Immunological Detection and Quantitation of Tryptophan Decarboxylase in Developing Catharanthus roseus Seedlings

l-Tryptophan decarboxylase (TDC) (EC 4.2.1.27) enzyme activity was induced in cell suspension cultures of Catharanthus roseus after treatment with a Pythium aphanidermatum elicitor preparation. The enzyme was extracted from lyophilized cells containing high levels of TDC and the protein was purified to homogeneity. The pure protein was used to produce highly specific polyclonal antibodies, and an enzyme-linked immunosorbent assay (ELISA) was developed to quantitate the level of TDC antigen during seedling development and in leaves of the mature plant. Western immunoblotting of proteins after SDS-PAGE with anti-TDC antibodies detected several immunoreactive proteins (40, 44, 54.8, 55, and 67 kilodaltons) which appeared at different stages during seedling development and in leaves of the mature plant. The major 54.8 and 55 kilodalton antigenic proteins in immunoblots appeared transiently between days 1 to 5 and 5 to 8 of seedling development, respectively. The 54.8 kilodalton protein was devoid of TDC enzyme activity, whereas the appearance of the 55 kilodalton protein coincided with the appearance of this decarboxylase activity. The minor immunoreactive proteins (40, 44, and 67 kilodaltons) appeared after day 5 of seedling development and in older leaves of the mature plant, and their relationship, if any, to TDC is presently unknown. Results suggest that the synthesis and degradation of TDC protein is highly regulated in Catharanthus roseus and that this regulation follows a preset developmental program.     

2',3'-dideoxycytidine permeation of the human erythrocyte membrane

The mechanism by which 2,3'-dideoxycytidine, an inhibitor of HIV-I infectivity, permeates the cell membrane was investigated. The influx of ddCyd into human erythrocytes was nonconcentrative. The initial velocity of both ddCyd influx and efflux was, in contrast to compounds that permeate the cell membrane via the nucleoside transporter, a linear function of nucleoside concentration in the 1 microM to 10 mM range and relatively insensitive to temperature. Furthermore, potent inhibitors of nucleoside transporter and other nucleosides were found to inhibit ddCyd influx only partially or not at all suggesting that ddCyd permeates the human erythrocyte membrane predominantly by nonfacilitated diffusion. This unusual characteristic seems to be due to the lack of 3'-hydroxyl moiety of ddCyd which appears to be an important determinant for the nucleoside carrier specificity rather than to lipid solubility itself. As far as permeation of the cell membrane is concerned ddCyd shares these properties with 2',3'-dideoxythymidine and 3'-azido-3'-deoxythymidine.     

Positive feedback of consumer population density on resource supply

Recent studies demonstrate positive density-dependent feedbacks between animal populations and their resource supply that result in increased individual fitness at high densities. Such feedbacks occur in both terrestrial and aquatic organisms not showing strong social organization. A number of different mechanisms are involved. Detecting positive feedbacks in natural populations may not be possible from simple correlations between resource abundance and animal population density in space or time, but experimental manipulation of resource supply or animal density can reveal their presence. Positive feedbacks may result in higher equilibrium densities of animal populations, alter the density range over which intraspecific competition is detectable, and offer a resource-based explanation for the evolution of gregariousness and social organization.     

Biosynthesis of glycosaminoglycan precursors: evidences for different tissue specific forms of UDP-glucose dehydrogenase

UDP-glucose dehydrogenase (UDPGDH) was extracted and partially purified from different rat tissues and the kinetic parameters and some properties of the enzyme were determined and compared. The pH optimum ranged between 8.6 and 9.4 for liver and kidney UDPGDH and between 8.4 and 8.6 for skin and lung UDPGDH. Liver and kidney enzymes showed a similar affinity for both UDPG and NAD. Lung and skin enzymes also showed similar affinity for both substrates, which differed however from that of liver and kidney UDPGDH. Both liver and kidney enzymes had a higher heat stability and a different electrophoretic mobility compared to skin and lung UDPGDH. These data suggest the existence of different tissue specific forms of the enzyme.     

The myelodysplastic syndrome: prognostic factors in relation to the "in vitro" clonogenic assay.

