Regulation of transcription of the mph(A) gene for macrolide 2'-phosphotransferase I in Escherichia coli: characterization of the regulatory gene mphR(A).

The synthesis of macrolide 2'-phosphotransferase I [Mph(A)], which inactivates erythromycin, is inducible by erythromycin. The expression of high-level resistance to erythromycin requires the mph(A) and mrx genes, which encode Mph(A) and an unidentified protein, respectively. We have studied the mphR(A) gene, which regulates the inducible expression of mph(A). An analysis of the synthesis of Mph(A) in minicells and results of a complementation test indicated that mphR(A) is located downstream from mrx and that its product, MphR(A), represses the production of Mph(A). DNA sequencing indicated that the mph(A), mrx, and mphR(A) genes exist as a cluster that begins with mph(A) and that the deduced amino acid sequence of MphR(A) can adopt an alpha-helix-turn-alpha-helix structure. To study the regulation of gene expression by MphR(A), we performed Northern blotting and primer extension. A transcript of 2. 9 kb that corresponded to the transcript of mph(A) through mphR(A) was detected, and its level was elevated upon exposure of cells to erythromycin. Gel mobility shift assays and DNase I footprinting indicated that MphR(A) binds specifically to the promoter region of mph(A), and the amount of DNA shifted as a results of the binding of MphR(A) decreased as the concentration of erythromycin was increased. These results indicate that transcription of the mph(A)-mrx-mphR(A) operon is negatively regulated by the binding of a repressor protein, MphR(A), to the promoter of the mph(A) gene and is activated upon inhibition of binding of MphR(A) to the promoter in the presence of erythromycin.

Suppression of apoptotic cell death progressed in vitro with incubation of the chorion laeve tissues of human fetal membrane by glucocorticoid.

In the trophoblast layer of the chorion laeve of human fetal membranes obtained by cesarean section at the month of normal parturition, cells with condensed nuclei could be observed by histochemical examination. Incubating fetal membranes at 37 degrees C in vitro in cultivation medium, the frequency of cells with condensed nuclei increased in the chorion laeve, associating with an increase in DNA fragmentation and the population of in situ TUNEL (terminal deoxynucleotidyl transferase (TdT)-mediated dUTP-X nick end labeling) staining-positive cells. The progressed apoptotic cell death in the chorion laeve in vitro was suppressed by incubation of the tissue in the presence of glucocorticoids, cortisone or hydrocortisone, which was also demonstrated by DNA fragmentation analysis and in situ TUNEL staining. These results reveal that a substantial proportion of trophoblast cells in the chorion laeve of human fetal membranes are induced to undergo apoptosis at the end of pregnancy, and that the apoptosis progresses rapidly in vitro as the incubation period increases. It is suggested that certain hormones such as glucocorticoid, may be related to the regulation of the apoptosis in human fetal membranes.

Testicular malignant lymphoma; imaging and diagnosis.

The sonographic and magnetic resonance imaging findings of three cases of surgically proven malignant lymphoma of the testis were reviewed and their diagnostic implications were evaluated. In all three cases ultrasonography showed striated hypoechoic bands with paralleling hyperechoic lines radiating peripherally from the mediastinum testis. We believe they represented blood vessels. On T1-weighted MR images, the tumor showed homogenous hyperintensity in one case and homogenous isointensity in the other two. On T2-weighted images the tumor showed homogenous hypointensity in two cases and homogenous isointensity in one. After Gd-DTPA administration, the tumor showed homogenous enhancement in two cases and inhomogenous enhancement in one. In two cases there were enhancing striated bands in the tumor. Despite the diffuse infiltration by the tumor, the basic architecture of the testis, epididymis, and spermatic cord was preserved. When MRI and ultrasonography show an infiltrative disease preserving the basic architecture of the testis and ultrasonography presents hypoechoic bands radiating peripherally from the mediastinum testis, a diagnosis of malignant lymphoma should be considered.

Nucleotide sequence and characterization of erythromycin resistance determinant that encodes macrolide 2'-phosphotransferase I in Escherichia coli.

The DNA fragment (3.3 kb) containing the erythromycin resistance determinant was cloned from Escherichia coli Tf481A and sequenced. Deletion and complementation analyses indicated that the expression of high-level resistance to erythromycin requires two genes, mphA and mrx, which encode macrolide 2'-phosphotransferase I and an unidentified hydrophobic protein, respectively.

Expression in Escherichia coli of a TetK determinant from Staphylococcus aureus.

The expression in Escherichia coli of a tet(K) gene, which originated in Staphylococcus aureus, was studied. The minimum inhibitory concentration (MIC) of tetracycline (TC) for E. coli cells that carried the tet(K) gene was only slightly higher than that for the recipient cells. This result indicated that the level of expression of the tet(K) gene in E. coli was very low. Insertion of a lac promoter into the upstream region of the tet(K) gene resulted in a slight increase in the MIC, from 12.5 to 50 micrograms/ml in the presence of isopropyl-beta-D-thiogalactopyronoside. An altered tet(K) gene, in which the initiation codon and the ribosome-binding site (RBS) were changed from TTG to ATG and from GAGG to GGAGG, respectively, and in which the distance between the RBS and the initiation codon was increased from 4 to 11 bases, was associated with high-level resistance to TC, with a MIC of 200 micrograms/ml. The MIC resembles that associated with expression of the tetA(B) gene of E. coli. These results indicate that the barrier to expression of the tet(K) gene in E. coli is located at the initiation of translation.

A case of Behçet's disease with Weber's syndrome.

A case of Behçet's disease with right hemiplegia and left oculomotor nerve involvement is described. Computed tomography of the brain with or without contrast, and nuclear magnetic resonance demonstrated a low attenuation mass lesion in the right mid brain. Lumbar puncture revealed there was moderate leukocytosis in the cerebrospinal fluid. Corticosteroids seemed effective in reducing her clinical symptoms, and our patient started walking one week after taking 50 mg of prednisolone. This case of neuro-Behçet's disease is the first with Weber's syndrome with documentation of the lesion by means of nuclear magnetic resonance.