Monitoring dental sterilizers' effectiveness using biological indicators.

The purpose of this study was to evaluate the effectiveness of dental office sterilizers as measured by their ability to kill bacterial spores present on biological indicator strips. The biological indicators used in this study contained two different spores, Bacillus stearothermophilus and Bacillus subtilis (Spordi, AMSCO/Medical Products). Ten spore test strips were sent to 87 dental offices; 51 sterilizers were tested. Office personnel were instructed to place four strips in the center of a normal sterilization load and process the load. The procedure was repeated on a second day. The processed strips, along with two unprocessed control strips, were returned by mail for laboratory culturing. The results indicated the overall failure rate (positive test) of sterlizers tested for both days was 51% at the culturing temperature of 37 degrees C and 33.3% at 55 degrees C. McNemar's test indicated a significant difference (p less than .03) in sterilization failures associated with the type and number of microorganisms present on the test strips. This study also showed that the more times a sterilizer was tested, the more likely a failure would occur. Overall, an alarming number of sterilizers (64.7%) were not effective in killing all the spores present on the indicator strips. When office personnel were given information for improving sterilizer performance, there was a noticeable reduction in sterilization failures following retesting.

2'(3')-O-L-Phenylalanyl derivatives of N2,5'-anhydroformycin and N4,5'-anhydroformycin: new substrates for ribosomal peptidyltransferase with a fixed anti and syn conformation of the base.

The 2'(3')-O-L-phenylalanyl-N2,5'-anhydroformycin (1c) and 2'(3')-O-L-phenylalanyl-N4,5'-anhydroformycin (2c), obtained by chemical synthesis, are substrates for ribosomal peptidyltransferase from Escherichia coli. Nucleoside 1c, which mimics an anti conformation of antibiotic formycin, has 80% of the acceptor activity of puromycin at 5 x 10(-4) M determined by the release of N-Ac-Phe residue from the 70 S ribosome-poly(U)-N-Ac-[14C]Phe-tRNA complex. The reaction product, 2'(3')-O-(N-acetyl)-L-phenylalanyl-L-phenylalanyl-N2,5'-anhydroformyc in (1d), was characterized by paper electrophoresis before and after alkaline hydrolysis. By contrast, nucleoside 2c, which resembles a syn conformation of formycin, exhibited only 20% of the acceptor activity of puromycin at 5 x 10(-4) M. The results which are in accord with previous models have shown that a substrate with its base in an anti conformation is preferable for the acceptor site of peptidyltransferase than the corresponding syn counterpart. Nevertheless, it is possible that an intermediate conformation, for example, high anti (amphi-minus), is an optimal arrangement for acceptor site substrates.

Analogues of 2'(3')-O-L-phenylalanyladenosine as substrates and inhibitors of ribosomal peptidyltransferase.

The chemical syntheses of 2'(3')-O-(L-3-amino-3-phenylpropionyl)adenosine (2e), the corresponding D stereoisomer 2f, 2'(3')-O-(DL-phenylglycyl)adenosine (2g), 2'(3')-O-(N-benzylglycyl)adenosine (2h), and 9(2-O-L-phenylalanyl-beta-D-xylofuranosyl)adenine (3b) are described. Compounds 2e-h were obtained by acylation of 5'-O-(4-methoxytrityl)adenosine with the appropriate N-benzyloxycarbonyl or N-tert-butoxycarbonyl amino acids with dicyclohexylcarbodiimide in pyridine. The corresponding reaction of N-(benzyloxycarbonyl)-D-phenylglycine led to an almost complete racemization of the aminoacyl residue (compounds 2c and 2g). Subsequent chromatographic separation and deprotection of intermediates 2a-d afforded the desired target derivatives 2e-h. Product 3b was obtained by a similar acylation of 9-(3,5-O-isopropylidene-beta-D-xylofuranosyl)adenine with N-(benzyloxycarbonyl)-L-phenylalanine, followed by deblocking. The NMR spectra of 2' and 3' isomers of stereoisomers 2a and 2b are discussed. Compounds 2g and 3b are both substrates and inhibitors of Escherichia coli ribosomal peptidyltransferase, although the activity of 3b is low. Derivatives 2e,f,h do not accept AcPhe from N-AcPhe-tRNA in a peptidyltransferase-catalyzed reaction, but they inhibit the puromycin reaction in the same system. The order of inhibitory activity is 2e greater than 2f greater than 2h. The implications of these findings for the mechanism of peptidyltransferase and comparison of the latter with the action of chymotrypsin are discussed.

