

Distinct light-mediated pathways regulate the biosynthesis and exchange of isoprenoid precursors during Arabidopsis seedling development

Plants synthesize an astonishing diversity of isoprenoids, some of which play essential roles in photosynthesis, respiration, and the regulation of growth and development. Two independent pathways for the biosynthesis of isoprenoid precursors coexist within the plant cell: the cytosolic mevalonic acid (MVA) pathway and the plastidial methylerythritol phosphate (MEP) pathway. In at least some plants (including Arabidopsis), common precursors are exchanged between the cytosol and the plastid. However, little is known about the signals that coordinate their biosynthesis and exchange. To identify such signals, we arrested seedling development by specifically blocking the MVA pathway with mevinolin (MEV) or the MEP pathway with fosmidomycin (FSM) and searched for MEV-resistant Arabidopsis mutants that also could survive in the presence of FSM. Here, we show that one such mutant, rim1, is a new phyB allele (phyB-m1). Although the MEV-resistant phenotype of mutant seedlings is caused by the upregulation of MVA synthesis, its resistance to FSM most likely is the result of an enhanced intake of MVA-derived isoprenoid precursors by the plastid. The analysis of other light-hyposensitive mutants showed that distinct light perception and signal transduction pathways regulate these two differential mechanisms for resistance, providing evidence for a coordinated regulation of the activity of the MVA pathway and the crosstalk between cell compartments for isoprenoid biosynthesis during the first stages of seedling development.     

Antigenicity of recombinant proteins after regioselective immobilization onto polyanhydride-based copolymers

We previously demonstrated that the introduction of a tag consisting of several contiguous lysines at the N- or C-terminus of a recombinant protein greatly improved the covalent grafting of the protein onto negatively charged maleic anhydride-alt-methyl vinyl ether (MAMVE) copolymer, under many different experimental conditions (Ladavière, C., et al. (1998) Bioconjugate Chem. 9, 655; Allard, L., et al. (2002) Biotechnol. Bioeng. 80, 341). The grafting efficiency was dependent on the charge and amine density of the tag, characteristics which were determined by the tag composition. The six lysine tag (Lys6) was found to be the most efficient (Allard, L., et al. (2001) Bioconjugate Chem. 12, 972). In the present work, the biological activity of Lys6-proteins covalently bound to polymer was investigated. N- or C-terminal Lys6-tagged HIV-1 p24 recombinant proteins (RK24H and RH24K) were grafted onto MAMVE, and the antigenicity each of the bioconjugates was evaluated using six monoclonal antibodies that recognized different epitopes distributed along the protein. We demonstrate that the position of the tag and the hydrolysis rate of the anhydride moieties of the polymer are the two main parameters involved in the conservation of the biological activity of the immobilized protein. We thus present a process which allows an efficient oriented immobilization of proteins onto copolymers with optimal biological activity that is suitable for the controlled production of active bioconjugates.