Asparaginase inhibition of adhesion of type 1-fimbriated and P-fimbriated Escherichia coli to epithelial cell receptors.

The 3D structures of Fim H and PapG proteins complexed with the host carbohydrate receptor demonstrate that both utilize binding-pocket asparagines for contact or stabilization with the carbohydrate. Pretreatment of whole bacteria with asparaginase resulted in decreased fimbriae-mediated attachment to urinary epithelial cells. Enzyme treatment of bacteria pre-adhered to epithelial cells removed more uropathogenic E. coli than the indigenous flora attached to them.

Peripheral transcatheter embolization with platinum microcoils.

Embolotherapy with platinum microcoils delivered through the Tracker-18 microcatheter (Target Therapeutics, San Jose, Calif) was performed in 16 patients when peripheral superselective catheterization with standard angiographic catheters was not possible. The Tracker-18 catheter could be directed distally into small peripheral vessels for delivery of the microcoils. These microcoils, with attached fiber strands, resulted in vascular occlusion within a few minutes in all cases. Embolotherapy was technically successful in all patients. The procedures were clinically successful in 15 of 16 patients (94%), and no complications were encountered. Embolization with platinum microcoils through the Tracker-18 catheter is useful when standard methods of embolization are not possible.

Vaginal reconstruction with rectus abdominis myocutaneous flap: CT findings.

The distally based rectus abdominis myocutaneous flap is used in a new technique for vaginal reconstruction after pelvic exenteration for malignant disease. We identified 27 patients who underwent this procedure, of whom eight had a total of 14 postoperative CT scans and two CT-directed biopsies. The myocutaneous flap appeared as a unilateral arcuate band of soft tissue extending from the linea alba to the rectal fascia or sacrum. Additional CT findings included asymmetric thinning of the ventral abdominal wall (7/8), fluid collections (2/8), vaginal breakdown (1/8), presacral soft-tissue thickening (6/8), and tumor recurrence (3/8). The postoperative CT scan reflects the altered anatomy produced by the surgery. Complications and recurrent disease can be recognized as deviations from the normal postoperative appearance.

3' noncoding region of phosphoenolpyruvate carboxykinase mRNA contains a glucocorticoid-responsive mRNA-stabilizing element.

The stabilization of phosphoenolpyruvate carboxykinase mRNA by glucocorticoids appears to result from the interaction of an induced factor with an RNA element located in the 3' noncoding sequence of the mRNA. This element can confer glucocorticoid-dependent stabilization upon a heterologous mRNA, and thus strategies developed to investigate the control of mRNA transcription can now be applied to the analysis of hormone-regulated mRNA stabilization.

Regulation of phosphoenolpyruvate carboxykinase gene transcription in H4IIE hepatoma cells: evidence for a primary role of the catalytic subunit of 3',5'-cyclic adenosine monophosphate-dependent protein kinase.

The purpose of these studies was to determine whether the catalytic subunit of cAMP-dependent protein kinase is involved in the regulation of P-enolpyruvate carboxykinase (PEPCK) gene transcription. Cyclic AMP analog pairs that preferentially stimulate either type I or type II protein kinase in a synergistic manner were used to compare regulation of mRNAPEPCK synthesis in H4IIE rat hepatoma cells with protein kinase activation in vitro. Type II protein kinase is predominant in H4IIE cells and analog pairs directed toward this isozyme resulted in a synergistic increase of mRNAPEPCK that was due to a corresponding enhancement of PEPCK gene transcription. When compared to a single analog the addition of a type II-directed analog pair reduced the total analog concentration required for maximal induction of transcription by about 30-fold. H4IIE cells have a small amount of type I kinase; pairs specific for this form of the enzyme were also effective, but to a lesser extent than those for the type II kinase. (Rp)-cAMPS, a cyclic nucleotide-dependent protein kinase antagonist, inhibited the agonist-induced increase of mRNAPEPCK in a concentration-dependent manner. The results indicate that the activation of PEPCK gene transcription by cAMP in H4IIE cells is mediated by cAMP-dependent protein kinase. Although the type II isozyme is primarily responsible, type I is also effective. These isozymes have identical catalytic subunits, hence this component presumably mediates the cAMP effect.