Effect of thiostrepton and 3'-terminal fragments of aminoacyl-tRNA on EF-Tu and ribosome-dependent GTP hydrolysis.

The effect of the antibiotics thiostrepton and micrococcin on EF-Tu-catalyzed (ribosome-dependent) GTP hydrolysis in the presence of A-Phe, C-A-Phe, or C-C-A-Phe (related to the sequence of the 3'-terminus of aminoacyl-tRNA)(System I) or by methanol ('uncoupled GTPase', System II) was investigated. In System I, thiostrepton increases the binding affinities of the effectors to the EF-Tu.GTP.70 S ribosome complex, as well as the extent of the GTP hydrolysis, while the KmGTP is virtually unchanged. Similarly, in the uncoupled system (System II) and in the absence of effectors, thiostrepton significantly increases VmaxGTP, whereas KmGTP remains unaffected. Micrococcin is without any effect in both systems. The 'uncoupled GTPase' (in System II) is also strongly inhibited by C-A-Phe. The results indicate the crucial role of the EF-Tu site which binds the aminoacylated C-C-A terminus of aminoacyl-tRNA in promoting GTP hydrolysis. It follows that the binding of the model effectors (such as C-C-A-Phe) to that site is favorably influenced by the interaction of thiostrepton with the 50 S ribosomal subunit, whereas thiostrepton, per se, does not influence the affinity of EF-Tu for GTP.

The peptidyltransferase center of Escherichia coli ribosomes: binding sites for the cytidine 3'-phosphate residues of the aminoacyl-tRNA 3'-terminus and the interrelationships between the acceptor and donor sites.

The substrate specificity of the acceptor site of peptidyltransferase of Escherichia coli 70 S ribosomes was investigated in Ac-Phe-tRNA . poly(U) . 70 S ribosome (system A) and tRNC-A-Phe . poly(U) . C-A-C-C-A-Phe . 70 S ribosome (system B) systems by using C-C-A-Gly, C-C-A-Phe, C-A-Gly and C-A-Phe as analogs of the 3'-terminus of aminoacyl-tRNA. It was found that an addition of CP residue to C-A-Gly and C-APhe resulted in an increase of the acceptor activity in system A; the increase is more remarkable for C-A-Gly than for C-A-Phe, while the acceptor activities of C-C-A-Gly and C-C-A-Phe are roughly similar. On the other hand, dramatically increased binding affinities of C-C-A-Phe and C-C-A-Gly relative to C-A-Phe and C-A-Gly for the A site of peptidyltransferase were observed in system B using an inhibition assay; C-C-A-Phe binds much more strongly than C-C-A-Gly. The results indicate the important role of the third CP residue and the aminoacyl moiety of the 3'-terminus of aminoacyl-tRNA in the interaction with the acceptor site of peptidyltransferase, as well as the existence of cooperative effects between A and P sites of peptidyltransferase. These effects, depending on an occupancy of P site, may significant the specificity of the peptidyltransferase A site.

Inhibition of the peptidyltransferase acceptor site by 2'(3')-O-cycloleucyl- and alpha-aminoisobutyryl derivatives of cytidylyl-(3'-5')adenosine.

