Recombinant phosphoribosyl pyrophosphate synthetases from <Emphasis Type="Italic">Thermus thermophilus</Emphasis> HB27: Isolation and properties

Two genes of T. thermophilus HB27, TT_C1184 and TT_C1274, encoding proteins with phosphoribosyl pyrophosphate synthetase activity were cloned in an expressing vector pET23d+. Escherichia coli strains overproducing two relevant proteins in soluble forms were obtained. The methods of isolation of thermophilic phosphoribosyl pyrophosphate synthetases Tth PRPPS1 and Tth PRPPS2 were developed. The activities of these enzymes were determined as a function of concentration of metal ions, inorganic phosphate, and temperature. The kinetic parameters for basic natural substrates were calculated; the substrate specificity for different carbohydrate 5-phosphates of D-series was studied. It was shown that the two proteins differ significantly in these characteristics. According to the results and comparison of amino acid sequences of new proteins with those of other phosphoribosyl pyrophosphate synthetases, both enzymes belong to class I PRPPS.     

Antiangiogenic and antivascular effects of a recombinant tumstatin-derived peptide in a corneal neovascularization model

Tumstatin, a cleavage fragment of collagen IV, is a potent endogenous inhibitor of angiogenesis. Tumstatin-derived peptide T8 possesses all angiostatic properties of full-length tumstatin and indirectly suppresses tumor growth. The potential of T8 to block pathological angiogenesis in the eye has not been explored yet. Here we assess antiangiogenic effects of a recombinant T8 peptide in rabbit corneal neovascularization models. The fusion protein consisting of T8 and thioredoxin was synthesized in a highly efficient Escherichia coli expression system, isolated using ion-exchange chromatography and cleaved with TEV (tobacco etch virus) protease. The target peptide was purified on an anion-exchange resin and by reversed phase high-performance liquid chromatography. The recombinant peptide suppressed the proliferation of basic fibroblast growth factor-induced SVEC-4-10 endothelial cells (simian virus 40-immortalized murine endothelial cells) and inhibited tube formation in these cells in a dose-dependent manner. In rabbit corneal neovascularization models T8 demonstrated the ability to prevent pathological angiogenesis (when injected simultaneously with the induction of neovascularization) and, moreover, to promote the regression of newly-formed blood vessels (when injected on day 8 after angiogenesis stimulation). Our results suggest that T8 may have a therapeutic potential in the treatment of ocular neovascular diseases.     

Formation of the Pigment Apparatus in Etiolated Barley Leaves under the Influence of Levulinic Acid

The effects of levulinic acid (LA) on the synthesis of pigments and the membrane system of etioplasts were studied in etiolated leaves of barley (Hordeum vulgare L.). Growing in the solution of LA during a six-day period, which started one day after the soaking of seeds, resulted in a retardation of leaf growth, more than a twofold decrease in the level of carotenoids and protochlorophyllide (Pd) in the leaf tissue, and suppression of the synthesis of long-wave form of Pd₆₅₅; these effects depended on the LA concentration. In etioplasts isolated from the seedlings treated with 50 M LA and containing predominantly a short-wave form of Pd with a peak of fluorescence at 632 nm (–196°C), there was a membrane fraction whose location in the sucrose density gradient was identical to that of prolamellar bodies (PLB) in the control plants. The content of Pd and carotenoids in this fraction calculated on a protein basis was 2.46 and 1.3 times lower than in control seedlings, while the relative content of Pd oxidoreductase (POR) essentially did not change. Thus, the suppression of Pd synthesis did not affect translocation of POR from the cytoplasm to the membranes of etioplasts. In the PLB membranes, there was no transfer of energy from the molecules of lipophilic fluorescent probe pyrene (excitation at 337, 278, and 296 nm) to Pd; however, under pigment deficiency, the production therein of pyrene excimer form at the expense of energy transfer from protein tryptophanyls (excitation at 278 and 296 nm) became more efficient, which indicated changes in protein–lipid interactions. The obtained results suggest that the short-wave form of Pd₆₃₂ accumulating in etioplasts under the suppressed synthesis of tetrapyrroles is not a free pigment.     

Pallister-Killian syndrome: rapid decrease of isochromosome 12p frequency during amniocyte subculturing. Conclusion for strategy of prenatal cytogenetic diagnostics.

Pallister-Killian syndrome (PKS) is characterized cytogenetically by mosaic tetrasomy of chromosome 12p. Routine prenatal diagnosis of PKS is still complicated because of the difficulties of discriminating between the supernumerary isochromosome 12p and the duplication 21q and because of the variable level of mosaicism. The frequency of cells with an extra metacentric chromosome i(12)(p10) is usually determined by tissue-limited or tissue-specific mosaicism. We demonstrated a decrease of the abnormal clone with extra i(12p) in the amniotic fluid cells of the PKS fetus during amniocyte subculturing. The rapid loss of the i(12p) in the course of amniocyte subculturing should be the focus of attention during prenatal karyotyping. This is especially necessary for cultures with slow growth, which require further interpretation of the result during cytogenetic diagnosis of PKS.     

Effect of Periodic Heat Shock on the Inner Membrane System of Etioplasts

Etiolated barley (Hordeum vulgare L.) seedlings were treated with heat shock (HS). The heat treatment was conducted daily for 1 h at 40°C over 6 days and led to shortening of leaves and coleoptiles, an increase in the etioplast volume and prothylakoid length, and to a decrease in the size of paracrystalline prolamellar bodies (PLB). As a result of HS treatment, stimulation of carotenoid and protochlorophyllide (Pchlide) synthesis as well as an increase in the relative content of the Pchlide short-wavelength form (Pchlide₆₃₀) were observed in the leaf tissue of seven-day-old seedlings 12 h after the last HS treatment. HS had no effect on the overall amount of Pchlide-oxidoreductase (POR) in leaves and PLB membranes and did not suppress the Pchlide photoreduction in vivo. PLB membranes, isolated from the HS-treated seedlings, possessed a higher Pchlide and carotenoid content as calculated on total protein basis. These membranes showed more intense protein fluorescence than PLB from untreated plants, whereas hydrophobicity of the microenvironment of the fluorescent amino-acid residues remained unchanged. Studies using pyrene (lipophilic fluorescent probe emitted in Pchlide and carotenoid absorption bands) showed that HS increases the fluidity of membrane lipids in PLB membranes and that the pigments accumulated in these membranes are located in the region of lipid–protein contact site. The results are discussed in relation to the adaptive role of protein–protein and pigment–protein–lipid interactions in etioplast membranes under stress.