Expression of ferredoxin-dependent glutamate synthase in dark-grown pine seedlings

Pine seedlings are able to accumulate chlorophylls and develop green plastids in a light-independent manner. In this work, we have characterized ferredoxin-dependent glutamate synthase (EC 1.4.7.1; Fd-GOGAT), a key enzyme in nitrogen interconversion during this process. Fd-GOGAT has been purified about 170-fold from cotyledons of maritime pine (Pinus pinaster). As occurs in angiosperms, the native enzyme is a single polypeptide with an apparent molecular mass of 163–168 kDa that is confined to the chloroplast stroma. Polyclonal antibodies generated against the purified enzyme were used to immunoscreen a gt11 expression library from Scots pine (Pinus sylvestris) seedlings and partial cDNA clones were isolated and characterized. The clone with the longest cDNA insert (pGOP44) contained the codification for the C-terminal (550 amino acids) of the pine Fd-GOGAT polypeptide. Immunological cross-reactivity and comparative amino sequence analysis revealed that Fd-GOGAT is a well conserved protein in higher plants. Western blot analyses showed that protein was expressed in chloroplast-containing pine tissues and this expression pattern was not affected by exogenously supplied nitrogen. Fd-GOGAT mRNA, polypeptide and enzyme activity accumulated in substantial amounts in dark-grown pine seedlings. The presence of a functional Fd-GOGAT may be important to provide the required glutamate for the biosynthesis of nitrogen compounds during chloroplast biogenesis in the dark.     

Subnucleolar location of fibrillarin and variation in its levels during the cell cycle and during differentiation of plant cells

The nucleolar protein fibrillarin has been studied in onion cells; it is detected as an Mr 37,000 protein by immunoblotting using a human autoimmune serum. Quantitative immunoelectron microscopy showed that most fibrillarin is localized in the transition zone between the fibrillar center (FC) and the dense fibrillar component (DFC) as well as in the priximal zone of the DFC, where the labeling shows a gradual decrease out-ward until it reaches insignificant levels in the distal zone of the DFC. Thus, fibrillarin is not uniformly distributed throughout the DFC of plant cells. This result supports the hypothesis that the morphologically homogeneous DFC may not be uniform in function; it is also in agreement with the hypothesized vectorial flow of ribosome biogenesis through the same compartments. Data are also presented showing that the amount of fibrillarin increase when nucleolar activity increases in G2, and probably decreases when nucleolar activity decreases during differentiation.     

Characterization of the phosphatidylinositol-specific phospholipase C-released form of rat osseous plate alkaline phosphatase and its possible significance on endochondral ossification

S-2-(3 aminopropylamino) ethylphosphorothioic acid (WR-2721) shown to surpass radical scavenging thiols in their radioprotective efficacy in cancer-type diseases has been tested for its protective potential in the reperfused heart. We investigated the radical scavenger properties of the compound in a radical generating systemin vitro as well as in isolated rat hearts subjected to 30 min ischaemia and 30 min reperfusion with the electron-paramagnetic resonance spin trap technique. The action on high-energy phosphates is observed by means of phosphorus-31 nuclear magnetic resonance (NMR) spectroscopy while its influence on left ventricular systolic segmental length change (SSLC) during 60 min reperfusion following 60 min regional ischaemia was assessed with a fibreoptic system in anaesthetized open-chest rats. WR-2721 (0.1 mM) reduced the vascular concentration of radical adduct in isolated hearts by up to 78% (275±84% of pre-ischaemic baseline values vs 1260±413%, p<0.05) between 5 and 12.5 min reperfusion. This was accompanied by a reduction of the left ventricular end diastolic pressure to pre-ischaemic values at 30 min of reperfusion (9±6 mmHg vs 42±8 mmHg in the absence of WR-2721, p<0.02). An accelerated recovery of creatine phosphate levels (78±5% of pre-ischaemia values vs 41±5% within 60 min reperfusion; p<0.05) was observed under similar conditions with NMR-spectroscopy, although the post-ischaemic tissue content of adenosine triphosphate was not affected. The administration of WR-2721 (0.5 mmol/kg body weight) ledin situ to an accelerated restoration of contractile activity in the post-ligated left ventricular area reflected by the post-ischaemic recovery of SSLC (64±10% of pre-ischaemic values compared with 27±6% in control animals 60 min following reperfusion; p<0.02). The present data confirm an effective reduction in the exposure of the reperfused heart to endogenously released free radicals by WR-2721, a partial preservation of high-energy phosphates and an improvement in post-ischaemic contractility and encourage further investigation of such favourable action in injured myocardium.     

Diversity, abundance or rare species as a target for the conservation of mammalian carnivores: a case study in Southern Spain

Management goals in protected areas and/or communities usually include diversity as one of the most valuable and confident criteria. Nevertheless, the use of diversity and related indices as a means of evaluating successful management practices could produce conflicting results. Here we report a case study in one of the most important European protected areas. After 6 years of intensive conservation management of the Don~ana National Park, the general abundance and numbers of the target single-species conservation plan (the Iberian lynx) increased, although carnivore community diversity and evenness decreased. This was a result of a disproportionate increase of an oportunistic native species, the red fox. We propose the combined use of diversity, richness and evenness indices when monitoring management practices such as those reported here.     

