Phytochrome-mediated control of grana and stroma thylakoid formation in plastids of mustard cotyledons

The etioplast»chloroplast transition in the cotyledons of mustard seedlings (Sinapis alba L.) has been studied by electron microscopy. It was found that the active form of phytochrome, established by a red light pulse pretreatment, increases the initial rate and eliminates the lag of grana and stroma thylakoid formation after the onset of white light 60 h after sowing. The effect of a pretreatment with 15 s red light pulses is fully reversible by 756 nm light pulses. This reversibility is lost within 5 min. Evidence is presented which suggests that the time course of grana and stroma thylakoid formation is not correlated with the time course of the dispersal of the prolamellar body. The different functions of phytochrome and chlorophyll in controlling thylakoid formation are discussed.     

Fast-Growing, Aerobic, Heterotrophic Bacteria from the Rhizosphere of Young Sugar Beet Plants

Fast-growing, aerobic, heterotrophic bacteria from the root surface of young sugar beet plants were inventoried. Isolation of the most abundant bacteria from the root surface of each of 1,100 plants between the second and tenth leaf stage yielded 5,600 isolates. These plants originated from different fields in Belgium and Spain. All isolates were characterized by sodium dodecyl sulfate-polyacrylamide gel electrophoresis of total cellular proteins. Comparison of protein fingerprints allowed us to inventory the bacteria of individual plants of different fields or leaf stages and to analyze the composition and variability of the rhizobacterial population of young sugar beet plants. Each field harbored a specific population of bacteria which showed a highly hierarchic structure. A small number of bacteria occurring frequently at high densities dominated in each field. The major bacteria were identified as Pseudomonas fluorescens, Xanthomonas maltophilia, Pseudomonas paucimobilis, and Phyllobacterium sp. The former three species showed a high genetic variability as they were represented by different protein fingerprint types on the same or different fields or leaf stages. Twinspan analysis and relative abundance plots showed that the structure and composition of the bacterial populations varied strongly over time. Pseudomonads were typically early colonizers which were later replaced by X. maltophilia or Phyllobacterium sp.     

Density distribution of 2′,3′-cyclic nucleotide 3′-phosphodiesterase and myelin proteins in particulate material from myelin deficient (mld) mutant and control brains

Total particulate material from control and myelin deficient (mld) brains was subjected to density centrifugation on a continuous sucrose gradient. Particles from control brains distributed in a bell-shaped mode with a peak density near 0.64 M-sucrose. In mld material only a slight elevation of optical density was observed near 0.8 M-sucrose. The highest specific activities of 2′,3′-cyclic nucleotide 3′-phosphodiesterase were observed at densities of 0.63 and 0.71 M-sucrose for mld and control brains, respectively. The peak of myelin basic protein in control fractions was near 0.60 M-sucrose. In mld fractions no peak was observed. Proteolipid and Wolfgram proteins had a maximum near 0.65 and 0.73 M-sucrose in control and mld fractions, respectively. The absence of myelin basic proteins in all the fractions makes it unlikely that, in mld mice, myelin basic proteins are synthesized but not incorporated into myelin.     

Distribution of lipid synthesizing enzymes, 2′,3′-cyclic nucleotide 3′-phosphodiesterase,and myelin proteins in rat forebrain subfractions during development

The distribution of UDP-galactose: ceramide galactosyltransferase (CGalT) was studied in subcellular fractions of rat forebrain during development using zonal centrifugation on linear gradients. Specialized subfractions: SN 1, a microsomal fraction, SN 4, a myelin-related fraction, and purified myelin were also used for this study. For comparison, two microsomal lipid synthesizing enzymes, a myelin-specific enzyme, 2,3-cyclic nucleotide 3-phosphodiesterase and myelin proteins were measured in the same subfractions. UDP-glucose: ceramide glucosyltransferase and cerebroside sulfotransferase were confined to microsomes. CGalT was ferase and cerebroside sulfotransferase were confined to microsomes. CGalT was localized in microsomes, but also in myelin and myelin-related fractions. The developmental change in distribution of CGalT in adult animals toward myelin containing fractions could indicate that the replacement of galactosylceramide in compact myelin could be carried out in close proximity to compact myelin (mesaxon, paranodal loops) rather than in the distant oligodendrocyte perikaryon.     