The object of this study was to determine whether the "in vitro" parameters of medullary and blood granulopoiesis in patients with MDS, furnish information of either prognostic or diagnostic value. This study covered 94 patients with MDS. All patients were studied at the onset of disease. In order to identify the factors related to patients' survival, Cox Multiple Regression analysis was performed by the BMD P2L program. When analyzing by means of actuarial curves the survival probability of patients with benign development versus those of malignant development (those who developed ANLL), the significance between both groups was p = 0.0001. Different variables of patients included in this study were analyzed and all showed great significances. Fab: p = 0.0022, disease evolution: p = 0.0001 and presence of blastic aggregates: p = 0.0011. Cox's regression analysis revealed that the only predictable survival variable is the presence of blastic colonies and/or clusters. Accordingly, two groups were constructed: favourable and unfavourable. In the favourable group, 40% of the patients belonged to the RA group, while in the unfavourable group, 55% belonged to the RAEB group. This study shows the validity of the elaboration of prognostic groups in MDS according to the presence of blastic colonies and/or clusters in CFUGM medullary and/or peripheral cultures. The "in vitro" myeloid progenitors culture techniques may therefore be advantageously applied in these disorders for formulating a diagnosis and predicting the patient's short term evolution.     

Medicago truncatula,a model plant for studying the molecular genetics of theRhizobium-legume symbiosis

Medicago truncatula has all the characteristics required for a concerted analysis of nitrogen-fixing symbiosis withRhizobium using the tools of molecular biology, cellular biology and genetics.M. truncatula is a diploid and autogamous plant has a relatively small genome, and preliminary molecular analysis suggests that allelic heterozygosity is minimal compared with the cross-fertilising tetraploid alfalfa (Medicago sativa). TheM. truncatula cultivar Jemalong is nodulated by theRhizobium meliloti strain 2011, which has already served to define many of the bacterial genes involved in symbiosis with alfalfa. A genotype of Jemalong has been identified which can be regenerated after transformation byAgrobacterium, thus allowing the analysis ofin-vitro-modified genes in an homologous transgenic system. Finally, by virtue of the diploid, self-fertilising and genetically homogeneous character ofM. truncatula, it should be relatively straightforward to screen for recessive mutations in symbiotic genes, to carry out genetic analysis, and to construct an RFLP map for this plant.     

Structure and expression of a gene from Arabidopsis thaliana encoding a protein related to SNF1 protein kinase

The AKin10 gene from Arabidopsis thaliana encoding a putative Ser/Thr protein kinase (PK) has been isolated and characterized. The AKin10-encoding gene is located on a genomic 5.4-kb BamHI fragment and contains ten introns, one being located in the 5' untranslated region. The deduced amino acid sequence of AKin10 is 65% identical over the catalytic domain to the yeast PK (SNF1). SNF1 is essential for the derepression of many glucose-repressible genes, including Suc2 which encodes invertase. Southern blot hybridization experiments suggested the presence of one copy of the gene per haploid genome of A. thaliana. Northern hybridization experiments indicated that this gene is expressed in roots, shoots and leaves. AKin10 may play an important role in a signal transduction cascade regulating gene expression and carbohydrate metabolism in higher plants.     

Effect of alpha-adrenoreceptors in the control of spontaneous motility and morphine withdrawal syndrome.

The effects of different alpha-2 agonists on the spontaneous motility in naive and morphine tolerant mice were studied. Clonidine caused a reduction at the lower (1-3 micrograms Kg-1 i.p.) and higher (100 micrograms Kg-1 i.p.) doses and no effect at 10-30 micrograms Kg-1 i.p. in naive mice, while an increase was found at the intermediate doses (10-30 micrograms Kg-1 i.p.) in morphine tolerant mice. The clonidine-induced inhibition on spontaneous motility at the lower and higher doses was prevented both in naive and tolerant mice by idazoxan pretreatment. In morphine-treated animals the increase induced by clonidine was antagonized by prazosin. The action of guanabenz and guanfacine on locomotion differed from clonidine, by producing inhibition only at higher doses (100-300 micrograms Kg-1 i.p.). Clonidine, but not guanfacine or guanabenz, prevented the withdrawal syndrome precipitated by naloxone. Thus the only alpha-2 agonistic properties do not appear sufficient to explain the prevention of morphine abstinence by clonidine in mice, which can represent a single model to screen anti-withdrawal drugs.     

Targeting of T7 RNA polymerase to tobacco nuclei mediated by an SV40 nuclear location signal

We have expressed two T7 RNA polymerase genes by electroporation into tobacco protoplasts. One of the genes was modified by inserting nucleotides encoding a viral nuclear localization signal (NLS) from the large T antigen of SV40. Both T7 RNA polymerase genes directed synthesis of a ca. 100 kDa protein in the electroporated protoplasts. T7 RNA polymerase activity was detected in extracts of protoplasts electroporated with both genes. Immunofluorescence analysis of these protoplasts indicated that only the polymerase carrying the NLS accumulated in the cell nucleus. These experiments suggest that mechanisms involved in the transport from the cytoplasm to the nucleus are similar in plant and animal cells. This system demonstrates the feasibility of T7 RNA polymerase-based approaches for the high-level expression of introduced genes in plant cells.