Specificity of GlcNAc-PI de-N-acetylase of GPI biosynthesis and synthesis of parasite-specific suicide substrate inhibitors.

The substrate specificities of Trypanosoma brucei and human (HeLa) GlcNAc-PI de-N-acetylases were determined using 24 substrate analogues. The results show the following. (i) The de-N-acetylases show little specificity for the lipid moiety of GlcNAc-PI. (ii) The 3'-OH group of the GlcNAc residue is essential for substrate recognition whereas the 6'-OH group is dispensable and the 4'-OH, while not required for recognition, cannot be epimerized or substituted. (iii) The parasite enzyme can act on analogues containing betaGlcNAc or aromatic N-acyl groups, whereas the human enzyme cannot. (iv) Three GlcNR-PI analogues are de-N-acetylase inhibitors, one of which is a suicide inhibitor. (v) The suicide inhibitor most likely forms a carbamate or thiocarbamate ester to an active site hydroxy-amino acid or Cys or residue such that inhibition is reversed by certain nucleophiles. These and previous results were used to design two potent (IC50 = 8 nM) parasite-specific suicide substrate inhibitors. These are potential lead compounds for the development of anti-protozoan parasite drugs.     

Mechanism of "uncoupled" tetrahydropterin oxidation by phenylalanine hydroxylase

Phenylalanine hydroxylase can catalyze the oxidation of its tetrahydropterin cofactor without concomitant substrate hydroxylation. We now report that this "uncoupled" tetrahydropterin oxidation is mechanistically distinct from normal enzyme turnover. Tetrahydropterins are oxygenated to 4a-carbinolamines only during catalytic events involving substrate hydroxylation. In the absence of hydroxylation tetrahydropterins are oxidized directly to quinonoid dihydropterins. Stoichiometry studies define a ratio of two tetrahydropterins oxidized per O2 consumed in uncoupled enzyme turnover, thus indicating the complete reduction of O2 to H2O. Complementary results establish the lack of H2O2 production by the enzyme when uncoupled and define a tetrahydropterin oxidase activity for the enzyme. Thus, the hydroxylating intermediate of phenylalanine hydroxylase may be discharged in two ways, by substrate hydroxylation or by electron abstraction. A mechanism is proposed for the uncoupled oxidation of tetrahydropterins by phenylalanine hydroxylase, and the significance of these findings is discussed.     

The rate of cleavage of GTP on the binding of Phe-tRNA.elongation factor Tu.GTP to poly(U)-programmed ribosomes of Escherichia coli

Interaction of Phe-tRNA.elongation factor Tu.GTP with poly(U)-programmed ribosomes containing an occupied P site can be described by a three-step kinetic mechanism. Initial binding is followed by the cleavage of GTP, and then a new peptide bond is formed. Rate constants controlling the first and third of these reactions are known, but only a lower limit for the rate constant of the cleavage step has been reported. We have determined this rate constant to be 20 s-1 at 5 degrees C, 30 s-1 at 15 degrees C, and 50 s-1 at 25 degrees C. This is much faster than the reverse step of the initial binding process and implies that the intrinsic accuracy of the ribosome in the initial selection step is sacrificed in favor of speed. The similarity of the kinetic and chemical mechanism of this GTP cleavage step with other nucleoside 5'-triphosphatases is discussed.     

Hydroperoxide-dependent epoxidation of 3,4-dihydroxy-3,4-dihydrobenzo[a]anthracene by ram seminal vesicle microsomes and by hematin

Addition of arachidonic acid to ram seminal vesicle microsomes oxidizes 3,4-dihydroxy-3,4-dihydrobenzo[a]anthracene (BA-3,4-diol) to five more polar products. Four of the products are identified by chromatographic and spectroscopic analysis as tetrahydrotetraols, which are solvolysis products of dihydrodiolepoxides. The fifth product is a 10-methyl ether formed by methanolysis of the anti-diolepoxide. Quantitation of the individual products indicates that anti-diolepoxides predominate over syn-diolepoxides by approximately 2:1. Identical product profiles are detected from the reaction of BA-3,4-diol with hematin and 13-hydroperoxy-octadecadienoic acid in the presence of Tween 20. No other products are detected in either system, which indicates that peroxyl radicals oxidize BA-3,4-diol exclusively by epoxidation of the 1,2-double bond. The stereochemical and regiochemical differences between oxidation of BA-3,4-diol by peroxyl radicals and cytochrome P-450 are dramatic and suggest that BA-3,4-diol is uniquely suited as a probe to quantitate peroxyl radical-dependent epoxidation in vitro and in vivo.     

