Aerosolization of P2Y(2)-receptor agonists enhances mucociliary clearance in sheep.
(9/594)
The purpose of this study was to determine whether aerosolized INS316 (UTP) stimulates lung mucociliary clearance (MCC) in sheep and, if so, to compare its effects with INS365, a novel P2Y(2)-receptor agonist. In the first series of studies, we used a previously described roentgenographic technique to measure tracheal mucus velocity (TMV), an index of MCC, before and for 4 h after aerosolization of INS316 (10(-1) M and 10(-2) M) and INS365 (10(-1) M and 10(-2) M), or normal saline in a randomized crossover fashion (n = 6). In a second series of studies, we compared the ability of these agents to enhance total lung clearance. For these tests, the clearance of inhaled technetium-labeled human serum albumin was measured serially over a 2-h period after aerosolization of 10(-1) M concentration of each agent (n = 7). Aerosolization of both P2Y(2)-receptor agonists induced significant dose-related increases in TMV (P < 0.05) compared with saline. The greatest increase in TMV was observed between 15 and 30 min after drug treatment. The highest dose (10(-1) M) of INS316 produced a greater overall stimulation of TMV than did INS365 (10(-1) M). Both compounds, compared with saline, induced a significant increase in MCC (P < 0.05) within 20 min of treatment. This enhancement in MCC began to plateau at 60 min. Although the response to INS316 started earlier, there was no significant difference between the clearance curves for the two compounds. We conclude that inhaled P2Y(2)-receptor agonists can increase lung MCC in sheep and that for P2Y(2)-receptor stimulation TMV accurately reflects changes in whole lung MCC. (+info)
Chitin biosynthesis during Blastocladiella zoospore germination: evidence that the hexosamine biosynthetic pathway is post-translationally activated during cell differentiation.
(10/594)
De novo construction of a chitinous cell wall accompanies Blastocladiella emersonii zoospore germination. At least an order of magnitude increase in total hexosamine occurs during germination. This increase is into polymer (chitin) and occurs on schedule in the presence of cycloheximide. Uridine-5'-diphospho-N-acetylglucosamine (UDPGlcNAc), both the end product of hexosamine biosynthesis and a substrate for chitin biosynthesis, is a potent inhibitor of the activity of the first pathway-specific enzyme of hexosamine biosynthesis in zoospore extracts. Certain uridine nucleotides, not perceptibly influencing the activity of the first enzyme per se, counteract the inhibitory effects of UDPGlcNAc. The concentration of UDPGlcNAc in the zoospore is sufficient to act as an inhibitor of the enzyme, but the amount of UDPGlcNAc is sufficient, by over an order of magnitude, to account for the chitin synthesized during germination. Both the production of UDPGlcNAc and its utilization for chitin synthesis appear to be post-translationally regulated in zoospores and during zoospore germination. (+info)
In vitro transcription of chromatin in the presence of a mercurated nucleotide.
(11/594)
Mercurated uridine triphosphate has been used to label transcripts of chicken reticulocyte chromatin made with Escherichia coli RNA polymerase. The mercury-labeled RNA product can be completely separated from endogenous RNA sequences in the chromatin by passage through a sulfhydryl Sepharose column. Globin cDNA hybridization to the transcript shows that only 2.6 x 10-5 of the transcript is globin RNA. In contrast to this result, erythrocte chromatin transcript contains less than one tenth as many globin RNA sequences. (+info)
Amino acid sequence at the FdUMP binding site of thymidylate synthetase.
(12/594)
Cyanogen bromide treatment of thymidylate synthetase of Lactobacillus casei, which had been converted to a ternary complex with [2-14c] FdUMP and 5,10-methylene-tetrahydrofolate followed by S-carboxymethylation, yielded at least four visible peptide bands, the largest with a molecular weight of about 13,000, on polyacrylamide gel electrophoresis in sodium dodecyl sulfate-urea. Identical results were obtained with enzyme that had all four of its cysteinyl residues S-carboxymethylated with iodo [I-14C] acetate in the absence of FdUMP and cofactor. In each case, only the second band from the top of the gel (CN2), with an approximate molecular weight of 10,000= was labeled. Analysis of CN2 that had been labeled with [2-14C] FdUMP and nonradioactive iodoacetate and of that labeled only with iodo[1-14C] acetate revealed that their amino-acid contents were almost identical except for the presence of two S-carboxymethyl (Cm)-cysteinyl residues in the latter peptide and only one in FdUMP-CN2. A nonapeptide was isolated from (Cm)2-CN2 after chymotrypsin digestion that contained the following sequence by dansyl-Edman analysis: Ala-Leu-Pro-Pro-[Cm-Cys]-His-Thr-Leu-Tyr. This peptide was found to be located on the NH2-terminal end of CN2. Automatic sequence analysis of the first 13 residues of (Cm)2-CN2 and of the FdUMP-containing CN2 yielded identical results except for the fifth, or cysteinyl, residue, which could not be identified in the latter peptide. These findings strongly suggest that FdUMP is linked to a cysteinyl residue in thymidylate synthetase that has been inactivated irreversibly by this nucleotide. (+info)
Immunochemical characterization of the anti-RNA antibodies found in scleroderma and systemic lupus erythematosus. II. Reactivity with hsa-coupled, uridine-containing, monophosphoric ribodinucleotides.
