Nuclei have been prepared from the oviduct of the adult laying hen which are capable of synthesizing large amounts of RNA for long periods of time. The time course of RNA synthesis is linear through 3 h of incubation after an initial burst of activity and is inhibited 60-70% by alpha-amanitin. Maximum synthetic activity requires the presence of serum albumin to stabilize the nuclei, high concentrations of the four ribonucleoside triphosphates, and an incubation temperature of 25 degrees C for continued linear synthesis beyond 30 min. The RNA synthesized in vitro is predominantly 10-20 S with a small proportion of higher molecular weight product. Much of the 10-20S RNA is probably transcribed by RNA polymerase II and is of a size comparable to ovalbumin mRNA. A fraction of this RNA appears to contain poly(A) sequences suggesting that there is some processing of the newly synthesized RNA. These nuclei may provide a useful system for studying the control of the transcription and maturation of ovalbumin mRNA in vitro.

Peptidyl (acyloxy)methyl ketones, previously established as potent irreversible inhibitors of the cysteine proteinase cathepsin B in vitro, were investigated and optimized for their inhibitory activity in vivo. Incorporation of polar or charged functional groups in the inhibitor structure afforded effective cathepsin B inhibition, following dosing to rats. The most effective inhibitor, Z-Phe-Lys-CH2OCO-(2,4,6-Me3)Ph (8), was found to give ED50 values of 18 mg/kg po (orally) and 5.0 mg/kg ip (intraperitoneally) at 4-5 h postdose, and 2.4 mg/kg sc (subcutaneously) at 24 h postdose, for liver cathepsin B inhibition (measured ex vivo). The subcutaneous route of administration of (acyloxy)methyl ketone 8 also provided potent cathepsin B inhibition in certain peripheral tissues (e.g., ED50 1.0 mg/kg for skeletal muscle, 0.1 mg/kg for heart). These investigations demonstrate that peptidyl (acyloxy)methyl ketones such as 8 have promise as tools for the characterization of in vivo biochemical processes and as therapeutic agents.

Tyrosine aminotransferase (L-tyrosine:2-oxoglutarate aminotransferase, EC 2.6.1.5) enzyme and mRNA activity were not detectable in day 20 fetal rat liver. Precocious induction of catalytic activity by in utero injection of dibutyryl cAMP was a direct consequence ofthe de novo appearance oftranslatable tyrosine aminotransferase mRNA. In contrast, in utero injection ofhydrocortisone acetate failed to elicit fetal liver enzyme activity. This failure was due to the inability of the steroid hormone to induce the appearance of tyrosine aminotransferase mRNA activity. In fetal rat liver explants, either compound was capable of stimulating the synthesis of adult levels of enzyme and mRNA. However, catalytic and mRNA activity comparable with that seen in vivo 24 hr after birth required the concerted action of both inducers.One of the central problems in the study of development is the determination of the factors responsible for differential gene expression and the appearance of tissue-specific proteins. The enzymatic differentiation of developing rat liver has proved to be a useful model for these studies. During development, most rat liver enzymes accumulate rapidly and in discrete groups or clusters: the late fetal cluster (16-22 days ofgestation), the neonatal cluster, and the late suckling cluster (1).Tyrosine aminotransferase (L-tyrosine:2-oxoglutarate aminotransferase, EC 2.6.1.5) is one of the enzymes appearing in the neonatal cluster (1). Enzyme activity, virtually absent in fetal rat liver, starts to increase 2 hr after birth, reaching a maximum of at least twice the adult level by 12 hr. The catalytic activity then decreases to adult level by 2 days after birth (2, 3). Premature appearance of tyrosine aminotransferase can be elicited in fetal rats by in utero injection of glucagon (4, 5) or dibutyryl cAMP (6); glucocorticoid injection does not precociously induce catalytic activity (4, 6). However, all three compounds induce enzyme activity in vitro (7-9).As part of our studies on the regulation of tyrosine aminotransferase gene expression, we are investigating the process responsible for the appearance of tyrosine aminotransferase in newborn rats. We present evidence that the appearance of enzyme activity after precocious induction in utero is a direct consequence of the de novo accumulation of mRNA coding for tyrosine aminotransferase. In addition, we have used fetal rat liver explants to correlate tyrosine aminotransferase mRNA and enzyme activity during induction by dibutyryl cAMP and hydrocortisone acetate.MATERIALS AND METHODS Chemicals. Cortef acetate (hydrocortisone acetate, 50 mg/ ml) was provided by Upjohn. BGJb medium, Fitton-Jackson modification, and penicillin/streptomycin (penicillin, 10,000 units/ml; streptomycin, 10,000 Ag/ml) were from GIBCO.Translation grade [3S]methionine (specific activity, 1350-1450 Ci/mmol; 1 Ci = 3.7 X 101°becquerels) was from Amersham. Cordycepin, protein A-Sepharose, and other biochemicals were from Sigma.Animals. Sprague-Dawley (CD) rats from Charles River Br...

The title compound 3-carboxyisoxazole 3 was synthesized by cycloaddition of carbethoxyformonitrile oxide to N-[4-(trifluoromethyl)phenyl]-3-pyrrolidino-2-butenamide (6) with spontaneous elimination of pyrrolidine followed by hydrolysis of the ethyl ester. Compound 3 was shown to be absorbed intact after oral administration to rats. Over 24 h, the compound was metabolized to yield plasma concentrations of the antiinflammatory agent 2-cyano-3-hydroxy-N-[4-(trifluoromethyl)phenyl]-2-butenamide (2), similar to those obtained following an equivalent dose of the established prodrug of 5-methyl-N-[4-(trifluoromethyl)phenyl]isoxazole-4-carboxamide (1).

Tyrosine aminotransferase messenger ribonucleic acid (mRNA) activity in rat liver was rapidly increased 3-6-fold following in vivo administration of hydrocortisone acetate, dibutyryladenosine cyclic 3',5'-phosphate, or the protein synthesis inhibitor cycloheximide. Treatment with the steroid hormone or cyclic nucleotide in combination with cycloheximide resulted in levels of tyrosine aminotransferase mRNA 10-20-fold greater than control values. These changes in mRNA activity were not accompanied by changes in albumin mRNA or total liver template activity. The rapid decline in tyrosine aminotransferase mRNA activity following cordycepin inhibition of de novo RNA synthesis was prevented by cycloheximide treatment. This protection was not observed when pactamycin was substituted for cycloheximide, demonstrating that the inhibition of protein synthesis per se was not responsible for the stabilization of tyrosine aminotransferase mRNA. Based upon the effects of cycloheximide and pactamycin on rat liver polysome structure, it is concluded that the cycloheximide-mediated increase in tyrosine aminotransferase mRNA activity is the result of stabilization of the mRNA molecule which renders the message less susceptible to inactivation and degradation in the cytoplasm. The action of cycloheximide is very specific for tyrosine aminotransferase, phosphoenolpyruvate carboxykinase, and probably several other mRNAs that code for minor liver proteins that turn over rapidly in response to hormonal or metabolic stimuli.