Mechanism of action of oxazolidinones: effects of linezolid and eperezolid on translation reactions.

The oxazolidinones are a new class of synthetic antibiotics with good activity against gram-positive pathogenic bacteria. Experiments with a susceptible Escherichia coli strain, UC6782, demonstrated that in vivo protein synthesis was inhibited by both eperezolid (formerly U-100592) and linezolid (formerly U-100766). Both linezolid and eperezolid were potent inhibitors of cell-free transcription-translation in E. coli, exhibiting 50% inhibitory concentrations (IC50s) of 1.8 and 2.5 microM, respectively. The ability to demonstrate inhibition of in vitro translation directed by phage MS2 RNA was greatly dependent upon the amount of RNA added to the assay. For eperezolid, 128 microg of RNA per ml produced an IC50 of 50 microM whereas a concentration of 32 microg/ml yielded an IC50 of 20 microM. Investigating lower RNA template concentrations in linezolid inhibition experiments revealed that 32 and 8 microg of MS2 phage RNA per ml produced IC50s of 24 and 15 microM, respectively. This phenomenon was shared by the translation initiation inhibitor kasugamycin but not by streptomycin. Neither oxazolidinone inhibited the formation of N-formylmethionyl-tRNA, elongation, or termination reactions of bacterial translation. The oxazolidinones appear to inhibit bacterial translation at the initiation phase of protein synthesis.

Estrogen sulfotransferase of the rat liver: complementary DNA cloning and age- and sex-specific regulation of messenger RNA.

Mammalian estrogen sulfotransferase (EST; EC 2.8.2.4) sulfurylates the hydroxyl group of estrogenic steroids by transferring the sulfate from a cosubstrate adenosine 3'-phosphate-5'-phosphosulfate. Sulfurylated steroids do not bind to the estrogen receptor with high affinity and, therefore, are hormonally inactive. We have purified rat liver EST and developed monoclonal antibody to this enzyme. By immunoscreening a lambda gt-11 expression library constructed from male rat liver cDNAs, the cDNA clone corresponding to EST was identified and isolated. A recombinant expression plasmid (pCMV5) containing this cDNA insert when transfected into COS-7 cells generated both immunologically and enzymatically active EST. With the help of this cDNA probe, we have explored the regulation of the EST mRNA in the liver and the possible role of this enzyme in sex hormone action. During the lifespan of male rats, only the young adult animals show hepatic androgen responsiveness. Also, estrogenic hormones strongly antagonize androgen action in the rat liver. Northern blot analysis of liver RNA derived from male rats of different ages shows that the androgen sensitivity of young adult animals is associated with a high expression of EST mRNA. During the same period, mRNA corresponding to dehydroepiandrosterone sulfotransferase is markedly (approximately 10-fold) down-regulated. Such a correlation is in concordance with the role of these enzymes in the maintenance of hepatic androgen sensitivity during young adult life by inactivating the estrogenic and sparing the androgenic steroids. Furthermore, the increase in the hepatic androgen sensitivity of androgen-treated female rats is also associated with the induction of EST.(ABSTRACT TRUNCATED AT 250 WORDS)

Androgen receptor messenger ribonucleic acid (mRNA) in the rat liver: changes in mRNA levels during maturation, aging, and calorie restriction.

By means of RNAase protection assay with an antisense cRNA probe, we have shown that the liver of the young adult male rat contains androgen receptor (AR) mRNA to a level of 4% compared to the prostate. Steady state levels of AR mRNA in the liver show both sex and age specificity. Compared to that of the male, the female liver contains a markedly reduced amount of AR mRNA. AR mRNA is almost undetectable in livers of prepubertal male (less than 35 days old) and senescent male (greater than 750 days old) rats. Both prepubertal and senescent animals are relatively insensitive to the androgenic induction of alpha 2u-globulin, a hepatic secretory protein. The age-dependent decline in hepatic androgen sensitivity and AR mRNA level can be delayed considerably by a 40% reduction in the dietary calorie intake. Analysis of poly(A)-containing RNA from two liver cell populations, hepatocytes and nonhepatocytes, revealed that only the hepatocytes that express alpha 2u-globulin gene contain AR mRNA. From these results and our earlier observation of in vitro induction of alpha 2u-globulin in isolated rat liver, we conclude 1) that androgen can act directly on hepatocytes to promote alpha 2u-globulin synthesis; 2) that changes in the hepatic androgen sensitivity during maturation and aging are reflections of the age-dependent expression of the receptor gene; and 3) that retardation of the age-dependent loss of androgen sensitivity by calorie restriction is due to a concomitant delay in the decline of the hepatic AR mRNA level.

