Genetic determinants of a nickel-specific transport system are part of the plasmid-encoded hydrogenase gene cluster in Alcaligenes eutrophus.

Nickel-deficient (Nic-) mutants of Alcaligenes eutrophus requiring high levels of nickel ions for autotrophic growth with hydrogen were characterized. The Nic- mutants carried defined deletions in the hydrogenase gene cluster of the indigenous pHG megaplasmid. Nickel deficiency correlated with a low level of the nickel-containing hydrogenase activity, a slow rate of nickel transport, and reduced activity of urease. The Nic+ phenotype was restored by a cloned DNA sequence (hoxN) of a megaplasmid pHG1 DNA library of A. eutrophus H16. hoxN is part of the hydrogenase gene cluster. The nickel requirement of Nic- mutants was enhanced by increasing the concentration of magnesium. This suggests that the Nic- mutants are impaired in the nickel-specific transport system and thus depend on the second transport activity which normally mediates the uptake of magnesium.     

In vivo analysis of glial cell phenotypes during a viral demyelinating disease in mice

C57 BL/6N mice injected intracranially with the A59 strain of mouse hepatitis virus exhibit extensive viral replication in glial cells of the spinal cord and develop demyelinating lesions followed by virus clearing and remyelination. To study how different glial cell types are affected by the disease process, we combine three-color immunofluorescence labeling with tritiated thymidine autoradiography on 1-micron frozen sections of spinal cord. We use three different glial cell specific antibodies (a) to 2',3' cyclic-nucleotide 3' phosphohydrolase (CNP) expressed by oligodendrocytes, (b) to glial fibrillary acidic protein (GFAP) expressed by astrocytes, and (c) the O4 antibody which binds to O-2A progenitor cells in the rat. These progenitor cells, which give rise to oligodendrocytes and type 2 astrocytes and react with the O4 antibody in the adult central nervous system, were present but rare in the spinal cord of uninfected mice. In contrast, cells with the O-2A progenitor phenotype (O4 + only) were increased in number at one week post viral inoculation (1 WPI) and were the only immunostained cells labeled at that time by a 2-h in vivo pulse of tritiated thymidine. Both GFAP+ only and GFAP+, O4+ astrocytes were also increased in the spinal cord at 1 WPI. Between two and four WPI, the infected spinal cord was characterized by the loss of (CNP+, O4+) oligodendrocytes within demyelinating lesions and the presence of O-2A progenitor cells and O4+, GFAP+ astrocytes, both of which could be labeled with thymidine. As remyelination proceeded, CNP immunostaining returned to near normal and tritiated thymidine injected previously during the demyelinating phase now appeared in CNP+ oligodendrocytes. Thus O4 positive O-2A progenitor cells proliferate early in the course of the demyelinating disease, while CNP positive oligodendrocytes do not. The timing of events suggests that the O-2A progenitors may give rise to new oligodendrocytes and to type 2 astrocytes, both of which are likely to be instrumental in the remyelination process.     

Physical Basis for Altered Stem Elongation Rates in Internode Length Mutants of Pisum

Biophysical parameters related to gibberellin (GA)-dependent stem elongation were examined in dark-grown stem-length genotypes of Pisum sativum L. The rate of internode expansion in these genotypes is altered due to recessive mutations which affect either the endogenous levels of, or response to, GA. The GA deficient dwarf L181 (ls), two GA insensitive semierectoides dwarfs NGB5865 and NGB5862 (Ika and Ikb, respectively) and the `slender' line L197 (la cry^[ill]), which is tall regardless of GA content, were compared to the wild-type tall cultivar, Torsdag. Osmotic pressure, estimated by vapor pressure osmometry, and turgor pressure, measured directly with a pressure probe, did not correlate with the differences in growth rate among the genotypes. Mechanical wall properties of frozen-thawed tissue were measured using a constant force assay. GA deficiency resulted in increased wall stiffness judged both on the basis of plastic compliance and plastic extensibility normalized for equal stem circumference. Plastic compliance was not reduced in the GA insensitive dwarfs, though Ika reduced circumference-normalized plasticity. In contrast, in vivo wall relaxation, determined by the pressure-block technique, differed among genotypes in a manner which did correlate with extension rates. The wall yield threshold was 1 bar or less in the tall lines, but ranged from 3 to 6 bars in the dwarf genotypes. The results with the ls mutant indicate that GA enhances stem elongation by both decreasing the wall yield threshold and increasing the wall yield coefficient. In the GA-insensitive mutants, Ika and Ikb, the wall yield threshold is substantially elevated. Plants possessing Ika may also possess a reduced wall yield coefficient.     

