EmtA, a rRNA methyltransferase conferring high-level evernimicin resistance.

Enterococcus faecium strain 9631355 was isolated from animal sources on the basis of its resistance to the growth promotant avilamycin. The strain also exhibited high-level resistance to evernimicin, a drug undergoing evaluation as a therapeutic agent in humans. Ribosomes from strain 9631355 exhibited a dramatic reduction in evernimicin binding, shown by both cell-free translation assays and direct-binding assays. The resistance determinant was cloned from strain 9631355; sequence alignments suggested it was a methyltransferase and therefore it was designated emtA for evernimicin methyltransferase. Evernimicin resistance was transmissible and emtA was localized to a plasmid-borne insertion element. Purified EmtA methylated 50S subunits from an evernimicin-sensitive strain 30-fold more efficiently than those from a resistant strain. Reverse transcription identified a pause site that was unique to the 23S rRNA extracted from resistant ribosomes. The pause corresponded to methylation of residue G2470 (Escherichia coli numbering). RNA footprinting revealed that G2470 is located within the evernimicin-binding site on the ribosome, thus providing an explanation for the reduced binding of the drug to methylated ribosomes.

Inactivation of p42 mitogen-activated protein kinase is required for exit from M-phase after cyclin destruction.

By using cycling Xenopus egg extracts, we have previously found that if mitogen-activated protein kinase (p42 MAPK) is activated on entry into mitosis (M-phase), the extract is arrested with condensed chromosomes and spindle microtubules. Here we show that these arrested extracts have high levels of M-phase promoting factor (MPF, Cyclin B/Cdc2) activity, stabilized levels of Cyclin B, and sustained M-phase-specific phosphorylations. We also examined the role of p42 MAPK in DNA damage checkpoint-arrested extracts that were induced to enter M-phase by the addition of Cdc25C protein. In these extracts, Cdc25C protein triggers the abrupt, premature activation of MPF and entry into M-phase. MPF activity then drops suddenly due to Cyclin B proteolysis, just as p42 MAPK is activated. Unexpectedly, however, M-phase is sustained, as judged by maintenance of M-phase-specific phosphorylations and condensed chromosomes. To determine if this M-phase arrest depended on p42 MAPK activation, we added PD98059 (PD), an inhibitor of p42 MAPK activation, to egg extracts with exogenous Cdc25. Both untreated and PD-treated extracts entered M-phase simultaneously, with a sharp peak of MPF activity. However, only PD-treated extracts subsequently exited from M-phase and entered interphase. In PD-treated extracts, p42 MAPK was not activated, and the transition to interphase was accompanied by the formation of decondensed nuclei and the disappearance of M-phase-specific phosphorylation of proteins. These results show that although entry into M-phase requires the activation of MPF, exit from M-phase even after cyclin destruction, is dependent on the inactivation of p42 MAPK.

Activation of the p42 mitogen-activated protein kinase pathway inhibits Cdc2 activation and entry into M-phase in cycling Xenopus egg extracts.

We have added constitutively active MAP kinase/ERK kinase (MEK), an activator of the mitogen-activated protein kinase (MAPK) signaling pathway, to cycling Xenopus egg extracts at various times during the cell cycle. p42MAPK activation during entry into M-phase arrested the cell cycle in metaphase, as has been shown previously. Unexpectedly, p42MAPK activation during interphase inhibited entry into M-phase. In these interphase-arrested extracts, H1 kinase activity remained low, Cdc2 was tyrosine phosphorylated, and nuclei continued to enlarge. The interphase arrest was overcome by recombinant cyclin B. In other experiments, p42MAPK activation by MEK or by Mos inhibited Cdc2 activation by cyclin B. PD098059, a specific inhibitor of MEK, blocked the effects of MEK(QP) and Mos. Mos-induced activation of p42MAPK did not inhibit DNA replication. These results indicate that, in addition to the established role of p42MAPK activation in M-phase arrest, the inappropriate activation of p42MAPK during interphase prevents normal entry into M-phase.

Mos-induced p42 mitogen-activated protein kinase activation stabilizes M-phase in Xenopus egg extracts after cyclin destruction.

