15N-labeling to determine chlorophyll synthesis and degradation in Synechocystis sp. PCC 6803 strains lacking one or both photosystems

Rates of chlorophyll synthesis and degradation were analyzed in Synechocystis sp. PCC 6803 wild type and mutants lacking one or both photosystems by labeling cells with ((15)NH(4))(2)SO(4) and Na(15)NO(3). Pigments extracted from cells were separated by HPLC and incorporation of the (15)N label into porphyrins was subsequently examined by MALDI-TOF mass spectrometry. The life time (tau) of chlorophyll in wild-type Synechocystis grown at a light intensity of 100 micromol photons m(-2) s(-1) was determined to be about 300 h, much longer than the cell doubling time of about 14 h. Slow chlorophyll degradation (tau approximately 200-400 h) was also observed in Photosystem I-less and in Photosystem II-less Synechocystis mutants, whereas in a mutant lacking both Photosystem I and Photosystem II chlorophyll degradation was accelerated 4-5 fold (tau approximately 50 h). Chlorophyllide and pheophorbide were identified as intermediates of chlorophyll degradation in the Photosystem I-less/Photosystem II-less mutant. In comparison with the wild type, the chlorophyll synthesis rate was five-fold slower in the Photosystem I-less strain and about eight-fold slower in the strain lacking both photosystems, resulting in different chlorophyll levels in the various mutants. The results presented in this paper demonstrate the presence of a regulation that adjusts the rate of chlorophyll synthesis according to the needs of chlorophyll-binding polypeptides associated with the photosystems.     

Regulation of the tetrapyrrole biosynthetic pathway leading to heme and chlorophyll in plants and cyanobacteria

Photosynthetic organisms synthesize chlorophylls, hemes, and bilin pigments via a common tetrapyrrole biosynthetic pathway. This review summarizes current knowledge about the regulation of this pathway in plants, algae, and cyanobacteria. Particular emphasis is placed on the regulation of glutamate-1-semialdehyde formation and on the channelling of protoporphyrin IX into the heme and chlorophyll branches. The potential role of chlorophyll molecules that are not bound to photosynthetic pigment-protein complexes ('free chlorophylls') or of other Mg-containing porphyrins in regulation of tetrapyrrole synthesis is also discussed.     

Synthesis of Novel Nucleoside 5′-Triphosphates and Phosphoramidites Containing Alkyne or Amino Groups for the Postsynthetic Functionalization of Nucleic Acids

A series of novel nucleoside 5′-triphosphates and phosphoramidites containing alkyne or amino groups for the postsynthetic functionalization of nucleic acids were designed and synthesized. For this purpose, the new 3-aminopropoxypropynyl linker group was used. It contains two alternative functional capabilities: an amino group for the reaction of amino–alkynyl-modified oligonucleotides with corresponding activated esters and an alkyne group for the copper(I)-catalyzed azide–alkyne cycloaddition (CuAAC) reaction. It was shown that a variety of methods of the attachment of the new linker can be used to synthesize a diversity of modified pyrimidine nucleosides.     

Effect of acetylcholine on the pituitrin induced osmotic flow of water through the wall of the frog urinary bladder]

The role of intercellular pathways in the ADH-dependent water transport was studied on the frog urinary bladder by means of acetylcholine (AC) and other cholinergic compounds. AC (10(-3) M) was found to cause a strong suppression of the pituitrin-stimulated water flow. Analogous effect was produced by AC on the osmotic flow stimulated by cyclic adenosine monophosphate (cAMP) and theolin. The antipituitrin effect was not reproduced either by nicotine, nor by potent M-cholinomimetic agents (methylfurmetide and F-2268), and was not prevented by M- and N-cholynolytic drugs (atropine, metacin, flaxedil, hexamethonium). However, the antipituitrin effect of AC was completely removed by the anticholinesterase drugs with different mode of action (eserine, proserine, armin, acridine iodmethylate, GD-42) in concentrations of 10(-6)--10(-3) M. It was concluded that the smooth muscles contraction with the subsequent closure of the intercellular spaces was not responsible for the antipituitrinic action of AC. This effect appears to be connected with cholinesterase activation. A possible role of the phosphoinositides in the water permeability regulation of the urinary bladder wall is discussed.     

Effect of acetylcholine and acetylcholinesterase on the activity of contractile vacuole of Amoeba proteus

Acetylcholine (ACh, 1 microM) stimulates activity of the contractile vacuole of proteus. The effect of ACh is not mimicked by its analogs which are not hydrolyzed by acetylcholinesterase (AChE), i. e., carbacholine and 5-methylfurmethide. The effect of ACh is not sensitive to the blocking action of M-cholinolytics, atropine and mytolone, but is suppressed by N-cholinolytic, tubocurarine. The inhibitors of AChE, eserine (0.01 microM) and armine (0.1 microM), suppress the effect of ACh on amoeba contractile vacuole. ACh does not affect activation of contractile vacuole induced by arginine-vasopressin (1 microM), but it blocks such effect of opiate receptors agonist, dynorphin A1-13 (0.01 microM). This effect of ACh is also suppressed by the inhibitors of AChE. These results suggest that, in the above-described effects of ACh, AChE acts not as an antagonist, but rather as a synergist.     

Synthesis of novel nucleoside 5'-triphosphates and phosphoramidites containing alkyne or amino groups for the postsynthetic functionalization of nucleic acids

A series of novel nucleoside 5'-triphosphates and phosphoramidites containing alkyne or amino groups for the postsynthetic functionalization of nucleic acids were designed and synthesized. For this purpose, the new 3-aminopropoxypropynyl linker group was used. It contains two alternative functional capabilities: an amino group for the reaction of amino-alkynyl-modified oligonucleotides with corresponding activated esters and an alkyne group for the copper(I)-catalyzed azide-alkyne cycloaddition (CuAAC) reaction. It was shown that a variety of methods of the attachment of the new linker can be used to synthesize a diversity of modified pyrimidine nucleosides.     

Stomatin is highly expressed in exosomes of different origin and is a promising candidate as an exosomal marker

Proteins involved in the organizing of lipid rafts can be found in exosomes, as shown for caveolin‐1, and they could contribute to exosomal cargo sorting, as shown for flotillins. Stomatin belongs to the same stomatin/prohibitin/flotillin/HflK/C family of lipid rafts proteins, but it has never been studied in exosomes except for extracellular vesicles (EVs) originating from blood cells. Here we first show the presence of stomatin in exosomes produced by epithelial cancer cells (non–small cell lung cancer, breast, and ovarian cancer cells) as well as in EVs from biological fluids, including blood plasma, ascitic fluids, and uterine flushings. A high abundance of stomatin in EVs of various origins and its enrichment in exosomes make stomatin a promising exosomal marker. Comparison with other lipid raft proteins and exosomal markers showed that the level of stomatin protein in exosomes from different sources corresponds well to that of CD9, while it differs essentially from flotillin‐1 and flotillin‐2 homologs, which in turn are present in exosomes in nearly equal proportions. In contrast, the level of vesicular caveolin‐1 as well as its EV‐to‐cellular ratio vary drastically depending on cell type.     

A study of lamellas of the web recombinant protein by atomic force microscopy

Lamellas formed on the mica by protein 1F9, a recombinant analogue of the web protein, have been studied by atomic force microscopy. It has been shown that the molecules of 1F9 dissolved in strong solvents are capable of aggregating on the mica surface to form lamellas less than 1 nm in height and more than 1 microm in length. A model of a plane zigzag has been constructed to describe the conformation of 1F9 molecules on the mica surface.