Differential effects of five types of antipathogenic plant peptides on model membranes

The effects of five antipathogenic plant peptides, wheat α-thionin, potato PTH1 defensin, barley LTP2 lipid transfer protein, and potato tuber DL1 and DL2 defensins, have been tested against phospholipid vesicles (liposomes). Wheat thionin very actively induces aggregation and leakage of negatively charged vesicles. LTP2 displays the same activities, although to a limited extent. Under certain conditions PTH1 and DL2 induce vesicle aggregation, but not leakage. Potato defensin DL1 failed to show any effect on liposomes. The same peptides have been assayed against a plant pathogenic bacterium, both the membrane-active and -inactive compounds having efficient antibacterial action.     

Lactase production in continuous culture by Trichoderma reesei Rut-C30

Summary Trichoderma reesei Rut-C30 was found to produce extracellular lactase when grown on lactose medium. Maximum enzyme levels in continuous culture were observed at dilution rates (D) between 0.02 and 0.027 hr^-1. The enzyme productivity reached 27.3 U/L hr at D = 0.027 hr^-1. Lactase synthesis appears to be inducible and subject to catabolite repression. Optimal growth temperature and pH for enzyme production were 28°C and pH 5. Maximum enzyme activity was observed at 63°C and pH 4.6. The apparent K_m, based on the nitrophenyl-galactopyranoside assay was estimated as 0.4 mM. The enzyme is suitable for lactose hydrolysis in acid whey.     

Characterisation of actI-homologous DNA encoding polyketide synthase genes from the monensin producer Streptomyces cinnamonensis

Summary Cloned DNA encoding polyketide synthase (PKS) genes from one Streptomyces species was previously shown to serve as a useful hybridisation probe for the isolation of other PKS gene clusters from the same or different species. In this work, the actI and actIII genes, encoding components of the actinorhodin PKS of Streptomyces coelicolor, were used to identify and clone a region of homologous DNA from the monensin-producing organism S. cinnamonensis. A 4799 by fragment containing the S. cinnamonensis act-homologous DNA was sequenced. Five open reading frames (ORFs 1–5) were identified on one strand of this DNA. The five ORFs show high sequence similarities to ORFs that were previously identified in the granaticin, actinorhodin, tetracenomycin and whiE PKS gene clusters. This allowed the assignment of the following putative functions to these five ORFS : a heterodimeric -ketoacyl synthase (ORF1 and ORF2), an acyl carrier protein (ORF3), a -ketoacyl reductase (ORF5), and a bifunctional cyclase/dehydrase (ORF4). The ORFs are encoded in the order ORFl-ORF2-ORF3-ORF5-ORF4, and ORFs-1 and -2 show evidence for translational coupling. This act-homologous region therefore appears to encode a PKS gene cluster. A gene disruption experiment using the vector pGM 160, and other evidence, suggests that this cluster is not essential for monensin biosynthesis but rather is involved in the biosynthesis of a cryptic aromatic polyketide in S. cinnamonensis. An efficient plasmid transformation system for S. cinnamonensis has been established, using the multicopy plasmids pWOR120 and pWOR125.     

Chromosome Interactions in DROSOPHILA MELANOGASTER. II. Total Fitness

In a large experiment, using nearly 200 population cages, we have measured the fitness of Drosophila melanogaster homozygous (1) for the second chromosome, (2) for the third chromosome, and (3) for both chromosomes. Twentyfour second chromosomes and 24 third chromosomes sampled from a natural population were tested. The mean fitness of the homozygous flies is 0.081 ± 0.014 for the second chromosome, 0.080 ± 0.017 for the third chromosome, and 0.079 ± 0.024 for both chromosomes simultaneously. Assuming that fitnesses are multiplicative (the additive fitness model makes no sense in the present case because of the large selection coefficients involved), the expected mean fitness of the homozygotes for both chromosomes is 0.0066; their observed fitness is more than ten times greater. Thus, it appears that synergistic interactions between loci are considerable; and that, consequently, the fitness function substantially departs from linearity. Two models are tentatively suggested for the fitness function: a "threshold" model and a "synergistic" model.—The experiments reported here confirm previous results showing that the concealed genetic load present in natural populations of Drosophila is sufficient to account for the selective maintenance of numerous polymorphisms (of the order of 1000).     

Volatile constituents of Cupressus dupreziana and the sesquiterpenes of Cupressus sempervirens

Analysis of wood essential oil of Cupressus dupreziana revealed 26 components: 13 monoterpenes and 13 sesquiterpenes. The main components were carv     

Trypanosoma cruzi: defined medium for continuous cultivation of virulent parasites.

A liquid medium was developed for the continuous cultivation of Trypanosoma cruzi. Among the several highly purified macromolecules tested only bovine liver catalase, horseradish peroxidase, lactoperoxidase, and bovine hemoglobin supported the continuous growth, at high yield, of mice-virulent Trypanosoma cruzi; other hemoproteins were inactive. Bovine liver catalase showed optimal Trypanosoma cruzi growth-promoting activity, parasites reaching 20 × 10⁶ parasites/ml (95% epimastigotes) at about 10 days in most of the 45 subpassages to date. Furthermore, this protein in the incubation medium provided all the amino acid requirements of actively growing parasites, thus eliminating the need for exogeneous free amino acids. Additional experiments revealed that the hemoprotein's growth-promoting activity was independent of any enzymatic activity and that reconstituting the exact protein composition by means of exogeneous amino acids did not support parasite multiplication, suggesting the importance of the primary structure of the active proteins for growth-promoting activity. These active macromolecules supported the multiplication of five different strains of Trypanosoma cruzi, but did not support Leishmania brasiliensis or Leishmania mexicana proliferation, suggesting species specificity.