Effect of aluminium on iron uptake and transferrin-receptor expression by human erythroleukaemia K562 cells.

Incubation of human erythroleukaemia K562 cells with Al-transferrin inhibited iron uptake from 59Fe-transferrin by about 80%. The inhibition was greater than that produced by a similar quantity of Fe-transferrin. Preincubation of cells for 6 h with either Al-transferrin or Fe-transferrin diminished the number of surface transferrin receptors by about 40% compared with cells preincubated with apo-transferrin. Al-transferrin did not compete significantly with Fe-transferrin for transferrin receptors and, when cells were preincubated for 15 min instead of 6 h, the inhibitory effect of Al-transferrin on receptor expression was lost. Both forms of transferrin also decreased the level of transferrin receptor mRNA by about 50%, suggesting a common regulatory mechanism. Aluminium citrate had no effect on iron uptake or transferrin-receptor expression. AlCl3 also had no effect on transferrin-receptor expression, but at high concentration it caused an increase in iron uptake by an unknown, possibly non-specific, mechanism. Neither Al-transferrin nor AlCl3 caused a significant change in cell proliferation. It is proposed that aluminium, when bound to transferrin, inhibits iron uptake partly by down-regulating transferrin-receptor expression and partly by interfering with intracellular release of iron from transferrin.     

Binding characteristics of reduced hepatic receptors for acetylated low-density lipoprotein and maleylated bovine serum albumin.

The binding characteristics of reduced hepatic membrane proteins for acetylated low-density lipoprotein (acetyl-LDL) and maleylated bovine serum albumin (Mal-BSA) have been examined. Two receptor activities were extracted from hepatic membranes in the presence of octyl beta-D-glucoside and beta-mercaptoethanol, and were separated by chromatography on Mal-BSA-Sepharose 4B. The receptors were revealed by ligand blotting. The active binding proteins had apparent molecular masses of 35 and 15 kDa in SDS/polyacrylamide gels. Equilibrium studies with protein-phosphatidylcholine complexes indicated that the reduced 35 kDa protein expresses two binding sites for Mal-BSA and one for acetyl-LDL, whereas the 15 kDa protein-phosphatidylcholine complex binds 131I-Mal-BSA and 131I-acetyl-LDL with a 4:1 stoichiometry. 131I-Mal-BSA binding was linear with both proteins, with a Kd of 4.8 nM at the 35 kDa protein and a Kd of 5.6 nM at the 15 kDa protein. The 35 kDa protein displayed saturable binding of 131I-acetyl-LDL with a Kd of 5 nM; the 15 kDa binding protein bound 131I-acetyl-LDL with a Kd of 2.3 nM. A 85 kDa protein was obtained by Mal-BSA-Sepharose chromatography when the hepatic membranes had been solubilized with Triton X-100 in presence of GSH/GSSG. This protein displayed saturable 131I-Mal-BSA binding with a Kd of 30 nM and 131I-acetyl-LDL binding with a Kd of 6.5 nM. The 131I-Mal-BSA binding capacity was four times higher than that of 131I-acetyl-LDL. Competition studies with the 35 kDa, 15 kDa and 85 kDa proteins binding Mal-BSA, acetyl-LDL, formylated albumin and polyanionic competitors provide evidence for the existence of more than one class of binding sites at the reduced binding proteins.     

Generation of rhythmic and arrhythmic behaviour of Crassulacean acid metabolism in Kalanchoë daigremontiana under continuous light by varying the irradiance or temperature: Measurements in vivo and model simulations

Leaves of Kalanchoë daigremontiana Hamet et Perr. at a photon flux density (PFD) above 220 mol·m^–2s^–1 (400–700 nm) or at leaf temperatures above 27.0 °C showed a rapid loss of rhythmicity, and a more or less pronounced damping-out of the endogenous circadian rhythm of CO₂ exchange under continuous illumination. This rhythm was reinitiated after reduction of the PFD by 90–120 mol·m^–2·s^–1 or reduction of leaf temperature by 3.5–11.0 °C under otherwise unchanged external conditions. The reduction in the magnitude of the external control parameter of the Crassulacean acid metabolism (CAM) rhythm (i.e. PFD or leaf temperature) set the phase of the new rhythm. The maxima of CO₂ uptake occurred about 5, 28, 51, 75 h after the reduction. Simulations with a CAM model under comparable conditions showed a similar behaviour. The influence of temperature on the endogenous CAM rhythm observed in K. daigremontiana in vivo could be simulated by incorporating into the model temperature-dependent switch modes for passive efflux of malate from the vacuole to the cytoplasm. Thus, the model indicates that tonoplast function plays an important role in regulation of the endogenous CAM rhythm in K. daigremontiana.Abbreviations CAM Crassulacean acid metabolism - PAR photosynthetically active radiation - PFD photon flux density This work was supported by a grant to F.B. and U.L. from Teilprojekt B5 in the Sonderforschungsbereich 199 of the Deutsche Forschungsgemeinschaft (Bonn, Germany) and by a grant to T. E. E. G. from the Sudienstiftung des deutschen Volkes (Bonn, Germany). Erika Ball is thanked for processing of time-course data for the analysis of Fourier spectra.     

