EFFECT OF TEMPERATURE ON TWO ENZYMES FROM A PSYCHROPHILIC CHLOROMONAS (CHLOROPHYTA)1

A psychrophilic green alga belonging to the Chloromonas genus and here named ANT1 was collected in Antarctica. The activities of two enzymes, nitrate reductase and argininosuccinate lyase, were measured at various temperatures and compared to the corresponding enzyme activities in the mesophilic species Chlamydomonas reinhardtii Dangeard. For both enzymes, the temperature for apparent optimal activity was about 20°C lower in ANT1 than in C. reinhardtii. The enzymes were also submitted to various heat treatments before measuring their activities. Both psychrophilic enzymes were more sensitive to heat than the corresponding mesophilic enzymes. It is worth stressing, however, that in both species nitrate reductase was much more sensitive to heat than argininosuccinate lyase, which probably indicates that the peculiar structure of each protein primarily determines its dependence to temperature. Secondary adaptations to low temperatures should then occur to confer the psychrophilic character.     

Characterization of DNA sequences encoding a novel isoform of the 55 kDa B regulatory subunit of the type 2A protein serine/threonine phosphatase of Arabidopsis thaliana

We have indentified a novel gene (AtB) encoding a previously uncharacterized isoform of the B regulatory subunit of the type 2A serine/threonine protein phosphatase (PP2A) of Arabidopsis, and show that mRNA derived from the AtB gene accumulates in all Arabidopsis organs. In addition, we examined the expression of the three genes encoding the A regulatory subunit of Arabidopsis PP2A and show these genes are expressed in all organs as well. Taken together, our results suggest a myriad of PP2A subunit combinations, possibly with distinct substrate specificities, may occur within each Arabidopsis cell.     

Elementary auxin response chains at the plasma membrane involve external abp1 and multiple electrogenic ion transport proteins

Studies of membrane electrical responses of isolated protoplasts to auxin have demonstrated the existence of elementary response chains to auxin at the plasma membrane, presently defined only by their uttermost ends. At one side, as demonstrated by several lines of evidence, the auxin perception unit involves proteins homologous to ZmER-abp1 (abp1), the most abundant auxin-binding protein from maize coleoptiles. At the other side, multiple ion transport proteins appear as targets of the auxin signal; the proton pump ATPase, an anion channel and potassium channels. We investigated early electrical responses to auxin at the plasma membrane of tobacco protoplasts. The work presented here will initially focus on abp1 and its functional role at the membrane. The C-terminus abp1 peptide (Pz151–163) was recently reported to modulate K⁺ currents at the plasma membrane of intact guard cells from broad bean [23] and induce plasma membrane hyperpolarisation of tobacco mesophyll protoplasts. These results further demonstrate that proteins involved in plasma membrane responses to auxin are related to maize abp1, and provide clues as to the region of the protein possibly involved in the interaction of abp1 with the plasma membrane. Secondly, this report concentrates on one of the targets of auxin, a voltage-dependent and ATP-regulated anion channel that we characterised on protoplasts from tobacco cell suspensions. This anion channel was specifically modulated by auxin, as already observed for the anion channel of guard cells [14]. Further work will be needed to assess if this auxin modulation involves a direct interaction between the hormone and the anion channel protein(s), or follows from the activation of a perception chain including abp1 homologues.     

Thermococcus chitonophagus sp. nov., a novel,chitin-degrading,hyperthermophilic archaeum from a deep-sea hydrothermal vent environment

From a hydrothermal vent site off the Mexican west coast (20°50′N, 109°06′W) at a depth of 2,600 m, a novel, hyperthermophilic, anaerobic archaeum was isolated. Cells were round to slightly irregular cocci, 1.2–2.5 μm in diameter and were motile by means of a tuft of flagella. The new isolate grew between 60 and 93°C (optimum: 85°C), from pH 3.5 to 9 (optimum: pH 6.7), and from 0.8 to 8% NaCl (optimum: 2%). The isolate was an obligate organotroph, using chitin, yeast extract, meat extract, and peptone for growth. Chitin was fermented to H₂, CO₂, NH₃, acetate, and formate. H₂S was formed in the presence of sulfur. The chitinoclastic enzyme system was oxygen-stable, cell-associated, and inducible by chitin. The cell wall was composed of a surface layer of hex- americ protein complexes arranged on a p6 lattice. The core lipids consisted of glycerol diphytanyl diethers and acyclic and cyclic glycerol diphytanyl tetraethers. The G+C content was 46.5 mol%. DNA/DNA hybridization and 16S rRNA sequencing indicated that the new isolate belongs to the genus Thermococcus, representing a new species, Thermococcus chitonophagus. The type strain is isolate GC74, DSM 10152. Received: 8 May 1995 / Accepted: 26 June 1995     