Insulin decreases H4IIE cell PEPCK mRNA by a mechanism that does not involve cAMP.

Insulin is thought to influence some metabolic events by decreasing the intracellular concentration of cyclic AMP (cAMP). To test whether this explains the repression of hepatic phosphoenolpyruvate carboxykinase (PEPCK) by insulin we measured intracellular cAMP, cAMP-dependent protein kinase, mRNAPEPCK, and PEPCK gene transcription in cultured Reuber H4IIE hepatoma cells treated with forskolin with and without insulin. In untreated cells, the concentration of cAMP was 2.9 pmol/mg of protein. Forskolin at 1, 10, and 50 microM increased the level of cAMP to 9.2, 35.8, and 115 pmol/mg of protein, respectively; 5 nM insulin had no significant effect on these cAMP concentrations. In untreated cells, the activity ratio of cAMP-dependent protein kinase was 0.43, and 50 microM forskolin increased this to 0.96; insulin had no effect on this ratio at times from 15-180 min. In untreated cells mRNAPEPCK bound 15 cpm of a 32P-labeled cDNA probe per microgram of total cellular RNA. Forskolin, at 1, 10, and 50 microM increased this to 48, 96, and 115 cpm/microgram RNA. Insulin (5 nM), in combination with 0, 1, 10, and 50 microM forskolin, decreased the concentration of mRNAPEPCK to 5, 8, 23, and 29 cpm/micrograms RNA, respectively. Finally, the rate of transcription of the PEPCK gene was 85, 168, 630, 823, and 884 parts per million (ppm) in H4IIE cells treated for 30 min with 0, 1, 5, 10, and 50 microM forskolin, respectively, while the corresponding rates in the presence of 5 nM insulin were 49, 45, 84, 85, and 136 ppm.(ABSTRACT TRUNCATED AT 250 WORDS)

Multihormonal regulation of phosphoenolpyruvate carboxykinase gene transcription. The dominant role of insulin.

We used a nuclear RNA transcript elongation assay to show that cAMP analogs and dexamethasone cause a selective increase of transcription of the P-enolpyruvate carboxykinase gene in H4IIE hepatoma cells. 8-(4-chlorophenylthio)-cAMP increased transcription within 5 min and the maximal rate, generally 10-15-fold above the basal rate, was attained by 30 min. This increase was of sufficient magnitude to account for the effect on mRNAPEPCK (for example, where PEPCK is phosphoenolpyruvate carboxykinase) accumulation. After the initial increase, and with continued presence of cAMP, transcription of this gene declined to a new steady-state level which was 2-3 times the basal value. The effect of cAMP analogs on P-enolpyruvate carboxykinase gene transcription was obtained in the absence of protein synthesis. This, and the rapidity of the response, indicates that the effect of cAMP is exerted directly on the P-enolpyruvate carboxykinase gene. Dexamethasone results in a specific, 6-fold increase of transcription, sufficient to account for the increase of mRNAPEPCK which follows treatment of H4IIE cells with this glucocorticoid. When 1 nM insulin was added to either untreated H4IIE cells, or cells first treated with a cAMP analog or dexamethasone, there was a marked reduction of cytoplasmic mRNAPEPCK. The inhibitory effect of insulin was readily reversible, as cells regained the basal level of mRNAPEPCK and full responsiveness to cAMP within 1 h after removing insulin. The transcript elongation assay was used to show that insulin inhibits transcription of the gene coding for mRNAPEPCK. The concentration of insulin required for 50% inhibition was 2-5 pM, whereas approximately 200 pM of proinsulin was required to achieve the same inhibition of transcription. This effect was specific, since insulin did not affect the synthesis of total RNA; it was rapid, as 5 nM insulin decreased the rate of P-enolpyruvate carboxykinase gene transcription by 50% within 15 min; and it also does not require ongoing protein synthesis. The magnitude and kinetics of the response suggest that the primary action of insulin in the regulation of P-enolpyruvate carboxykinase synthesis is exerted at the level of mRNAPEPCK transcription. The insulin-mediated inhibition of mRNAPEPCK transcription was noted in untreated cells and in cells first treated with 8-(4-chlorophenylthio)-cAMP, dexamethasone, or both of these agents. Hence, among these compounds, insulin is the dominant regulatory molecule.