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Isoelectric focusing and molecular hybridization with a TEM DNA probe were used to screen for TEM P-lactamase in 328 bacterial isolates representing 11 gram-negative genera. The TEM enzyme was detected in 50% of isolates, and nine additional types of I-lactamase could be identified in 36.9% of isolates. The TEM gene was detected in 53.6% of isolates. The results obtained by both methods were concordant in 92.7% of the entire sample. In situ colony hybridization with a specific probe therefore appears to be a convenient method to screen rapidly for the presence of homologous genetic sequences among a large number of isolates. Positive hybridization was observed for 16 isolates in which no TEM I8-lactamase was detected by isoelectric focusing. The significance of this hybridization remains to be determined.1-Lactamases are the major determinants of resistance to 1-lactam antibiotics by most gram-negative bacteria (30).The need to demonstrate 3-lactamase production by organisms isolated in the clinical laboratory has become increasingly important in recent years with the emergence of new 3-lactamase-producing strains and new types of ,-lactamases (26). These enzymes can be characterized and classified into a variety of types by several criteria (24, 25), including substrate profiles, inhibition assays, molecular weight, and particularly isoelectric point (23). The number of plasmid-determined 1-lactamases is currently greater than 20 (22,26,37). New screening methods will be useful for the examination of the P-lactamase profile and for studies of the evolution of resistance mechanisms. New methodologies such as amino acid sequencing, molecular cloning, and nucleic acid hybridization are expected to allow appropriate and easier classification of ,B-lactamases. Amino acid sequencing has already yielded a differentiation of Plactamases into only three classes (1). Molecular cloning of the structural genes that encode various P-lactamases has been completed in many cases. Because some of these genes do not show significant homology, it was predicted in 1983 that DNA hybridization of ,B-lactamase genes will be a new tool for the molecular epidemiology of antibiotic resistance (32 MATERIALS AND METHODSBacterial strains. Gram-negative bacteria (328 strains) were isolated from different patients in the Besancon Hospital within 3 months (from May to July 1985). They belonged to the following 11 genera: Escherichia (96 strains), Proteus (60 strains), Klebsiella (41 strains), Providencia (33 strains), Serratia (27 strains), Acinetobacter (24 strains), Pseudomonas (23 strains), Enterobacter (12 strains), Citrobacter (7 strains), Hafnia (4 strains), and Achromobacter (1 strain).The MIC was determined by a disk diffusion method. The Pseudomonas strains were resistant to ticarcillin (MIC, >128 p,g/ml), and all the other strains were resistant to ampicillin (MIC, >16 ,ug/ml).Reference bacterial strains, which were representative of 11 3-lactamase classes, were kindly supplied by several investigators. They are listed in Table 1. ...

The c-fos gene expression is rapidly induced by various mitogenic agents and protein synthesis inhibitors in many cell types. Estradiol-17 beta can induce c-fos gene expression in breast cancer cell lines and in the uterus in vivo, but not in cultured guinea-pig endometrial cells. Using this model, we investigated whether a protein synthesis inhibitor, cycloheximide, could induce the c-fos gene and permit a superinduction by estrogens. In the presence of cycloheximide (10 micrograms/ml), protein synthesis was inhibited at 95% within the first hour. From 190 min after the addition of estradiol-17 beta or diethylstilbestrol (10(-8) M) and cycloheximide (10 micrograms/ml), there was a significant increase (ranging from 3- to 5-fold) of the c-fos messenger RNA level (2.2 kilobase in size), compared with the level in cells treated with cycloheximide alone. Nonestrogenic steroid hormones and estradiol-17 alpha were unable to induce c-fos gene expression in the presence of cycloheximide. The effect of estradiol-17 beta observed in the presence of cycloheximide was completely abolished by 4-hydroxy-tamoxifen or by Ly 156758 or by ICI 164384 (10(-6) M). The c-fos mRNAs were rather stable in cells treated with cycloheximide for 2 h (half-life = 51 +/- 6 min) and there was no further increase in the c-fos messenger RNA stability after the addition of cycloheximide plus estradiol-17 beta (half-life = 40 +/- 3 min). The overall results suggest a response at the transcriptional level. In conclusion, cycloheximide transmits activating signals to the c-fos gene which act as priming elements to allow the estrogen action in cultured guinea-pig endometrial cells.