A small camel-milk protein rich in cysteine/half-cystine

A small protein (M_r about 14 000) rich in cysteine/half-cystine has been isolated from camel milk by exclusion chromatography and reverse-phase high-performance liquid chromatography. The N-terminal amino acid sequence shows a region with several positional identities with and -caseins, which however lack cysteine residues; postions 16–20 are identical and involve the serine residues that have been found to be phosphorylated in -caseins.     

Somatic embryogenesis from pea embryos and shoot apices

Conditions were defined for plant regeneration via somatic embryogenesis in pea, using explants from immature zygotic embryos or from shoot apices. For the induction of somatic embryos, an auxin (picloram or 2,4-dichlorophenoxyacetic acid) was required. Embryogenic callus originated from embryonic axis tissue of immature embryos and from the axillary-bud region and the plumula of shoot apices. A clear effect of embryo size on somatic embryogenesis was shown. There were differences in frequency of somatic embryogenesis among the five genotypes used in the study. Additions of BA to auxin-containing medium reduced embryo production. Histological examinations confirmed the embryogenic nature of the immature embryo cultures and revealed that somatic embryos originated from the meristematic areas near the callus surface.Abbreviations BA benzyladenine - 2,4-D 2,4-dichlorophenoxyacetic acid - NAA naphthaleneacetic acid - picloram 4-amino-3,5,6-trichloropicolinic acid     

Display of functional nucleic acid polymerase on Escherichia coli surface and its application in directed polymerase evolution

We report a first of its kind functional cell surface display of nucleic acid polymerase and its directed evolution to efficiently incorporate 2′-O-methyl nucleotide triphosphates (2′-OMe-NTPs). In the development of polymerase cell surface display, two autotransporter proteins (Escherichia coli adhesin involved in diffuse adherence and Pseudomonas aeruginosa esterase A [EstA]) were employed to transport and anchor the 68-kDa Klenow fragment (KF) of E. coli DNA polymerase I on the surface of E. coli. The localization and function of the displayed KF were verified by analysis of cell outer membrane fractions, immunostaining, and fluorometric detection of synthesized DNA products. The EstA cell surface display system was applied to evolve KF for the incorporation of 2′-OMe-NTPs and a KF variant with a 50.7-fold increased ability to successively incorporate 2′-OMe-NTPs was discovered. Expanding the scope of cell-surface displayable proteins to the realm of polymerases provides a novel screening tool for tailoring polymerases to diverse application demands in a polymerase chain reaction and sequencing-based biotechnological and medical applications. Especially, cell surface display enables novel polymerase screening strategies in which the heat-lysis step is bypassed and thus allows the screening of mesophilic polymerases with broad application potentials ranging from diagnostics and DNA sequencing to replication of synthetic genetic polymers.     

Regulation of colony morphology and biofilm formation in Shewanella algae

Bacterial colony morphology can reflect different physiological stages such as virulence or biofilm formation. In this work we used transposon mutagenesis to identify genes that alter colony morphology and cause differential Congo Red (CR) and Brilliant Blue G (BBG) binding in Shewanella algae, a marine indigenous bacterium and occasional human pathogen. Microscopic analysis of colonies formed by the wild-type strain S. algae CECT 5071 and three transposon integration mutants representing the diversity of colony morphotypes showed production of biofilm extracellular polymeric substances (EPS) and distinctive morphological alterations. Electrophoretic and chemical analyses of extracted EPS showed differential patterns between strains, although the targets of CR and BBG binding remain to be identified. Galactose and galactosamine were the preponderant sugars in the colony biofilm EPS of S. algae. Surface-associated biofilm formation of transposon integration mutants was not directly correlated with a distinct colony morphotype. The hybrid sensor histidine kinase BarA abrogated surface-associated biofilm formation. Ectopic expression of the kinase and mutants in the phosphorelay cascade partially recovered biofilm formation. Altogether, this work provides the basic analysis to subsequently address the complex and intertwined networks regulating colony morphology and biofilm formation in this poorly understood species.     

Recognition of peat-forming plant communities from their peat deposits in two south Swedish bog complexes

Peat samples, 3 015 from 103 boring points, on two mires (Åkhult mire, Store Mosse mire) south Sweden, have been subjected to macrofossil analysis. Based on plant remains, 9 peat groups were distinguished in the field. A further classification using phytosociological methods revealed 29 peat types. The affinities between the peat types were determined from TABORD classification and a Reciprocal Averaging ordination. The primary floristic differentiation is correlated with a gradient from treeless-to wooded stands, which coincides largely with the mire expanse-mire margin gradient. The poor-rich gradient seems to parallel the treeless-wooded gradient as well and may reflect the natural conditions in this mire before it was affected by man. The hummock-mud-bottom gradient is easy to distinguish in peat, formed by bog communities, but is not distinct in peat formed by fen communities and impossible to detect in peat dominated by wood remains. The amount of identifiable remains depends on the decomposition, which is determined by (1) the period of time the plant litter stays in the acrotelm, and (2) the nutrient status. The decomposition is greatest in fen-peat with abundant wood remains. This probably depends on a good supply of oxygen caused by greater horizontal water movements and better nutrient status.     

