Thomas Peterbauer

Biochemistry and physiology of raffinose family oligosaccharides and galactosyl cyclitols in seeds

Raffinose family oligosaccharides (RFOs) are of almost ubiquitous occurrence in plant seeds. They accumulate during seed development and disappear rapidly during germination. The biosynthesis of raffinose, the first member of the series, proceeds by addition of a galactosyl unit to sucrose. Galactinol, a galactosyl derivative of myo-inositol, acts as a galactosyl donor. It is synthesized from UDP-D-galactose and myo-inositol. Stachyose, verbascose and ajugose, the next higher RFOs, are either synthesized by galactinol-dependent galactosyltransferases or by transfer of galactosyl units between two RFO molecules. In seeds, the metabolism of methylated inositols, such as D-ononitol and D-pinitol, is linked with the RFO pathway. In contrast to myoinositol, these cyclitols are galactosylated by transfer of galactosyl residues from galactinol and not from UDP-Dgalactose. However, the resulting galactosyl cyclitols can replace galactinol as galactosyl donors for the biosynthesis of stachyose. These recently discovered branches of the RFO pathway are active in seeds of a range of crop species, especially in legumes. We focus here on the biochemistry and molecular biology of the enzymes of RFO and galactosyl cyclitol biosynthesis. The metabolic control and hormonal regulation of the pathway during seed development and germination is discussed. The controversial role of � -galactosidases, which are believed to hydrolyse RFOs during germination, is reviewed critically.

Proliferation of aligned mammalian cells on laser-nanostructured polystyrene.

Biomaterial surface chemistry and nanoscale topography are important for many potential applications in medicine and biotechnology as they strongly influence cell function, adhesion and proliferation. In this work, we present periodic surface structures generated by linearly polarized KrF laser light (248 nm) on polystyrene (PS) foils. These structures have a periodicity of 200-430 nm and a depth of 30-100 nm, depending on the angle of incidence of the laser beam. The changes in surface topography and chemistry were analysed by atomic force microscopy (AFM), advancing water contact-angle measurements, Fourier-transform infrared spectroscopy using an attenuated total reflection device (ATR-FTIR) and X-ray photoelectron spectroscopy (XPS). We show that the surface laser modification results in a significantly enhanced adhesion and proliferation of human embryonic kidney cells (HEK-293) compared to the unmodified polymer foil. Furthermore, we report on the alignment of HEK-293 cells, Chinese hamster ovary (CHO-K1) cells and skeletal myoblasts along the direction of the structures. The results indicate that the presence of nanostructures on the substrates can guide cell alignment along definite directions, and more importantly, in our opinion, that this alignment is only observed when the periodicity is above a critical periodicity value that is cell-type specific.

Functional expression of a cDNA encoding pea (Pisum sativum L.) raffinose synthase, partial purification of the enzyme from maturing seeds, and steady-state kinetic analysis of raffinose synthesis

Abstract. Raffinose (O-α-D-galactopyranosyl-(1→6)-O-α-D-glucopyranosyl-(1↔2)-O-β-D-fructofuranoside) is a widespread oligosaccharide in plant seeds and other tissues. Raffinose synthase (EC 2.4.1.82) is the key enzyme that channels sucrose into the raffinose oligosaccharide pathway. We here report on the isolation of a cDNA encoding for raffinose synthase from maturing pea (Pisum sativum L.) seeds. The coding region of the cDNA was expressed in Spodoptera frugiperda Sf21 insect cells. The recombinant enzyme, a protein of glycoside hydrolase family 36, displayed similar kinetic properties to raffinose synthase partially purified from maturing seeds by anion-exchange and size-exclusion chromatography. Apart from the natural galactosyl donor galactinol (O-α-D-galactopyranosyl-(1→1)-L-myo-inositol), p-nitrophenyl α-D-galactopyranoside, an artificial substrate, was utilized as a galactosyl donor. An equilibrium constant of 4.1 was determined for the galactosyl transfer reaction from galactinol to sucrose. Steady-state kinetic analysis suggested that raffinose synthase is a transglycosidase operating by a ping-pong reaction mechanism and may also act as a glycoside hydrolase. The enzyme was strongly inhibited by 1-deoxygalactonojirimycin, a potent inhibitor for α-galactosidases (EC 3.2.1.22). The physiological implications of these observations are discussed.

