A novel callose synthase from pollen tubes of Nicotiana

Pollen-tube cell walls are unusual in that they are composed almost entirely of callose, a (1,3)--linked glucan with a few 6-linked branches. Regulation of callose synthesis in pollen tubes is under developmental control, and this contrasts with the deposition of callose in the walls of somatic plant cells which generally occurs only in response to wounding or stress. The callose synthase (uridine-diphosphate glucose: 1,3--d-glucan 3--d-glucosyl transferase, EC 2.4.1.34) activities of membrane preparations from cultured pollen tubes and suspension-cultured cells of Nicotiana alata Link et Otto (ornamental tobacco) exhibited different kinetic and regulatory properties. Callose synthesis by membrane preparations from pollen tubes was not stimulated by Ca²⁺ or other divalent cations, and exhibited Michaelis-Menten kinetics only between 0.25 mM and 6 mM uridine-diphosphate glucose (K _m 1.5–2.5 mM); it was activated by -glucosides and compatible detergents. In contrast, callose synthesis by membrane preparations from suspension-cultured cells was dependent on Ca²⁺, and in the presence of 2 mM Ca²⁺ exhibited Michaelis-Menten kinetics above 0.1 mM uridine-diphosphate glucose (K _m 0.45 mM); it also required a -glucoside and low levels of compatible detergent for full activity, but was rapidly inactivated at higher levels of detergent. Callose synthase activity in pollen-tube membranes increased ten fold after treatment of the membranes with trypsin in the presence of detergent, with no changes in cofactor requirements. No increase in callose synthase activity, however, was observed when membranes from suspension-cultured cells were treated with trypsin. The insoluble polymeric product of the pollen-tube enzyme was characterised as a linear (1,3)--d-glucan with no 6-linked glucosyl branches, and the same product was synthesised irrespective of the assay conditions employed.Abbreviations Ara l-arabinose - CHAPS 3-[(3-cholamidopropyl)dimethylammonia]-1-propane sulphonic acid - DAP diphenylamine-aniline-phosphoric acid stain - Gal d-galactose - Glc d-glucose - Man d-mannose - Mes 2-(N-morpholino)ethane sulphonic acid - Rha d-rhamnose - Rib d-ribose - TFA trifluoroacetic acid - UDPGlc uridine-diphosphate glucose - Xyl d-xylose This research was supported by funds from a Special Research Centre of the Australian Research Council. H.S. was funded by a Melbourne University Postgraduate Scholarship and an Overseas Postgraduate Research Studentship; S.M.R. was supported by a Queen Elizabeth II Research Fellowship. We thank Bruce McGinness and Susan Mau for greenhouse assistance, and Deborah Delmer and Adrienne Clarke for advice and encouragement throughout this project.     

THE FLAGELLAR APPARATUS OF HYMENOMONAS CORONATA (PRYMNESIOPHYTA)1

The ultrastructure of Hymenomonas coronata Mills was reinvestigated to determine the microarchitecture of the flagellar apparatus. Cell morphology and flagellar apparatus structure are very similar to those of Pleurochrysis. Some important variations occur. First, a crystalline root (= compound root) is absent on microtubular root 1. Second, a two-stranded microtubular root emanates at a right angle from microtubular root 2. Third, a fibrous root emanates from the dorsal region between the basal bodies and extends to the cell's right, paralleling microtubular root 3. These similarities and variations in flagellar apparatus characters are discussed in reference to known variations in the Prymnesiophyta.     

Strategies for the identification and purification of ligands for orphan biomolecules

The isolation of related genes with evolutionary conserved motifs by the application ofpolymerase chain reaction-based molecular biology techniques, or from database searchingstrategies, has facilitated the identification of new members of protein families. Many of theseprotein molecules will be involved in protein–protein interactions (e.g. growth factors,receptors, adhesion molecules), since such interactions are intrinsic to virtually every cellularprocess. However, the precise biological function and specific binding partners of these novelproteins are frequently unknown, hence they are known as orphan molecules.Complementary technologies are required for the identification of the specific ligands orreceptors for these and other orphan proteins (e.g., antibodies raised against crude biologicalextracts or whole cells). We describe herein several alternative strategies for the identification,purification and characterisation of orphan peptide and protein molecules, specifically thesynergistic use of micropreparative HPLC and biosensor techniques.     

Glucan synthesis by intact cotton fibres fed with different precursors at the stages of primary and secondary wall formation