The involvement of phytochrome in controlling chlorophyll and 5-aminolevulinate formation in a gymnosperm seedling (Pinus sylvestris)

Chlorophyll a (Chl a) accumulation in the cotyledons of Scots pine seedlings (Pinus sylvestris L.) is much higher in the light than in darkness where it ceases 6 days after germination. When these darkgrown seedlings are treated with continuous white light (3,500 lx) a 3 h lag phase appears before Chl a accumulation is resumed. The lag phase can be eliminated by pretreating the seedlings with 7 h of weak red light (0.14 Wm^-2) or with 14 red light pulses separated by relatively short dark periods (<100 min). The effect of 15s red light pulses can be fully reversed by 1 min far-red light pulses. This reversibility is lost within 2 min. In addition, the amount of Chl a formed within 27 h of continuous red light is considerably reduced by the simultaneous application of far-red (RG 9) light. It is concluded that phytochrome (P_fr) is required not only for the elimination of the lagphase but also to maintain a high rate of Chl a accumulation in continuous light. Since accumulation of 5-aminolevulinate (ALA) responds in the same manner as Chl a accumulation to a red light pretreatment it is further concluded that ALA formation is the point where phytochrome regulates Chl biosynthesis in continuous light. No correlation has been found between ALA and Chl a formation in darkness. This indicates that in a darkgrown pine seedling ALA formation is not rate limiting for Chl a accumulation.Abbreviations Chl chlorophyll(ide) - PChl protochlorophyll(ide) - ALA 5-aminolevulinate - P_r the red absorbing form of phytochrome - P_fr the far-red absorbing form of phytochrome - P_tot total phytochrome ([P_r]+[P_fr])     

Intracellular concentrations of phenylalanine,tyrosine and α-aminobutyric acid in 13 homozybotes and 19 heterozygotes for phenylketonuria (PKU) compared with 26 normals

Summary Intracellular concentrations of phenylalanine, tyrosine, -aminobutyric acid, and seven other aminoacids (glycine, alanine, valine, cystine, methionine, isoleucine, leucine) were measured in lymphocytes of 13 homozygotes and 19 heterozygotes for phenylketonuria and in lymphocytes of 26 normals. Intracellular concentrations for phenylalanine, tyrosine, and -aminobutyric acid were significantly higher in homo- and heterozygotes than in normals (P<0.001; P<0.01). For the other seven aminoacids there were no or only questionable differences. Between homo-and heterozygotes there was no difference in any of the aminoacids. The intracellular phenylalanine: tyrosine ratio was essentially the same in all three groups of individuals. There was no correlation between intracellular phenylalanine above or below 10nmol/10⁶ cells and IQ in heterozygotes. The same is true for phenylalanine: tyrosine ratio greater or smaller than 1. In homozygotes there was no correlation between intracellular phenylalanine and age—to which DQ/IQ is correlated. There was no significant difference in intracellular phenylalanine between homozygotes with blood levels above and below 908 mol/l (15 mg/100 ml) at the time of blood sampling and no correlation between intra- and extracellular phenylalanine concentrations.Among the 26 normals there were only two with intracellular phenylalanine above 10 nmol/10⁶ cells, both showing phenylalanine loading test curves suggestive of heterozygosity.The results are discussed and important functions of the cell wall are proposed. The formation of an abnormal unknown intracellular metabolite being the real noxious agent could explain the incomparably different degrees of brain dysfunction in individuals with equal though elevated intracellular phenylalanine concentrations, i.e., homozygotes and heterozygotes for PKU.     

