Degradation and release from the thylakoid membrane of Photosystem II subunits after UV-B irradation of the liverwort Conocephalum conicum

The effect of ultraviolet-B (UV-B) radiation on the amount of various Photosystem (PS) II subunits has been studied in the thalloid liverwort Conocephalum conicum. UV-B irradiation led to a drastic decrease of the reaction center proteins D1 and D2 and the outer light harvesting antenna (LHC II). A minor reduction was found in the levels of the CP 43 polypeptide of the inner antenna and the 33, 23 and 16 kDa extrinsic polypeptides of PS II. During UV-B irradiation, the extrinsic polypeptides accumulated in the soluble protein fraction, but D1 and D2 were not dedectable. Streptomycin, but not cycloheximide inhibited the repair process of PS II, indicating that only protein synthesis in the chloroplast is necessary for recovery. This indicates that the extrinsic proteins of PS II dissociate from the membrane during UV-B treatment and reassociate with PS II in the course of the repair process. We conclude that the reaction center core is a target of UV-B radiation in C. concicum. The extrinsic proteins of PS II are not directly affected by UV-B, but their release is the consequence of UV-B-induced degradation of the D1 and D2 proteins.     

Characterization of serum {beta}-glucuronyltransferase involved in chondroitin sulfate biosynthesis

We studied a glucuronyltransferase involved in chondroitin sulfate(CS) biosynthesis in a preparation obtained from fetal bovineserum by heparin-Sepharose affinity chromatography. This enzymetransferred GlcA from UDP-GlcA to the nonreducing GalNAc residuesof polymeric chondroitin. It required Mn²⁺ for maximal activityand showed a sharp pH optimum between pH 5.5 and 6.0. The apparentKm value of the glucuronyltransferase for UDP-GlcA was 51 µM.The specificity was investigated using structurally definedacceptor substrates, which consisted of chemically synthesizedtri-, penta-, and heptasaccharide-serines and various odd-numberedoligosaccharides with a GalNAc residue at the nonreducing terminus,prepared from chondroitin and CS by chondroitinase ABC digestionfollowed by mercuric acetate treatment. The enzyme utilizeda heptasaccharide-serine GalNAcß1-4GlcAß1-3GalNAcß1-4GlcAß1-3Galß1-3Galß1-4Xylß1-O-Serand a pentasaccharide-serine GalNAcß 4GlcAß1-3Galß1-3Galß1-4Xylß1-O-Seras acceptors. In contrast, neither a trisaccharide-serine Galß1-3Galß1-4Xylß1-O-Sernor an     

Fluorescence correlation spectrometry of the interaction kinetics of tetramethylrhodamin alpha-bungarotoxin with Torpedo californica acetylcholine receptor

Fluorescence correlation spectroscopy (FCS) is suited to determine low concentrations (10(-8) M) of slowly interacting molecules with different translational diffusion coefficients on the level of single molecule counting. This new technique was applied to characterize the interaction dynamics of tetramethylrhodamin labelled alpha-bungarotoxin (B( *)) with the detergent solubilized nicotinic acetylcholine receptor (AChR) of Torpedo californica electric organ. At pseudo-first-order conditions for AChR, the complex formation with B( *) is monophasic. The association rate coefficient of the monoliganded species AChR . B is k(ass)' = 3.8 . 10(3) s(-1) at 293 K (20 degrees C). The dissociation of bound B( *) from the monomer species AChR . B( *) . B (and AChR . B(2)( *)), initiated by adding an excess of nonlabelled alpha-bungarotoxin (B), is biphasic suggesting a three state cascade for the B-sites: R(alpha) --> R(alpha)' --> R(alpha)' with the exchange dissociation constants: (k(diss)')(B) = 5.5(+/-1) . 10(-5) s(-1) and (k(diss)')(B) = 3(+/-1) . 10(-6) s(-1) at 293 K. The data are consistent with dissociative intermediate steps of ligand exchange on two different interconvertible conformations of one binding site. The dissociation of bound B( *) by excess of the neurotransmitter acetylcholine (ACh) is biphasic. At [ACh] = 0.1 M both B( *) are released from the AChR . B(2)( *) species. The mechanism involves associative ternary intermediates (AChR . B( *)A, AChR . B( *)A(2) and AChR . B(2)( *)A(2)). The equilibrium constants (K(A)) and dissociation rate constants (k(-A)) for ACh in the ternary complex state R(alpha)' and R(alpha)', respectively, are K(A)' = 1.1 . 10(-2) M and k(-A)' = 3 . 10(5) s(-1) and K(A)' = 7.5 . 10(-2) M and k(-A)' = 2 . 10(6) s(-1). It is of physiological importance that the FCS data indicate that the AChR monomer species (M(r) = 290 000), which normally at [ACh] 1 mM only binds one ACh molecule, does bind two ACh molecules at [ACh] 0.1 M.     