Loss of androgenic induction of alpha 2u-globulin gene family in the liver of NIH black rats.

Unlike all known strains of rat, the androgen-inducible alpha 2u-globulin gene family is totally silent in the liver of NIH black (NB) rats. No endocrinological or reproductive abnormalities are apparent, and the mRNA for the androgen-repressible hepatic protein SMP-2 is normally regulated in these animals. Furthermore, immunoblot analysis shows a normal level of the male-specific cytoplasmic androgen-binding protein. Cross-breeding of the NB male and Sprague-Dawley female shows that the hybrid male in the F-1 generation regains the androgen-dependent expression of alpha 2u-globulin in the liver. These results along with the observation of high constitutive level of alpha 2u-globulin mRNA in the preputial gland of NB rats indicate a tissue- and gene-specific regulatory defect which prevents androgenic induction of alpha 2u-globulin in the liver.

Purification and immunochemical characterization of the cytoplasmic androgen-binding protein of rat liver.

The cytoplasmic androgen-binding (CAB) protein of the male rat liver has been implicated to play a role in the androgen-dependent regulation of alpha 2u-globulin synthesis. The liver of the adult male rat contains about 50 fmol of specific high-affinity androgen-binding activity per milligram of total cytosolic protein. Photoaffinity labeling with [3H]R-1881 followed by SDS-polyacrylamide gel electrophoresis and autoradiography shows that the CAB is a 31-kilodalton protein. By means of DEAE-cellulose chromatography and preparative SDS-polyacrylamide gel electrophoresis, we have purified the CAB protein to electrophoretic homogeneity and have raised polyclonal rabbit antiserum that is monospecific to this protein. In the sucrose density gradient, the antiserum reacted with the androgen-binding component of the male liver cytosol prelabeled with tritiated dihydrotestosterone. Western blot analysis of the liver cytosol showed that the antiserum recognizes only the 31-kDa androgen-binding component. Such immunoblotting also showed that unlike the young adult, the androgen-insensitive states during prepuberty and senescence are associated with a marked reduction in the hepatic concentration of the immunoreactive CAB protein. No immuno-chemical cross-reactivity between CAB and another androgen-binding component of Mr 29K (which is associated with androgen insensitivity during prepuberty and senescence) was observed. The latter finding favors the possibility that 31- and 29-kDa androgen-binding components may have distinct sequence structure.

Rapid postexposure decay of alpha 2u-globulin and hyaline droplets in the kidneys of gasoline-treated male rats.

Unleaded gasoline induces nephropathy, characterized by rapid accumulation of hyaline (protein resorption) droplets in epithelial cells of the renal proximal convoluted tubules, only in male rats. The hepatic synthesis of the male rat-specific protein alpha 2u-globulin, a constituent of renal hyaline droplets, is unaltered by gasoline treatment (Olson et al., 1987). Renal alpha 2u-globulin content increased to 210% of control within 18 h of a single oral dose of gasoline (2.0 ml/kg); maximal levels (320% of control) were attained following gasoline administration for 3 d. Increases in renal alpha 2u-globulin caused by gasoline were accompanied by concurrent proliferation of hyaline droplets. However, within 3 d of terminating gasoline administration renal alpha 2u-globulin content decreased to the same level as that in unexposed rats, although renal hyaline droplet number returned to pretreatment levels somewhat more slowly. The conjoint effect of postexposure recovery and estradiol (an inhibitor of hepatic alpha 2u-globulin synthesis) administration was also determined in male rats. On postexposure d 3, 6, and 9, estradiol treatment (1 mg/kg, sc, 4 d, starting on d 9 of gasoline treatment) decreased renal alpha 2u-globulin content to 75%, 59%, and 48%, respectively, of that in rats allowed to recover from gasoline with no hormone treatment. Hepatic alpha 2u-globulin content in estradiol-treated rats was decreased by 74%, 97%, and 96% at the same intervals. Estradiol treatment during recovery from gasoline also appeared to increase the removal of accumulated hyaline droplets from the renal cortex. Thus, accumulation of alpha 2u-globulin-containing hyaline droplets after subacute exposure of male rats to gasoline is rapidly reversible, dependent on continuous exposure to gasoline and maintenance of the normal rate of hepatic alpha 2u-globulin synthesis. These results emphasize the dynamic state of renal cortical hyaline droplets and suggest strongly that gasoline hydrocarbons cause hyaline droplet accumulation by prolonging the half-time of degradation of alpha 2u-globulin.