Ultrastructure of Lycopersicon peruvianum leaf explants and streptomycin-resistant shoots on medium containing streptomycin

The effect of streptomycin on morphogenic explants of Lycopersicon peruvianum Mill. was examined microscopically at both the light and ultrastructural level. Early stages in shoot regeneration from leaf explants were distinguished as meristematic tissue at both levels. Small starch grains were observed in the plastids in this tissue but not in plastids in regenerated shoots. In the presence of streptomycin, adventitious shoot regeneration from sensitive leaf strips was inhibited. Large layered bodies were observed within the plastids of sensitive leaf tissue, suggesting the disruption of thylakoid membrane formation. Streptomycin resistant L. peruvianum lines, as well as a chlorophyll-deficient line, were also examined microscopically. The chloroplasts of newly regenerated streptomycin resistant shoots contained well developed internal membranes and conspicuous starch grains. Cells containing a mixture of resistant and sensitive plastids were not observed. The plastids in chlorophyll-deficient tissue completely lacked thylakoid membranes, although small vesicles and intraplastid bodies were seen within the stroma.Abbreviations NMU N-methyl-N-nitrosourea     

Beta1 integrin-mediated T cell adhesion and cell spreading are regulated by calpain

To investigate the function of calpain in T cells, we sought to determine the role of this protease in cellular events mediated by beta1 integrins. T cell receptor cross-linked or phorbol ester-stimulated T cells binding to immobilized fibronectin induce the translocation of calpain to the cytoskeletal/membrane fraction of these cells. Such translocation of calpain is associated with proteolytic modification of protein tyrosine phosphatase 1B, increased cellular adhesion, and dramatic alterations in cellular morphology. However, affinity-related increases in T cell adhesion induced by the anti-beta1 integrin antibody 8A2 occur in a calpain-independent manner and in the absence of morphological shape changes. Furthermore, calpain undergoes activation in response to either alpha4beta1 or alpha5beta1 integrin binding to fibronectin in appropriately stimulated T cells, and calpain II as well as protein tyrosine phosphatase 1B accumulates at sites of focal contact formation. Inhibition of calpain activity not only inhibits the proteolytic modification of protein tyrosine phosphatase 1B, but also decreases the ability of T cells to adhere to and spread on immobilized fibronectin. Thus, we describe a potential regulatory role for calpain in beta1 integrin-mediated signaling events associated with T cell adhesion and cell spreading on fibronectin.     

Homeologous plastid DNA transformation in tobacco is mediated by multiple recombination events.

Efficient plastid transformation has been achieved in Nicotiana tabacum using cloned plastid DNA of Solanum nigrum carrying mutations conferring spectinomycin and streptomycin resistance. The use of the incompletely homologous (homeologous) Solanum plastid DNA as donor resulted in a Nicotiana plastid transformation frequency comparable with that of other experiments where completely homologous plastid DNA was introduced. Physical mapping and nucleotide sequence analysis of the targeted plastid DNA region in the transformants demonstrated efficient site-specific integration of the 7.8-kb Solanum plastid DNA and the exclusion of the vector DNA. The integration of the cloned Solanum plastid DNA into the Nicotiana plastid genome involved multiple recombination events as revealed by the presence of discontinuous tracts of Solanum-specific sequences that were interspersed between Nicotiana-specific markers. Marked position effects resulted in very frequent cointegration of the nonselected peripheral donor markers located adjacent to the vector DNA. Data presented here on the efficiency and features of homeologous plastid DNA recombination are consistent with the existence of an active RecA-mediated, but a diminished mismatch, recombination/repair system in higher-plant plastids.     

Restoring lakes by using artificial plant beds: habitat selection of zooplankton in a clear and a turbid shallow lake

1. Return of large‐bodied zooplankton populations is of key importance for creating a shift from a turbid to a clear‐water state in shallow lakes after a nutrient loading reduction. In temperate lakes, recovery is promoted by submerged macrophytes which function as a daytime refuge for large zooplankton. However, recovery of macrophytes is often delayed and use of artificial plant beds (APB) has been suggested as a tool to enhance zooplankton refuges, thereby reinforcing the shift to a clear‐water state and, eventually, colonisation of natural plants. 2. To further evaluate the potential of APB in lake restoration, we followed the day–night habitat choices of zooplankton throughout summer in a clear and a turbid lake. Observations were made in the pelagic and littoral zones and in APB in the littoral representing three different plant densities (coverage 0%, 40% and 80%). 3. In the clear lake, the zooplankton (primarily Daphnia) were mainly found in the pelagic area in spring, but from mid‐May they were particularly abundant in the APB and almost exclusively so in mid‐June and July, where they appeared in extremely high densities during day (up to 2600 ind. L⁻¹). During night Daphnia densities were overall more equally distributed between the five habitats. Ceriodaphnia was proportionally more abundant in the APB during most of the season. Cyclopoids were more abundant in the high APB during day but were equally distributed between the five habitats during night. 4. In the turbid lake, however, no clear aggregation was observed in the APB for either of the pelagic genera (Daphnia and Bosmina). This may reflect a higher refuge effect in the open water due to the higher turbidity, reduced ability to orient to plant beds and a significantly higher fish density (mainly of roach, Rutilus rutilus, and perch, Perca fluviatilis) in the plant beds than in the clear lake. Chydorus was found in much higher proportions among the plants, while cyclopoids, particularly the pelagic Cyclops vicinus, dominated in the pelagic during day and in the pelagic and high density plants during night. 5. Our results suggest that water clarity is decisive for the habitat choice of large‐bodied zooplankton and that introduction of APB as a restoration measure to enhance zooplankton survival is only a useful tool when water clarity increases following loading reduction. Our results indicate that dense APB will be the most efficient.