2'(3')-O-(N-Benzyloxycarbonylcycloleucyl)adenosine (1a) was prepared by esterification of 5'-O-(4-methoxytrityl)adenosine with N-benzyloxycarbonylcycloleucine in the presence of dicyclohexylcarbodiimide and subsequent deprotection in acidic medium. The compound 1a was separated into pure 2'- and 3'-isomers using HPLC; these isomers were found to undergo an easy interconversion. Compound 1a was coupled with N-dimethylaminomethylene-2',5'-di-O-tetrahydropyranylcytidine 3'-phosphate in the presence of dicyclohexylcarbodiimide to give, after subsequent deblocking, cytidylyl(3' leads to 5')2'(3')-O-cycloleucyladenosine (1c). Compound 1c, as well as the related cytidylyl(3' leads to 5')2'(3')-O-(alpha-aminoisobutyryl)adenosine (1d), inhibited the peptidyltransferase catalyzed transfer of an AcPhe residue to puromycin in the Ac[14C]Phe-tRNA . poly(U) . 70 S E. coli ribosome system. A half of the maximum inhibition of AcPhe-puromycin formation (at 10(-5) M puromycin) was achieved at 9.5 . 10(-6) M of compound 1c and 9 . 10(-5) M of compound 1d, respectively. The inhibition of the puromycin reaction by compound 1d shows a mixed-type of inhibition kinetics. Further, none of the compounds 1c and 1d was an acceptor in the peptidyltransferase reaction. Both compounds 1c and 1d inhibited the binding of C-A-C-C-A[14C]Phe to the A site of peptidyltransferase in a system containing tRNAPhe . poly(U) . 70 S E. coli ribosomes, in which compound 1d was a much stronger inhibitor than 1c. These results indicate that the derivatives such as compounds 1c and 1d which contain an anomalous amino acid with a substituent in lieu of alpha-hydrogen can interfere with the peptidyltransferase A site; however, they are not acceptors in the peptidyltransferase reaction probably due to a misfit of the alpha-substituent.

Analogues of chloramphenicol: circular dichroism spectra, inhibition of ribosomal peptidyltransferase, and possible mechanism of action.

Circular dichroism spectra of series of chloramphenicol derivatives la-r were measured in water at pH 7. Compounds 1a-o exhibit two positive Cotton effects at 310--340 and 240--260 nm, respectively, and a weaker negative Cotton effect at 280--300 nm. In analogues 1c, 11, and 1m there is only a minimum between the two positive Cotton effects. Derivatives 1p--r possess a strong negative Cotton effect at ca. 280 nm. Compounds 1a--r were examined as inhibitors of the puromycin reaction with Escherichia coli 70S ribosome-poly(U)-N-AcPhe-tRNA complex. Analogues 11, 1n, lo, and lq are potent competitive inhibitors of puromycin comparable to or better than chloramphenicol (1b). Compounds 1k and 1m are less active, whereas 1d--g and 1j are only moderately effective. The rest of the analogues have marginal or no activity. The results are compared with previous biological data and discussed in terms of a retro-inverso relationship of chloramphenicol (1b) to the aminoacyl moiety of puromycin (aminoacyl-tRNA) and to a hypothetical transition state of peptide bond formation.

Chemical aminoacylation of transfer ribonucleic acid. The reaction of Escherichia coli tRNA with ethyl N-benzyloxycarbonylorthoglycinate.

The alpha-carbethoxypentadecyltrimethylammonium (Septonex) salt of tRNA (Ib) was condensed with ethyl N-benzyloxycarbonylorthoglycinate (II) in dimethylformamide in vacuo and in the presence of H3PO4 as catalyst. Pancreatic RNAase degradation and phenylalanine acceptor activity showed a 55--60% conversion to the 2',3'-cyclic orthoglycinate derivative of tRNA (IIIb). The orthoester grouping of IIIb was quantitatively hydrolyzed in 80% formic acid at 0 degrees C for 15 min to give 2'(3')-O-(N-benzyloxycarbonyl)glycyl tRNA (IVb). The latter was stripped at pH 8.8 to give tRNA whose behavior on DEAE cellulose column and gel electrophoresis was similar to that of starting tRNA. The phenylalanine acceptor activity amounted to almost 80% of the starting tRNA.