Simple and versatile methods for the fabrication of arrays of live mammalian cells

Single-step methods for the generation of patterned surfaces on hydrogels are presented. Poly(vinyl alcohol) films covalently bonded on glass cover slips and commercially available hydrogel-coated polystyrene plates were used as cell-repellent surfaces. Cell-adhesive domains were created by spotting dilute solutions of sodium hypochlorite onto the surfaces. Alternatively, domains supporting cell attachment were created by exposure to UV light from a xenon excimer lamp, employing a contact mask. Rat skeletal myoblast cells, HEK 293 human embryonic kidney cells and Caco-2 colon carcinoma cells adhered and spread exclusively on modified areas. The surfaces are durable for weeks under cell culture conditions and re-usable after removal of the cells by trypsin treatment. Arrays of adhesive spots seeded with cells at a low density permitted dynamic monitoring of cell proliferation. Selected colonies can be harvested from the surfaces by means of local trypsination. Thus, these techniques may provide useful tools for the isolation of clonal cell populations. Additionally, we demonstrate the possibility of surface-mediated gene delivery from the micro patterns. We show that DNA, complexed with a lipid reagent, can be adsorbed on modified poly(vinyl alcohol) coatings, resulting in spatially controlled adhesion and reverse transfection of HEK 293 cells.

Enzymatic breakdown of raffinose oligosaccharides in pea seeds

Both alkaline and acidic α-galactosidases (α-d-galactoside galactohydrolases, E.C.3.2.1.22) isolated from various plant species have been described, although little is known about their co-occurrence and functions in germinating seeds. Here, we report on the isolation of two cDNAs, encoding for α-galactosidases from maturing and germinating seeds of Pisum sativum. One was identified as a member of the acidic α-galactosidase of the family 27 glycosyl hydrolase cluster and the other as a member of the family of alkaline α-galactosidases, which are highly homologous to seed imbibition proteins (SIPs). PsGAL1 transcripts, encoding for the ACIDIC α-GALACTOSIDASE, were predominately expressed during seed maturation and acidic enzyme activities were already present in dry seeds, showing little changes during seed germination. Compartmentation studies revealed that acidic α-galactosidases were located in protein storage vacuoles (PSVs). PsAGA1, encoding for the ALKALINE α-GALACTOSIDASE, was only expressed after radicle protrusion, when about 50% of RFOs have already been broken down. RFO breakdown was markedly decreased when the translation of the alkaline enzyme was inhibited, providing evidence that PsAGA1 indeed functioned in RFO degradation. Based on these data, we present an integrated model of RFO breakdown by two sequentially active α-galactosidases in pea seeds.

Dynamics of spreading and alignment of cells cultured in vitro on a grooved polymer surface

We used mechanically embossed polyester films to analyze the dynamics of cell alignment and cell-specific factors modulating the response of Chinese hamster ovary (CHO) cells and of a rat myogenic cell line to the surface topography. The films used had grooves with a periodicity of approximately 750 nm and a depth of 150 nm. Both cell lines responded to the topographical feature. On unpatterned control areas, cells of both lines showed a random distribution with orientation angles close to 45°. Both cell types exhibited an elongated morphology on the patterned surface. CHO cells typically showed bipolar spreading. Their contact area increased almost exclusively along the groove direction. Likewise, freshly seeded rat myoblasts displayed protrusions emerging in parallel with the grooves. However, myoblasts frequently had more than two sites with plasma protrusions pulling the cells along different grooves. They could also develop lamellipodia expanding without a preferred direction and long filopodia.