Partial reversal of alpha 2u globulin gene expression by thyroxine in the liver of diabetic rats.

Synthesis of alpha 2u globulin and its mRNA has been used as an index to monitor the effect of thyroxine on specific gene expression in the liver of hypoinsulinemic male rats. Administration of a physiological dose of thyroxine can partially reverse (to approximately 30% of the normal control) the marked reduction (more than 90%) in the hepatic levels of alpha 2u globulin and its mRNA during streptozotocin-induced diabetes. Estimation of newly synthesized alpha 2u globulin RNA transcripts from the native chromatin of isolated liver nuclei by "nuclear runoff experiments" showed that thyroxine can elevate the rate of transcription of alpha 2u globulin gene in the diabetic rat. Hypoinsulinemic diabetes is also found to be associated with an approximately 35% reduction in the thyroid hormone receptor level as compared to the normal control. The stimulatory effect of thyroxine on the synthesis of alpha 2u globulin and its mRNA was also evident in spontaneous diabetic Wistar "BB" rats. From these studies it can be concluded that severe hypoinsulinemia can cause a decrease in thyroid hormone action at the level of specific gene expression.

Effect of anterior hypothalamic deafferentation and continuous growth hormone infusion on the hepatic synthesis of alpha 2u-globulin in the male rat.

Anterior hypothalamic deafferentation and infusion of human GH (hGH) in the normal male rat caused a marked reduction in the hepatic concentration of alpha 2u-globulin, an androgen-dependent protein. Although s.c. injections of hGH (twice-daily) resulted in more than a 50% reduction in the hepatic level of alpha 2u-globulin, the same dose of hGH when administered continuously through osmotic minipumps caused a threefold greater inhibition. The decreased hepatic concentration of alpha 2u-globulin after hGH administration was associated with corresponding changes in the hepatic level of translatable alpha 2u-globulin messenger RNA. Continuous infusion of hGH through osmotic minipumps and removal of the anterior hypothalamic influence on GH secretion by deafferentation also caused a marked reduction in the cytoplasmic androgen-binding activity of the rat liver. These results suggest that alterations in the level and pattern of GH secretion may influence hepatic androgen-binding activity and alpha 2u-globulin synthesis.

Monoclonal antibodies to alpha 2u-globulin and immunocytofluorometric analysis of alpha 2u-globulin-synthesizing hepatocytes during androgenic induction and aging.

Stable hybridomas generated by fusion of spleen cells from hyperimmunized mice and mouse myeloma cells were cloned to prepare monoclonal antibodies to alpha 2u-globulin, an androgen-dependent urinary protein of hepatic origin. One of these monoclonal antibodies was used as a probe for immunocytofluorometric analysis of alpha 2u-globulin producing hepatocytes during androgenic induction and aging through fluorescence-activated cell sorting (FACS). FACS patterns of hepatocytes from mature male rats that produce high levels of alpha 2u-globulin showed tow distinct peaks, arbitrarily designated as peak I (weakly fluorescent) and peak II (brightly fluorescent). In the mature male rat, peak II represented about 40% of the total hepatocytes, and the fluorescence intensity of this subpopulation decreased in direct correspondence with the gradual decline of alpha 2u-globulin synthesis during aging. Similarly the androgenic induction of this protein in ovariectomized female rats was associated with an increase in the fluorescence intensity of the hepatocyte subpopulation under peak II rather than an increase in the relative number of these cells. From these results we conclude that the androgen-dependent synthesis of alpha 2u-globulin and its alteration during aging are confined to a specific subpopulation of hepatocytes within the liver.