Regeneration of roots, shoots and embryos: physiological, biochemical and molecular aspects

When the proper stimuli are given, somatic plant cells may form adventitious embryos, roots or shoots. The three pathways of regeneration show apparent similarities. They consist of three analogous phases: 1) dedifferentiation (during which the tissue becomes competent to respond to the organogenic/embryogenic stimulus), 2) induction (during which cells become determined to form either a root, a shoot or an embryo), and 3) realization (outgrowth to an organ or an embryo). The first phase may involve a period of callus growth (indirect regeneration), but often cells present in the explant become competent without cell division or without cell division at a large scale (direct regeneration). In an explant, only very few cells show the organogenic/embryogenic response. In direct regeneration, the three regenerative pathways start from cells in different tissues. This is most obvious when the different types of regeneration occur in the same explant. The hormonal trigger for the dedifferentiation phase is a general one, probably auxin. During the induction phase, each pathway requires specific hormonal triggers. During the realization phase, hormones should be absent or at low concentration. The successive steps in the regeneration process coincide with events on the molecular and biochemical levels, but so far no coherent picture has emerged. In particular during the early stages of regeneration, research on these levels is hampered by a technical problem, viz., the very low proportion of cells that participate in the process of regeneration. New methods may overcome this problem. This revised version was published online in July 2006 with corrections to the Cover Date.     

Molecular Forensic Profiling of Cryptosporidium Species and Genotypes in Raw Water

The emerging concept of host specificity of Cryptosporidium spp. was exploited to characterize sources of fecal contamination in a watershed. A method of molecular forensic profiling of Cryptosporidium oocysts on microscope slides prepared from raw water samples processed by U.S. Environmental Protection Agency Method 1623 was developed. The method was based on a repetitive nested PCR-restriction fragment length polymorphism-DNA sequencing approach that permitted the resolution of multiple species/genotypes of Cryptosporidium in a single water sample.     

A free carboxylate oxygen in the side chain of position 674 in transmembrane domain 7 is necessary for TSH receptor activation.

A specific H-bonding network formed between the central regions of transmembrane domain 6 and transmembrane domain 7 has been proposed to be critical for stabilizing the inactive state of glycoprotein hormone receptors. Many different constitutively activating TSH receptor point mutations have been identified in hyperfunctioning thyroid adenomas in the lower portion of transmembrane domain 6. Position D633 in transmembrane domain 6 of the human TSH receptor is the only one in which four different constitutively activating amino acid exchanges have been identified. Further in vitro substitutions led to constitutive activation of the TSH receptor (D633Y, F, C) as well as to the first inactivating TSH receptor mutation in transmembrane domain 6 without changes of membrane expression or TSH binding (D633R). Molecular modeling of this inactivating TSH receptor mutation revealed potential interaction partners of R633 in transmembrane domain 3 and/or transmembrane domain 7, presumably via hydrogen bonds that could be responsible for locking the TSH receptor in a completely inactive state. To further elucidate the H-bond network that most likely maintains the inactive state of the TSH receptor, we investigated these potential interactions by generating TSH receptor double mutants designed to break up possible H bonds. We excluded S508 in transmembrane domain 3 as a possible interaction partner of R633. In contrast, a partial response to TSH stimulation was rescued in a receptor construct with the double-substitution D633R/N674D. Our results therefore confirm the H bond between position 633 in transmembrane domain 6 and 674 in transmembrane domain 7 suggested by molecular modeling of the inactivating mutation D633R. Moreover, the mutagenesis results, together with a three-dimensional structure model, indicate that for TSH receptor activation and G protein-coupled signaling, at least one free available carboxylate oxygen is required as a hydrogen acceptor atom at position 674 in transmembrane domain 7.     