Study of sucrose and mannitol transport in plasma-membrane vesicles from phloem and non-phloem tissues of celery (Apium graveolens L.) petioles

The mature petiole of celery is an organ with versatile sink/source capacities where sucrose and mannitol are unloaded from and reloaded into the phloem cells. Plasma-membrane vesicles were purified by twophase partitioning either from phloem strands isolated from mature petioles of celery (Apium graveolens L.) or from mature petioles devoid of vascular bundles. Both types of vesicle were comparable in purity (more than 86% of plasma-membrane origin), size (135 nm diameter) and orientation (72% right-side-out). Plasma-membrane vesicles from phloem tissues had a higher vanadate-sensitive ATPase activity than plasma-membrane vesicles from petioles. Plasma-membrane vesicles from phloem tissues accumulated mannitol and sucrose in response to an artificial proton-motive force, in agreement with the existence of proton/substrate carriers. Plasma-membrane vesicles from petioles devoid of vascular bundles accumulated only mannitol following application of an artificial proton-motive force. The data suggest the volvement of apoplasmic transport events. The pathway for sucrose uptake in storage parenchyma cells is discussed in the light of the available physiological data.     

Amino acid sequence of the ribosomal protein HS23 from the halophilicHaloarcula marismortui and homology studies to other ribosomal proteins

The ribosomal protein HS23 from the 30S subunit of the extreme halophilicHaloarcula marismortui, belonging to the group of archaea, was isolated either by RP-HLPLC or two-dimensional polyacrylamide gel electrophoresis. The complete amino acid sequence was determined by automated N-terminal microsequencing. The protein consists of 123 residues with a corresponding molecular mass of 12,552 Da as determined by electrospray mass spectroscopy; the pI is 11.04. Homology studies reveal similarities to the eukaryotic ribosomal protein S8 fromHomo sapiens, Rattus norvegicus, Leishmania major, andSaccharomyces cerevisiae.Abbreviations H. marismortui Haloarcula marismortui - PVDF polyvinylidene difluoride - PTH phenylthiohydantoin - RP-HPLC reversed-phase high-performance liquid chromatography - TFA trifluoro acetic acid - TP30 total protein mixture from the 30S ribosomal subunit ofH. marismortui     

An Essential Role for CD44 Variant Isoforms in Epidermal Langerhans Cell and Blood Dendritic Cell Function

Upon antigen contact, epidermal Langerhans cells (LC) and dendritic cells (DC) leave peripheral organs and home to lymph nodes via the afferent lymphatic vessels and then assemble in the paracortical T cell zone and present antigen to T lymphocytes. Since splice variants of CD44 promote metastasis of certain tumors to lymph nodes, we explored the expression of CD44 proteins on migrating LC and DC. We show that upon antigen contact, LC and DC upregulate pan CD44 epitopes and epitopes encoded by variant exons v4, v5, v6, and v9. Antibodies against CD44 epitopes inhibit the emigration of LC from the epidermis, prevent binding of activated LC and DC to the T cell zones of lymph nodes, and severely inhibit their capacity to induce a delayed type hypersensitivity reaction to a skin hapten in vivo. Our results demonstrate that CD44 splice variant expression is obligatory for the migration and function of LC and DC.     

Lysine-ketoglutarate reductase and saccharopine dehydrogenase from Arabidopsis thaliana: nucleotide sequence and characterization

We isolated the gene encoding lysine-ketoglutarate reductase (LKR, EC 1.5.1.8) and saccharopine dehydrogenase (SDH, ED 1.5.1.9) from an Arabidopsis thaliana genomic DNA library based on the homology between the yeast biosynthetic genes encoding SDH (lysine-forming) or SDH (glutamate-forming) and Arabidopsis expressed sequence tags. A corresponding cDNA was isolated from total Arabidopsis RNA using RT-PCR and 5 and 3 Race. DNA sequencing revealed that the gene encodes a bifunctional protein with an amino domain homologous to SDH (lysine-forming), thus corresponding to LKR, and a carboxy domain homologous to SDH (glutamate-forming). Sequence comparison between the plant gene product and the yeast lysine-forming and glutamate-forming SDHs showed 25% and 37% sequence identity, respectively. No intracellular targeting sequence was found at the N-terminal or C-terminal of the protein. The gene is interrupted by 24 introns ranging in size from 68 to 352 bp and is present in Arabidopsis in a single copy. 5 sequence analysis revealed several conserved promoter sequence motifs, but did not reveal sequence homologies to either an Opaque 2 binding site or a Sph box. The 3-flanking region does not contain a polyadenylation signal resembling the consensus sequence AATAAA. The plant SDH was expressed in Escherichia coli and exhibited similar biochemical characteristics to those reported for the purified enzyme from maize. This is the first report of the molecular cloning of a plant LKR-SDH genomic and cDNA sequence.