Activation of latent phenolase during spinach leaf senescence

The observed increase of phenolase activity and of its rate of activation during spinach leaf senescence is due to reduced binding of latent phenolase to the thylakoid membranes and not to de novo synthesis. The same amount of phenolase which is active in isolated thylakoid membranes from senescent leaves can be found in the membranes of non-senescent leaves after activation of latent enzyme. Tracer experiments give evidence that one multiple form which is responsible for the bulk activity in senescent leaves, is synthesized before, but not after the onset of senescence, indicating that pre-existing latent phenolase is converted to easily activating forms.     

Serological studies on phenolase from spinach leaves

Antibodies were prepared against phenolase Form X, one of the electrophoretically fast-moving forms (VII-X) which are spontaneously liberated from thyl     

In vitro hemocompatibility testing of biomaterials according to the ISO 10993-4

The development of synthetic materials, textured polymers and metals and their increasing use in medicine make research of biomaterials' hemocompatibility very relevant. Problems arise from the polymorphism and diversity of the different materials, the static and dynamic test models and the patients' individual biologic factors. First, methods, models, tests as well as preanalytical factors have to be standardized according to the current knowledge in medicine laid down in the ISO 10993 part 4. The routine controls used in clinical chemistry and hematology have to be performed. Information about normal ranges (mean value, standard deviation, 95% confidence interval) should be provided. Tests have to be performed within a minimal delay of usually 2 h since some properties of blood change rapidly following collection. Various conditions (depending on the wall shear rate) were simulated within the centrifugation system and a Chandler system. Qualities and aspects of hemocompatibility such as platelet activation, oxidative burst, hemolysis, fibrinolysis, fibrin formation, generation of thrombin, contact activation, and complement activation were analysed and the results were entered non-dimensionally into a non-dimensional score system, where 0 points stand for the best and 65 points for the worst evaluation. We found a good correlation between the total score and contact activation, thrombin generation and leukocyte activation in a low shear stress system and a good correlation between the total score and thrombin generation, hemolysis and platelet activation in the high shear stress system. Further on the effect of additives and sterilization procedures can be measured. The concepts presented underline the relevance/importance of an efficient diagnostic approach to hemocompatibility that takes account of clinical and socio-economic concerns.     

Root phloem-specific expression of the plasma membrane amino acid proton co-transporter AAP3

Amino acids are regarded as the nitrogen 'currency' of plants. Amino acids can be taken up from the soil directly or synthesized from inorganic nitrogen, and then circulated in the plant via phloem and xylem. AtAAP3, a member of the Amino Acid Permease (AAP) family, is mainly expressed in root tissue, suggesting a potential role in the uptake and distribution of amino acids. To determine the spatial expression pattern of AAP3, promoter-reporter gene fusions were introduced into Arabidopsis. Histochemical analysis of AAP3 promoter-GUS expressing plants revealed that AAP3 is preferentially expressed in root phloem. Expression was also detected in stamens, in cotyledons, and in major veins of some mature leaves. GFP-AAP3 fusions and epitope-tagged AAP3 were used to confirm the tissue specificity and to determine the subcellular localization of AtAAP3. When overexpressed in yeast or plant protoplasts, the functional GFP-AAP3 fusion was localized in subcellular organelle-like structures, nuclear membrane, and plasma membrane. Epitope-tagged AAP3 confirmed its localization to the plasma membrane and nuclear membrane of the phloem, consistent with the promoter-GUS study. In addition, epitope-tagged AAP3 protein was localized in endodermal cells in root tips. The intracellular localization suggests trafficking or cycling of the transporter, similar to many metabolite transporters in yeast or mammals, for example, yeast amino acid permease GAP1. Despite the specific expression pattern, knock-out mutants did not show altered phenotypes under various conditions including N-starvation. Microarray analyses revealed that the expression profile of genes involved in amino acid metabolism did not change drastically, indicating potential compensation by other amino acid transporters.     

Genome sequence of the plant-pathogenic bacterium Dickeya dadantii 3937

Dickeya dadantii is a plant-pathogenic enterobacterium responsible for the soft rot disease of many plants of economic importance. We present here the sequence of strain 3937, a strain widely used as a model system for research on the molecular biology and pathogenicity of this group of bacteria.     