(13/594)
Sera from patients with scleroderma have been found to have anti-RNA antibodies which react with human serum albumin (HSA)-coupled uridine and uridine monophosphate (UMP) and are inhibited by uracil, uridine and UMP. Scleroderma sera react uniformly with 5'-polyuridylic acid (poly(U)) and fail to react with polyadenylic, polyuridylic acid poly(A) - poly(U)) which is also indicative of their uracil specificity. Anti-RNA antibodies found in systemic lupus erythematosus (SLE) are immunochemically different from those found in scleroderma in that, instead of being uniformly specific to uracil, they are markedly heterogeneous and may react with uracil, uridine and/or UMP. SLE sera frequently react with poly(A) - poly(U), indicating also their ability to recognize the double helical structure of double-stranded RNA. Thirty-seven scleroderma and thirty-four SLE sera from as many patients with either of these conditions were tested against HSA-coupled, uridine-containing monophosphoric dinucleotides in an attempt to characterize further their anti-RNA antibodies. Scleroderma sera were found to react primarily with dinucleotides in which uridine was the base proximal to the carrier protein and, except for sera that also contained antibodies to adenosine which reacted with UpA, they failed to react with dinucleotides in which uridine was in a terminal position only. Reaction with dinucleotides in which uridine was proximal to the carrier protein could be inhibited by uracil but not by the corresponding terminal base. Some lupus sera were found to react with both dinucleotides that contain the same bases in opposite sequence, e.g. ApU and UpA, while others were found to react with only one of the sequences. They were also found to react more frequently with dinucleotides in which HSA was coupled to a base other than uridine, suggesting that the reaction is primarily due to anti-DNA antibodies. Because immunization with dinucleotides coupled to protein prepared by the same method we have used, yields higher specificity to the base attached to the carrier protein, our findings suggest that, in scleroderma, a single event, akin to that of immunization with a purified antigen, gives rise to the anti-RNA antibodies, whereas in systemic lupus erythematosus there is a considerably wider immunological aberration. (+info)
Nucleotide pools and regulation of ribonucleic acid synthesis in yeast.
(14/594)
Nucleotide pools were measured in growing and amino acid-starved Saccharomyces cerevisiae. During amino acid starvation there are neither significant changes in the endogeneous nucleoside triphosphate pool levels nor measurable synthesis of guanosine 5'-diphosphate, 3'-diphosphate. Stable ribonucleic acid synthesis does not appear to be regulated by changes in the triphosphate pools or by the unusual nucleotide guanosine 5'-diphosphate, 3'-diphosphate. (+info)
Properties of ATP and UTP analogues with P-S-C-5' bonds.
(15/594)
Analogues of ATP and UTP bearing C-5'-S-P ester bonds were found not to be substrates but weak competitive inhibitors of Escherichia coli RNA polymerase. The K-i values of the analogues obtained in the transcription of poly[d(A-T)] or poly(dT) under various conditions are in the order of millimolar. Evidence was derived from proton magnetic resonance spectra that nucleotides with C-5'-S-P bonds do not exist in gauche-gauche conformation normally adopted by natural occurring nucleotides. This leads us to assume that the gauche-gauche conformation is an essental prerequisite for substrates of RNA polymerase. Ado-5'-S-PPP substituted for ATP as substrate of hexokinase from yeast rather effectively thus indicating that a distinct stereochemical orientation of the alpha-phosphate ester bond is not a stringent requirement for substrates of this enzyme. (+info)
Synthesis of viral and rRNA in bacteriophage R17 infection of a stringent strain of Escherichia coli.
(16/594)
A previous paper (1973) indicated that infection with bacteriophage R17 permits the synthesis of RNA and spermidine in Escherichia coli (CP78 in the absence of the exogenous essential amino acid, arginine. We have now isolated RNA formed under such conditions and analyzed the newly synthesized species by agarose-acrylamide electrophoresis. It has been shown that infection of the stringent cells in the absence of exogenous arginine resulted in a marked incorporation of uracil into rRNA, as well as into R17 RNA. It was shown that, although the organism was nonauxotrophic for uracil, addition of [-14C]uracil resulted in the rapid formation of TUP, the specific radioactivity of which approached that of the exogenous uracil. This indicated that the incorporation of exogenous uracil into rRNA in R17 infection of the stringent strain reflected a true stimulated synthesis of this nucleic acid. Infection of the essentially isogenic relaxed strain, CP79, under the same conditions inhibited the RNA synthesis to a much less extent than the inhibition caused during the normal infection. These observations provide another example of the close correlation between synthesis of spermidine and of host RNA, even in cells infected by an RNA bacteriophage. (+info)