Electroporative deformation of salt filled lipid vesicles

Membrane electroporation, vesicle shape deformation and aggregation of small, NaCl-filled lipid vesicles (of radius a = 50 nm) in DC electric fields was characterized using conductometric and turbidimetrical data. At pulse durations t_E≤ 55 ± 5 ms the increase in the conductivity of the vesicle suspension is due to the field-induced efflux of electrolyte through membrane electropores. Membrane electroporation and Maxwell stress on the vesicle membrane lead to vesicle elongation concomitant with small volume reduction (up to 0.6% in an electric field of E = 1 MV m^–1). At t_E > 55 ± 5 ms, further increases in the conductivity and the optical density suggest electroaggregation and electrofusion of vesicles. The conductivity changes after the electric pulse termination reflect salt ion efflux through slowly resealing electropores. The analysis of the volume reduction kinetics yields the bending rigidity κ = (4.1 ± 0.3) ⋅ 10^–20 J of the vesicle membrane. If the flow of Na⁺ and Cl^– ions from the vesicle interior is treated in terms of Hagen-Poiseuille's equation, the number of permeable electropores is N = 39 per vesicle with mean pore radius r_p = 0.85 ± 0.05 nm at E = 1 MVm^–1 and t_E≤ 55 ± 5 ms. The turbidimetric and conductometric data suggest that small lipid vesicles (a ≤ 50 nm) are not associated with extensive membrane thermal undulations or superstructures. In particular with respect to membrane curvature, the vesicle results are suggestive for the design and optimization of electroporative delivery of drugs and genes to cell tissue at small field strengths (≤1 MVm^–1) and large pulse durations (≤100 ms). Received: 8 July 1997 / Accepted: 15 September 1997     

Heat-shock proteins induce heavy-metal tolerance in higher plants

Cell cultures of Lycopersicon peruvianum L. stressed with CdSO₄ (10^–3M) show typical changes in the ultrastructure, starting with the plasmalemma and later on extending to the endoplasmic reticulum and the mitochondrial envelope. Part of the membrane material is extruded, with the formation of osmiophilic droplets which increase in size and number during the stress period. After 4 h, about 20 of the cells are dead. A short heat stress preceeding the heavy-metal stress induces a tolerance effect by preventing the membrane damage. The cells show a normal ultrastructure with one exception: cytoplasmic heat-shock granules are formed. This protective effect can be abolished by cycloheximide. Cadmium uptake is not markedly influenced by the heat stress. Cadmium is found together with sulfur in small deposits in the vacuoles of stressed cells. The precipitates contain an excess of sulfur, evidently due to the stress-induced formation of phytochelatins. The role in heavy-metal tolerance of heat-shock proteins in the plasmalemma (HSP70) and in cytoplasmic heat-stress granules (HSP17, HSP70) is discussed.Abbreviations EDX energy dispersive analysis of X-rays - ESI electron-spectroscopic imaging - HM heavy metal - HSG heat-stress granules - HSP heat-shock protein - MNDO modified neglect of diatomic overlap This work was supported by the Ministerium für Wissenschaft und Forschung des Landes Sachsen-Anhalt and the Deutsche Forschungsgemeinschaft.     

Sequence-specific DNA double-strand breaks induced by triplex forming 125I labeled oligonucleotides.

A triplex-forming oligonucleotide (TFO) complementary to the polypurine-polypyrimidine region of the nef gene of the Human Immunodeficiency Virus (HIV) was labeled with 125I at the C5 position of a single deoxycytosine residue. Labeled TFO was incubated with a plasmid containing a fragment of the nef gene. Decay of 125I was found to cause double-strand breaks (DSB) within the nef gene upon triplex formation in a sequence specific manner. No DSB were detected after incubation at ionic conditions preventing triplex formation or when TFO was labeled with 32P instead of 125I. Mapping DSB sites with single base resolution showed that they are distributed within 10 bp of a maximum located exactly opposite the position of the [125I] IdC in the TFO. We estimate that on average the amount of DSB produced per decay is close to one.