Discovery of a mandelonitrile hydrolase from Bradyrhizobium japonicum USDA110 by rational genome mining

A mandelonitrile hydrolase bll6402 from Bradyrhizobium japonicum USDA110 was predicted by rational genome mining, i.e. combining traditional genome mining with functional analysis of the genetic organization of the putative nitrilase gene within the chromosome of microorganisms. This putative gene was cloned and over-expressed in Escherichia coli, and the encoded protein was purified to give a nitrilase with a molecular mass of about 37kDa. The molecular weight of the holoenzyme was about 455kDa, suggesting that nitrilase bll6402 self-aggregated to the active form with native structure being 12 subunits of identical size. This nitrilase was most active toward mandelonitrile with V(max) and K(m) for mandelonitrile being 44.7U/mg and 0.26mM, respectively. The k(cat) and overall catalytic efficiency k(cat)/K(m) were 27.0s(-1) and 1.04x10(5)M(-1)s(-1), indicating that nitrilase bll6402 is very active for the hydrolysis of mandelonitrile to mandelic acid. Nitrilase bll6402 also effectively hydrolyzed several mandelonitrile derivatives.     

Transformation of chicken embryo retinal melanoblasts by a temperature-sensitive mutant of Rous sarcoma virus.

Retinal melanoblasts were transformed by a temperature-sensitive mutant of Rous sarcoma virus (ts-RSV). At the permissive temperature for transformation, the cells cease melanin synthesis, degrade their melanosomes and release much of their accumulated melanin into the medium. At the nonpermissive temperature, the cells assume an epithelioid morphology, actively synthesize melanin and become difficult to distinguish from normal uninfected control cultures. Both the transformed phenotype and the differentiated cell phenotype are temperature-dependent. Infected retinal melanoblasts which are incubated at the nonpermissive temperature and which accumulate a large amount of melanin are unable to transform in response to a temperature shift; instead, the cells degenerate and die. Retinal melanoblasts can be infected by subgroups A, B, C and D of RSV; however, their level of susceptibility to infection is about 1/40 compared to fibroblasts. Cultures infected by ts-RSV produce virus at both temperatures, suggesting that cell phenotype does not regulate virus synthesis.     

The effect of 5-bromodeoxyuridine on the differentiation of chick embryo pigment cells

Cultures of (a) dispersed presumptive melanoblasts from chick somites and (b) organ cultures of retinal melanoblasts were grown in control medium and medium containing 5-bromodeoxyuridine (BUdR). The somite cell cultures at time zero had no evidence of melanogenic organelles; the eye cultures, from embryos of the same stage, stage 16–18, contained cells showing the initial stages of melanogenesis. In the presence of BUdR, presumptive trunk melanoblasts failed to pigment, while controls became heavily pigmented, whereas cells in the retinal pigment epithelium made melanin but in reduced amounts when compared with controls. These results suggest different methods of control over synthetic programs depending upon whether synthesis has, or has not, been initiated when the cells are exposed to BUdR.     

Inactivation of the chloroplast ATP synthase gamma subunit results in high non-photochemical fluorescence quenching and altered nuclear gene expression in Arabidopsis thaliana

The nuclear atpC1 gene encoding the gamma subunit of the plastid ATP synthase has been inactivated by T-DNA insertion mutagenesis in Arabidopsis thaliana. In the seedling-lethal dpa1 (deficiency of plastid ATP synthase 1) mutant, the absence of detectable amounts of the gamma subunit destabilizes the entire ATP synthase complex. The expression of a second gene copy, atpC2, is unaltered in dpa1 and is not sufficient to compensate for the lack of atpC1 expression. However, in vivo protein labeling analysis suggests that assembly of the ATP synthase alpha and beta subunits into the thylakoid membrane still occurs in dpa1. As a consequence of the destabilized ATP synthase complex, photophosphorylation is abolished even under reducing conditions. Further effects of the mutation include an increased light sensitivity of the plant and an altered photosystem II activity. At low light intensity, chlorophyll fluorescence induction kinetics is close to those found in wild type, but non-photochemical quenching strongly increases with increasing actinic light intensity resulting in steady state fluorescence levels of about 60% of the minimal dark fluorescence. Most fluorescence quenching relaxed within 3 min after dark incubation. Spectroscopic and biochemical studies have shown that a high proton gradient is responsible for most quenching. Thylakoids of illuminated dpa1 plants were swollen due to an increased proton accumulation in the lumen. Expression profiling of 3292 nuclear genes encoding mainly chloroplast proteins demonstrates that most organelle functions are down-regulated. On the contrary, the mRNA expression of some photosynthesis genes is significantly up-regulated, probably to compensate for the defect in dpa1.