Reversible alteration of hepatic messenger RNA species for peroxisomal and non-peroxisomal proteins induced by the hypolipidaemic drug Wy-14,643.

Extensive peroxisomal proliferation in the hepatic parenchymal cells was observed when male rats were given a diet containing 0.1% Wy-14,643 [( 4-chloro-6-(2,3-xylidino)-2-pyrimidinylthio]acetic acid), a potent lipid-decreasing drug. This drug also caused a marked increase in the concentrations of the mRNA species coding for four proteins with Mr 77000, 61000, 43000 and 31000, and a similar decrease in the concentrations of three mRNA species coding for proteins of Mr 25000, 24000 and 19000. Specific immunoprecipitation studies identified the proteins of Mr 19000, 43000 and 77000 as alpha 2u-globulin, 3-ketoacyl-CoA thiolase (EC 2.3.1.16) and enoyl-CoA hydratase (EC 4.2.1.17) respectively. Comparisons of the Mr values suggest that the 61000- and 31000-Mr proteins may be equivalent to two additional peroxisomal enzymes, namely catalase (Mr 61000) and uricase (Mr 31000). The identity of the mRNA species coding for the 25000- and 24000-Mr proteins is at present unknown.

Interacting role of thyroxine and growth hormone in the hepatic synthesis of alpha 2u-globulin and its messenger RNA.

Hypophysectomy completely abolishes and thyroidectomy results in a 90% reduction in the hepatic content of alpha 2u-globulin and its mRNA in the male rat. Thyroid hormone is also known to be required for the synthesis and secretion of pituitary growth hormone. In the hypothyroid rat either thyroxine or growth hormone was found to increase the activity and number of sequences of the mRNA for alpha 2u-globulin (measured by translational assay and hybridizational analysis with a cloned cDNA probe) to the euthyroid level. Treatment of hypophysectomized rats with a hormone combination containing growth hormone but not thyroxine increased the hepatic level of the mRNA for alpha 2u-globulin to that of normal animals. From these results we conclude that thyroxine indirectly influences the hepatic concentration of the mRNA for alpha 2u-globulin through its effect on pituitary growth hormone. Although administration of growth hormone to hypothyroid animals raised the hepatic concentration of alpha 2u-globulin mRNA to the euthyroid level, synthesis of alpha 2u-globulin remained low (50% of the normal). Complete recovery of alpha 2u-globulin synthesis required thyroxine. Therefore, in addition to an indirect effect on the hepatic level of alpha 2u-globulin mRNA, thyroxine also directly influences the synthesis of this protein. This direct effect of thyroxine on alpha 2u-globulin synthesis seems to be exerted at a step distal to the formation of mature mRNA.

Pretranslational regulation of alpha 2u-globulin in rat liver by growth hormone.

Hypophysectomy is known to cause complete suppression of the hepatic synthesis alpha 2u-globulin. The effect of hypophysectomy on the synthesis of alpha 2u-globulin can be reversed by multiple hormone treatment. The role of pituitary growth hormone in the multihormonal regulation of alpha 2u-globulin in rat liver was examined in the hypophysectomized male rats with and without growth hormone supplementation. Daily treatment of hypophysectomized rats with 5 alpha-dihydrotestosterone, corticosterone, thyroxine, and growth hormone for 8 days caused about 80% recovery in the hepatic content of alpha 2u-globulin and its corresponding mRNA as determined by radioimmunoassay, in vitro translation, and liquid hybridization with a cloned cDNA probe. However, omission of growth hormone from the treatment regimen failed to raise hepatic alpha 2u-globulin and its mRNA to more than 5% of the normal control. The possible effect of growth hormone on the translation of the mRNA for alpha 2u-globulin was examined with cultured hepatocytes derived from growth hormone-deficient rats. Culture of these cells in the presence of growth hormone for 24 h did not turn on the synthesis of alpha 2u-globulin. These results indicate that growth hormone regulates the synthesis of alpha 2u-globulin by acting at a step antecedent to mRNA translation.