Stachyose in the cytosol does not influence freezing tolerance of transgenic Arabidopsis expressing stachyose synthase from adzuki bean

We expressed the stachyose synthase from adzuki bean (Vigna angularis) in Arabidopsis thaliana, under the control of the constitutive CaMV 35S promoter. Transgenic lines had only trace amounts of stachyose under normal growth conditions but accumulated stachyose to similar levels as raffinose upon cold acclimation. Stachyose production did not alter the freezing tolerance of cold acclimated rosette leaves. Non-aqueous fractionation of sub-cellular compartments revealed that in cold acclimated plants, raffinose but not stachyose accumulated to a proportion higher than the compartment size fraction in the plastids. Since both oligosaccharides are synthesized in the cytosol, this provides evidence that the so far unknown raffinose transporter of the Arabidopsis chloroplast envelope does not efficiently transport stachyose. The failure of stachyose to influence freezing tolerance in Arabidopsis supports the hypothesis that raffinose family oligosaccharides might function in protecting the thylakoid but not the plasma membrane during freezing.

Coupled and independent contributions of residues in IS6 and IIS6 to activation gating of CaV1.2.

Voltage dependence and kinetics of CaV1.2 activation are affected by structural changes in pore-lining S6 segments of the α1-subunit. Significant effects are induced by either proline or threonine substitutions in the lower third of segment IIS6 (“bundle crossing region”), where S6 segments are likely to seal the channel in the closed conformation (Hohaus, A., Beyl, S., Kudrnac, M., Berjukow, S., Timin, E. N., Marksteiner, R., Maw, M. A., and Hering, S. (2005) J. Biol. Chem. 280, 38471–38477). Here we report that S435P in IS6 results in a large shift of the activation curve (-25.9 ± 1.2 mV) and slower current kinetics. Threonine substitutions at positions Leu-429 and Leu-434 induced a similar kinetic phenotype with shifted activation curves (L429T by -6.6 ± 1.2 and L434T by -12.1 ± 1.7 mV). Inactivation curves of all mutants were shifted to comparable extents as the activation curves. Interdependence of IS6 and IIS6 mutations was analyzed by means of mutant cycle analysis. Double mutations in segments IS6 and IIS6 induce either additive (L429T/I781T, -34.1 ± 1.4 mV; L434T/I781T, -40.4 ± 1.3 mV; L429T/L779T, -12.6 ± 1.3 mV; and L434T/L779T, -22.4 ± 1.3 mV) or nonadditive shifts of the activation curves along the voltage axis (S435P/I781T, -33.8 ± 1.4 mV). Mutant cycle analysis revealed energetic coupling between residues Ser-435 and Ile-781, whereas other paired mutations in segments IS6 and IIS6 had independent effects on activation gating.

Identification of a digalactosyl ononitol from seeds of adzuki bean (Vigna angularis)

A digalactosyl ononitol was isolated from seeds of adzuki bean (Vigna angularis [Willd.] Ohwi et Ohasi). Analysis of hydrolysis products and NMR spectroscopy established its structure as O-alpha-D-galactopyranosyl-(1-->6)-O-alpha-D-galactopyranosyl-(1-->3)-4-O-methyl-D-myo-inositol.

Bacteriophage-encoded toxins: the lambda-holin protein causes caspase-independent non-apoptotic cell death of eukaryotic cells.

The bacteriophage‐encoded holin proteins are known to promote bacterial cell lysis by forming lesions within the cytoplasmic membrane. Recently, we have shown that the bacteriophage λ‐holin protein exerts cytotoxic activity also in eukaryotic cells accounting for a reduced tumour growth in vivo. In order to elucidate the mechanisms of λ‐holin‐induced mammalian cell death, detailed biochemical and morphological analyses were performed. Colocalization analyses by subcellular fractionation and organelle‐specific fluorescence immunocytochemistry indicated the presence of the λ‐holin protein in the endoplasmic reticulum and in mitochondria. Functional studies using the mitochondria‐specific fluorochrome JC‐1 demonstrated a loss of mitochondrial transmembrane potential in response to λ‐holin expression. Morphologically, these cells exhibited unfragmented nuclei but severe cytoplasmic vacuolization representing signs of oncosis/necrosis rather than apoptosis. Consistently, Western blot analyses indicated neither an activation of effector caspases 3 and 7 nor cleavage of the respective substrate poly(ADP‐ribose) polymerase (PARP) in an apoptosis‐specific manner. These findings suggest that the λ‐holin protein mediates a caspase‐independent non‐apoptotic mode of cell death.