Transmembrane topology of FRO2, a ferric chelate reductase from Arabidopsis thaliana

Iron uptake in Arabidopsis thaliana is mediated by ferric chelate reductase FRO2, a transmembrane protein belonging to the flavocytochrome b family. There is no high resolution structural information available for any member of this family. We have determined the transmembrane topology of FRO2 experimentally using the alkaline phosphatase fusion method. The resulting topology is different from that obtained by theoretical predictions and contains 8 transmembrane helices, 4 of which build up the highly conserved core of the protein. This core is present in the entire flavocytochrome b family. The large water soluble domain of FRO2, which contains NADPH, FAD and oxidoreductase sequence motifs, was located on the inside of the membrane.Electronic Supplementary Material Supplementary material is available to authorised users in the online version of this article at This work was supported by grants from the Swedish Research Council (VR) to S. Al-Karadaghi and Hans Hebert.     

Assembly and function of a spore coat-associated transglutaminase of Bacillus subtilis

The assembly of a multiprotein coat around the Bacillus subtilis spore confers resistance to lytic enzymes and noxious chemicals and ensures normal germination. Part of the coat is cross-linked and resistant to solubilization. The coat contains epsilon-(gamma-glutamyl)lysyl cross-links, and the expression of the gene (tgl) for a spore-associated transglutaminase was shown before to be required for the cross-linking of coat protein GerQ. Here, we have investigated the assembly and function of Tgl. We found that Tgl associates, albeit at somewhat reduced levels, with the coats of mutants that are unable to assemble the outer coat (cotE), that are missing the inner coat and with a greatly altered outer coat (gerE), or that are lacking discernible inner and outer coat structures (cotE gerE double mutant). This suggests that Tgl is present at various levels within the coat lattice. The assembly of Tgl occurs independently of its own activity, as a single amino acid substitution of a cysteine to an alanine (C116A) at the active site of Tgl does not affect its accumulation or assembly. However, like a tgl insertional mutation, the tglC116A allele causes increased extractability of polypeptides of about 40, 28, and 16 kDa in addition to GerQ (20 kDa) and affects the structural integrity of the coat. We show that most Tgl is assembled onto the spore surface soon after its synthesis in the mother cell under sigma(K) control but that the complete insolubilization of at least two of the Tgl-controlled polypeptides occurs several hours later. We also show that a multicopy allele of tgl causes increased assembly of Tgl and affects the assembly, structure, and functional properties of the coat.     

Functional screening of G protein-coupled receptors by measuring intracellular calcium with a fluorescence microplate reader

Ligand binding studies reveal information about affinity to G protein-coupled receptors (GPCRs) rather than functional properties. Increase in intracellular Ca(2+) appears to represent a universal second messenger signal for a majority of recombinant GPCRs. Here, we exploit Ca(2+) signaling as a fast and sensitive functional screening method for a number of GPCRs coupled to different G proteins. Ca(2+) fluorescence measurements are performed using Oregon Green 488 BAPTA-1/AM and a microplate reader equipped with an injector. Buffer alone or test compounds dissolved in buffer are injected into a cell suspension, and fluorescence intensity is recorded for 30 s. Each of the GPCRs tested--G(q)-coupled P2Y(2), G(s)-coupled dopamine D1 and D5, G(i)-coupled dopamine D2L, and G(q/11)-coupled muscarinic acetylcholine M1--yielded a significant rise in intracellular free [Ca(2+)] on agonist stimulation. Agonist stimulation was dose dependent, as shown for ATP or UTP stimulation of P2Y(2) receptors (EC(50) = 1 microM), SKF38393 stimulation of hD1 and hD5 (EC(50) = 18.1 nM and 2.7 nM), and quinpirole at hD2L (EC(50) = 6.5 nM). SCH23390 (at hD1 and hD5) and spiperone, haloperidol, and clozapine (at hD2L) competitively antagonized the Ca(2+) response. Furthermore, the Ca(2+) assay served to screen suramin analogs for antagonistic activity at P2Y(2) receptors. Screening at dopamine receptors revealed LE300, a new lead for a dopamine receptor antagonist. Advantages of the assay include fast and simple 96- or 384-well plate format (high-throughput screening), use of a visible light-excitable fluorescent dye, applicability to a majority of GPCRs, and simultaneous analysis of distinct Ca(2+) fluxes.     

HNCCH-TOCSY,a triple resonance experiment for the correlation of backbone 13Cα and 15N resonances with aliphatic side-chain proton resonances and for measuring vicinal 13CO, 1Hβ coupling constants in proteins

Summary A 3D triple resonance experiment has been designed to provide intraresidual and sequential correlations between amide nitrogens and -carbons in uniformly ¹³C¹⁵N-labeled proteins. In-phase ¹³C magnetization is transferred to the aliphatic side-chain protons via the side-chain carbons using a CC-TOCSY mixing sequence. Thus, the experiment alleviates the resonance assignment process by providing information about the amino acid type as well as establishing sequential connectivities. Leaving the carbonyl spins untouched throughout the transfer from ¹³C to ¹H leads to E.COSY-type cross peaks, from which the ³J_{H co} coupling constants can be evaluated. The pulse sequence is applied to oxidized Desulfovibrio vulgaris flavodoxin.