Previously, we have shown that the addition of a constitutively-active mitogen-activated protein kinase kinase protein (MAPKK = MEK) to cycling Xenopus egg extracts activates the p42MAPK pathway, leading to a G2 or M-phase cell cycle arrest. The stage of the arrest depends on the timing of p42MAPK activation. If p42MAPK is activated prior to M-phase, or after exit from M-phase, the extract is arrested in G2. If p42MAPK is activated during entry into M-phase, the extract is arrested in M-phase. In this study, we show that the addition of recombinant Mos protein (which directly phosphorylates and activates MEK) to cycling egg extracts has the same effect as those described for MEK. The addition of Mos to the extract at the start of incubation leads to a G2 arrest with large interphase nuclei with intact nuclear envelopes. If Mos is added at later times, however, the activation of p42MAPK leads to an M-phase arrest with condensed chromosomes and mitotic arrays of microtubules. Moreover, the extent of M-phase specific phosphorylations is shown by the sustained presence of phosphoproteins that are detected by the monoclonal antibody MPM-2. Unexpectedly, in certain M-phase arrested extracts, histone H1 kinase activity levels reach a peak on entry into M-phase but then fall abruptly to interphase levels. When these extracts are analyzed by immunoblotting, Cyclin B2 is destroyed in those samples containing low maturation promoting factor activity (MPF, cyclin B/Cdc2), yet chromosomes remain condensed with associated mitotic arrays of microtubules and M-phase-specific phosphorylations are sustained. These results suggest that although MPF is required for entry into M-phase, once established, M-phase can be maintained by the p42MAPK pathway after the proteolysis of mitotic cyclins.

Cloning and nucleotide sequence of the gyrA gene from Campylobacter fetus subsp. fetus ATCC 27374 and characterization of ciprofloxacin-resistant laboratory and clinical isolates.

The gyrA gene of Campylobacter fetus subsp. fetus, which encodes the A subunit of DNA gyrase, was cloned, and its nucleotide sequence was determined. An open reading frame of 2,586 nucleotides which encodes a polypeptide of 862 amino acids with an Mr of 96,782 was identified. C. fetus subsp. fetus GyrA is most closely related to Campylobacter jejuni GyrA, with 73% homology at the nucleotide level and 78% identity between polypeptides. The next most closely related GyrA was that from Helicobacter pylori, with both DNA homology and amino acid identity of 63%. The gyrA and gyrB (DNA gyrase B subunit) genes were located on the genomic map of C. fetus subsp. fetus ATCC 27374 and shown to be separate. A clinical isolate of C. fetus subsp. fetus and a laboratory-derived mutant of ATCC 27374, both resistant to ciprofloxacin, had identical mutations within the quinolone resistance determining region. In both mutants a G-->T transversion, corresponding to a substitution of Asp-91 to Tyr in GyrA, was linked to ciprofloxacin resistance, giving MICs of 8 to 16 micrograms/ml.

Effect of naturally occurring visual acuity differences between two eyes in stereoacuity.

Stereoacuity was measured in 30 subjects with a naturally occurring visual acuity (VA) difference between the eyes. The stereoacuity was measured by a modified Howard's apparatus using the staircase method and VA was measured with log MAR charts. Stereoacuity was worse in subjects with a large VA difference between the two eyes; the correlation between stereoacuity and VA difference was significant (r = 0.76, P < 0.001). Neither the VA of the worse eye nor of the better eye contributed to the reduction in stereoacuity. The deterioration was more obvious if VA difference between the two eyes was one line or more (correlation coefficient, r= 0.88, P < 0.001). This study also reinforces the use of a > or = 70% stereothreshold when attempt stereoacuity results to compare with other studies.

An analysis of multiple congenital anomaly syndromes in a Chinese population.

During an eight-year period, 4,890 index patients and their families were referred to the Clinical Genetic Service. Of the probands, 2,096 were diagnosed as suffering from multiple congenital anomaly syndromes. These represented 42.9% of the total. Ten commonest conditions accounted for 50% of patients suffering from this category of genetic diseases. A multidisciplinary approach was emphasized in the diagnosis and management.

Solvent effects on response factors for polynuclear aromatic hydrocarbons determined by capillary gas chromatography using splitless injections.

When standard solutions of polynuclear aromatic hydrocarbons (PAHs) were analyzed by capillary column gas chromatography using splitless injections, response factors were observed to be dependent on the solvent used to prepare the standard. This report presents the response factors for 16 individual PAHs in 5 commonly used solvents: acetonitrile, methanol, toluene, isooctane, and cyclohexane. To minimize quantitation errors due to differences in transfer efficiency, samples and standards of PAHs should be prepared in the same solvent.

Chemical derivatization analysis of pesticide residues. IX. Analysis of phenol and 21 chlorinated phenols in natural waters by formation of pentafluorobenzyl ether derivatives.