Role of nuclear proteins on [H]actinomycin D binding during lymphocyte mitogenesis

The uptake of [³H]actinomycin D ([³H]AD) by ConA-stimulated lymphocytes was followed during 96 h of incubation and correlated with the level of nuclear proteins in the nucleus, DNA synthesis and the degree of AD-induced inhibition of RNA and DNA synthesis. During the first 48 h there is a parallel increase of drug binding to cells and a rising level of non-histone proteins (NHP) in the nucleus. During the next 48 h, DNA synthesis occurs, drug uptake decreases and the nuclear level of NHP continues to rise. The level of histones remains constant during 96 h. The variations in cellular [³H]AD uptake during 96 h are not due to changes in cell membrane permeability, since similar variations in drug binding are observed in isolated cell nuclei. NHP, obtained as 0.25 M NaCl extracts of cell nuclei, increase binding of [³H]AD to nuclei isolated from non-stimulated lymphocytes, while histones have no such effect. NHP extracted with phenol, after washing the nuclei with salt and acid solutions, or extracted with 0.25 M NaCl from non-stimulated and stimulated lymphocytes and Chang liver cells are equally active to bind [³H]AD to nuclei of non-stimulated lymphocytes. NHP from Chang cells, purified by DNA-cellulose chromatography using calf thymus DNA, stimulated [³H]AD binding to lymphocyte nuclei, indicating that the drug-binding activity is due to proteins binding to DNA. NHP increase binding of [³H]AD to pure DNA in the absence of histones. The degree of [³H]AD binding to ConA-stimulated lymphocytes during 96 h correlated with the degree of inhibition of RNA and DNA synthesis by AD.     

Approach for an equation of state for adsorbed protein surfaces.

A surface equation of state for polymer interfaces is obtained by equating the chemical potentials of the solvent in the bulk and surface phases. This equation of state contains the fraction of the surface covered with polymer and the surface activity coefficient as parameters. These parameters may be obtained by measuring the thermodynamic modulus of elasticity. Moreover, if the amount of protein in the interface is known the degree of folding in the surface may be evaluated. The present theory was applied to adsorbed beta-lactoglobulin in surfaces. The results are in agreement with qualitative equations and results from other sources.     

Interleukin-1 beta and tumor necrosis factor alpha inhibit migration activity of chondrogenic progenitor cells from non-fibrillated osteoarthritic cartilage

Introduction

The repair capability of traumatized articular cartilage is highly limited so that joint injuries often lead to osteoarthritis. Migratory chondrogenic progenitor cells (CPC) might represent a target cell population for in situ regeneration. This study aims to clarify, whether 1) CPC are present in regions of macroscopically intact cartilage from human osteoarthritic joints, 2) CPC migration is stimulated by single growth factors and the cocktail of factors released from traumatized cartilage and 3) CPC migration is influenced by cytokines present in traumatized joints.

Methods

We characterized the cells growing out from macroscopically intact human osteoarthritic cartilage using a panel of positive and negative surface markers and analyzed their differentiation capacity. The migratory response to platelet-derived growth factor (PDGF)-BB, insulin-like growth factor 1 (IGF-1), supernatants obtained from in vitro traumatized cartilage and interleukin-1 beta (IL-1β) as well as tumor necrosis factor alpha (TNF-α) were tested with a modified Boyden chamber assay. The influence of IL-1β and TNF-α was additionally examined by scratch assays and outgrowth experiments.

Results

A comparison of 25 quadruplicate marker combinations in CPC and bone-marrow derived mesenchymal stromal cells showed a similar expression profile. CPC cultures had the potential for adipogenic, osteogenic and chondrogenic differentiation. PDGF-BB and IGF-1, such as the supernatant from traumatized cartilage, induced a significant site-directed migratory response. IL-1β and TNF-α significantly reduced basal cell migration and abrogated the stimulative effect of the growth factors and the trauma supernatant. Both cytokines also inhibited cell migration in the scratch assay and primary outgrowth of CPC from cartilage tissue. In contrast, the cytokine IL-6, which is present in trauma supernatant, did not affect growth factor induced migration of CPC.

Conclusion

These results indicate that traumatized cartilage releases chemoattractive factors for CPC but IL-1β and TNF-α inhibit their migratory activity which might contribute to the low regenerative potential of cartilage in vivo.