The biosynthesis of the lantibiotics epidermin,gallidermin, Pep5 and epilancin K7

Lantibiotics are antibiotic peptides that contain the rare thioether amino acids lanthionine and/or methyllanthionine. Epidermin, Pep5 and epilancin K7 are produced by Staphylococcus epidermidis whereas gallidermin (6L-epidermin) was isolated from the closely related species Staphylococcus gallinarum. The biosynthesis of all four lantibiotics proceeds from structural genes which code for prepeptides that are enzymatically modified to give the mature peptides. The genes involved in biosynthesis, processing, export etc. are found in gene clusters adjacent to the structural genes and code for transporters, immunity functions, regulatory proteins and the modification enzymes LanB, LanC and LanD, which catalyze the biosynthesis of the rare amino acids. LanB and LanC are responsible for the dehydration of the serine and threonine residues to give dehydroalanine and dehydrobutyrine and subsequent addition of cysteine SH-groups to the dehydro amino acids which results in the thioether rings. EpiD, the only LanD enzyme known so far, catalyzes the oxidative decarboxylation of the C-terminal cysteine of epidermin which gives the C-terminal S-aminovinylcysteine after addition of a dehydroalanine residue.Abbreviations Dha 2,3-didehydroalanine - Dhb 2,3-didehydrobutyrine - Lan lanthionine - Melan methyllanthionine     

Keratin Degradation by Fervidobacterium pennavorans, a Novel Thermophilic Anaerobic Species of the Order Thermotogales

From a hot spring of the Azores islands a novel thermophilic bacterium belonging to the Thermotogales order was isolated. This strain, which grows optimally at 70(deg)C and pH 6.5, is the first known extreme thermophile that is able to degrade native feathers at high temperatures. The enzyme system converts feather meal to amino acids and peptides. On the basis of physiological, morphological, and 16S rDNA studies the new isolate was found to be a member of the Thermotogales order and was identified as Fervidobacterium pennavorans. The strain was highly related to Fervidobacterium islandicum and Fervidobacterium pullulanolyticum. The cell-bound keratinolytic enzyme system was purified 32-fold by detergent treatment with CHAPS (3-[(3-cholamidopropyl)-dimethyl-ammonio]-1-propanesulfonate) and sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The enzyme was characterized as a serine protease with a molecular mass of 130 kDa and an isoelectric point of 3.8. Optimal activity was measured at 80(deg)C and pH 10.0. Furthermore, 19 anaerobic thermophilic archaea and bacteria belonging to the orders Thermococcales, Thermoproteales, Thermotogales, and Clostridiales (growth temperatures between 60 and 105(deg)C) were tested for their abilities to grow on feathers and produce heat-stable keratinolytic enzymes. None of the tested extremophilic microorganisms was able to attack the substrate in a native form.     

Increase in total protein following infection of CV-1 cells with SV40 virus as assayed by flow cytometry

Summary The changes in cell size and total protein were determined for G1-arrested, contact-inhibited CV-1 cells infected with Simian virus 40 (SV40). The assays used were the Biorad total protein assays (Bradford and DC protein assays) on a standard number of cells, total protein as assayed by fluorescein isothiocyanate (FITC) and SR101 by flow cytometry, orthoganol (90°) light scatter by flow cytometry, and direct microscopic measurement with an ocular micrometer. Uninfected CV-1 cells and two cell lines with variations in DNA content (diploid vs. tetraploid) were used as controls for the studies presented. The results demonstrated a 40–60% increase in total protein at 32 to 42 h postinfection. These increases were similar to values obtained as control cells progress through the cell cycle. At later times postinfection (>42 h), total protein decreased due to cellular changes resulting from viral replication and cell death.