A sensitive, isomer-specific method is described for the simultaneous and quantitative analysis of 22 phenols (phenol, 18 chlorophenols, and 3 chloroalkylphenols) in natural waters. The sample was acidified to pH less than or equal to 2, extracted with dichloromethane, evaporated, and dissolved in acetone. The phenol extract was then reacted with pentafluorobenzyl bromide (PFBBr) to give the PFB ether derivatives. After silica gel column cleanup, the ethers were chromatographed on a 12 m OV-1 fused silica capillary column attached to an electron capture detector (ECD). The detection limit was 0.1 ppb for 1 L samples. Recoveries of phenols from pH 2 water samples fortified at 10, 1, and 0.1 ppb were greater than or equal to 80% in most cases except for phenol which was only 30 to 35% recovered. Coefficients of variation were between 2 and 10% for all phenols. However, phenol recovery was quantitative when the sample volume was reduced to 100 mL. Because ECD sensitivities to the 22 phenol PFB ethers were similar, this method is most suitable for simultaneous screening of nonchlorinated and monochlorinated phenols as well as other higher chlorophenols at trace levels.

Chemical derivatization analysis of pesticide residues. VIII. Analysis of 15 chlorophenols in natural water by in situ acetylation.

A rapid analytical method is presented for quantitative analysis of 15 chlorophenols in natural waters by in situ acetylation. In the presence of KHCO3, phenols in water are acetylated by acetic anhydride directly without pre-extraction. The resultant acetates are extracted by petroleum ether and analyzed by electron capture gas chromatography. The investigation to optimize the conditions for in situ acetylation of these phenols is also described. This method has been validated and shown to be applicable over a range from 100 to 0.01 ppb with a 1 L water sample.

Analysis of pesticide residues by chemical derivatization. V. Multiresidue analysis of eight phenoxyalkanoic acid herbicides in natural waters.

A multiresidue method is presented for the gas-liquid chromatographic determination of phenoxyalkanoic acid herbicides in natural waters. Extraction efficiencies of different organic solvents are considered in developing a solvent extraction scheme for these herbicides from water. Reactions for derivatizing these compounds by using pentafluorobenzyl bromide, boron trichloride-2-chloroethanol, and dicyclohexylcarbodiimide-2-chloroethanol were studied in order to obtain extracts with low blanks to provide the lowest detection limits. Advantages and disadvantages of the 3 methods are discussed. Retention times are twice as long for the pentafluorobenzyl (PFB) esters as for the 2-chloroethyl (2-Cl) esters under the same conditions, although electron capture sensitivity to the former was greater. The PFB esters are easier to form, but the 2-Cl reaction is more specific for these herbicides. Solutions from the boron trichloride reaction gave the cleanest blanks.

Interlaboratory quality study of the analysis of water for pesticides.

An interlaboratory quality control study was conducted to provide information to 5 participating Canadian laboratories on their capability in analyzing natural waters for 10 pesticides. The study was designed to identify the quality of each laboratory's working standards for 15 organochlorine pesticides, and the accuracy and precision of each laboratory's analytical procedures. Fifteen samples were provided, of which 3 were standards prepared for direct injection into an electron capture gas chromatograph. Instructions were provided to use these 3 standards for quantitating the pesticide content (8-150 ng/L) in 5 pairs of 1 L water samples. These paired samples contained 10 organochlorine pesticides approaching natural levels. Results of the study indicated that participants were fairly precise and achieved the designed ratio 1.5 for concentrations in the paired samples.

A study of different analytical extraction methods for nondetrital heavy metals in aquatic sediments.

4.0 N HNO3-0.7 N HCl, 0.5 N HCl, 1 N hydroxylamine hydrochloride-25% acetic acid and 0.05 N EDTA were investiagted as extractants of nondetrital heavy metals from the sediments of the Rideau River. A comparison of these partial extraction methods with a total extraction method, showed that the 4.0 N HNO3-0.7 N HCl attacks the silicate crystal lattice considerably. 1 N hydroxylamine hydrochloride-25% acetic acid was found to be unsuitable for copper extraction. Both of the other two methods were suitable for the simultaneous extraction of Cd, Cu, Pb, Zn, Ni, Cr, Co, Mn, Fe, and Al from the organic adsorbed and precipitated phases of sediments. They both gave a measure of nondetrital heavy metals in sediments and are thus useful to environmental contamination studies.

Gas-liquid chromatographic determination of 3-trifluoromethyl-4-nitrophenol in natural waters.

A procedure for the analysis of 3-trifluoromethyl-4-nitrophenol (TFM) in natural waters is described. The lampricide is extracted from acidified water samples on the macroreticular resin XAD-7 and eluted from the column with ethyl ether. The ether extract is dried, concentrated, and partitioned with potassium carbonate. TFM is acetylated in the aqueous alkaline solution and the acetate derivative is extracted into benzene for analysis by electron capture gas-liquid chromatography. Recoveries of TFM from natural waters exceeded 90% and as little as 0.01 mug TFM can be quantitated in a 1 L sample.