Seed clusters of individual locules from fruit capsules of Gossypium arboreum L. with adhering intact fibres were fed with radioactive uridinediphosphoglucose (UDPG), guanosinediphosphoglucose (GDPG), glucose and sucrose. The incorporation into high molecular weight glucans of the fibres was studied. For primary wall fibres, UDPG at 1 mM was by far the best precursor, whereas sucrose was the best precursor for secondary wall fibres. No competition was observed between the incorporation of glucose from UDPG and from sucrose when the two were fed simultaneously to secondary wall fibres, indicating that their metabolic pathways are well separated when they are fed from the apoplast. Inhibitors of respiratory ATP-formation strongly inhibited incorporation of sucrose but not that of UDPG. Sucrose incorporation was studied at five different stages of development of the cotton fibres. At the stage of most intense secondary wall formation the incorporation rate was about 300 times that during primary wall formation (24 days post anthesis (DPA)). Incorporation from 1 mM UDPG or GDPG by secondary wall fibres (35 DPA) was less than twice that of primary wall fibres (22 DPA), indicating that the two sugar nucleotides are not readily used as precursors for secondary wall cellulose when they are fed to the exterior of intact cells. The high molecular weight non-cellulosic glucans formed from UDPG and sucrose at 5 and 1,000 M were solubilized in strongly alkaline solutions or dimethyl-sulfoxide (DMSO) and were partially characterized by degradation with an exo--1,3-glucanase. After feeding for one hour, at most 1/3 of the radioactivity in high molecular weight material was found in cellulose and at least 2/3 in -1,3-glucan. The proportions varied little for fibres in the age range of 30 to 48 DPA when sucrose was the precursor although the total incorporation varied by a factor of about four. The fact that at all stages of secondary wall formation -1,3-glucan is synthesized at a very high rate, but that the total amount in the cell wall does not exceed 2% in the later stages of wall formation, can be interpreted in terms of a high turnover of this polysaccharide if it is assumed that wound effects are negligible in the system under study.Abbreviations UDPG uridinediphosphoglucose - GDPG guanosinediphosphoglucose - HEPES N-2-hydroxyethylpiperazine-N-2-ethansulphonic acid - DMSO dimethyl-sulfoxide - DNP 2,4-dinitrophenol - DPA days post anthesis     

Extraction and analysis of superoxide free radicals (·O−2) from whole mammalian liver

Extraction of whole lobes of normal rat liver with dimethyl sulphoxide (DMSO) under N₂ gives extracts which contain 5—10 μmol/l·O^?₂ (50-100 nmol·O^?₂ per 10 ml extract per 4 g liver; 1.25-2.50 nmol·O^?₂ per millilitre per gram liver). Evidence for ·O^?₂ in the extracts is given by: (1) electron spin resonance signals (ESR), (2) differential pulse polarography (DPP), (3) chemiluminescence (CL), and (4) nitroblue tetrazolium reduction (NBT). All tests yield results identical with those obtained with authentic ·O^?₂. Extraction of ·O^?₂ is enhanced by tetrabutyl ammonium ion, and is maximal at 1-3 min. These results raise the possibility that substantial amounts of ·O^?₂ are normally sequestered in protective membranous sites in vivo.     

Effects of cell heterogeneity on production of polypeptide growth factors and mesoderm-inducing activity by Xenopus laevis XTC cells

The Xenopus laevis XTC cell line has been analyzed for the production of polypeptide growth factors and mesoderm-inducing activity. By the use of specific biological assays, it is shown that XTC cells produce a growth factor functionally related to the platelet-derived growth factor (PDGF) and two transforming growth factor (TGF)β-like activities. Mesoderm-inducing activity, as measured on X. laevis ectodermal explants from stage 10 embryos, was found to coelute on a Bio-Gel P-100 column with one of the TGFβ-like activities at an apparent molecular weight of 6–10 kDa. Analysis of the DNA content from XTC cells by flow cytometry demonstrated that the cell line is heterogeneous and consists of both tetraploid and diploid cells. Cloning of the XTC cells and selecting single-cell colonies on the basis of their ability to grow in soft agar resulted in the isolation of several homogeneous, morphologically different clonal derivatives. Analysis of conditioned medium from these clonal derivatives showed that only one of them, the only diploid line among six investigated, produced a strong heat- and acid-stable mesoderm-inducing activity that induced notochord and muscle formation in stage 10 X. laevis ectodermal expiants. The relation between this activity and a recently described TGFβ-like mesoderm-inducing factor obtained from XTC-conditioned medium will be discussed. In conclusion, a clonal cell line derived from X. laevis XTC cells which provides a good source for further characterization of mesoderm-inducing factors has been established.     

Heritability and components of phenotypic expression in skin reflectance of Mestizos from the Peruvian Lowlands

Skin reflectance was measured on the inner upper arm and forehead of a sample of 209 Mestizos ranging in age from 2 to 64 years living in the town of Lamas in the Eastern Peruvian Lowlands. The sample consisted of 43 father-son, 42 father-daughter, 62 mother-son, and 70 mother-daughter pairs. The sample also consisted of 57 brother-brother, 60 sister-sister and 139 brother-sister pairs. The reflectance measurements were made with a Photovolt Reflection Meter, model 670. Stepwise polynomial regression techniques were used to derive standardized residual values. Then using these residual values parent-offspring, sibling intraclass correlations and components of the phenotypic expression of skin reflectance were calculated. The study indicates that 1) the parent-offspring and sibling correlation coefficients conformed with the theoretical correlations expected assuming polygenic inheritance; 2) the husband-wife correlations indicate a high degree of assortative mating for skin color, but despite this effect the parent-offspring and sibling correlation coefficients are lower than the values expected under the influence of autosomal genes; 3) estimates of heritability and components of phenotypic expression indicate that about 55% of the total variability in skin reflectance could be attributed to the influence of additive genetic factors; and 4) there is no evidence of X-linkage in the inheritance of skin color.