Endoproteolytic processing of Sst2, a multidomain regulator of G protein signaling in yeast.

Regulators of G protein signaling (RGS proteins) constitute a large family of G protein-binding proteins. All RGS proteins contain a conserved core domain that can accelerate G protein GTPase activity. In addition, many family members contain a unique N-terminal domain of unknown function. Here, we demonstrate that the RGS protein in yeast, Sst2, is proteolytically processed in vivo to yield separate but functional N-terminal and RGS core domain fragments. In whole cell lysates, the full-length SST2 product (82 kDa) as well as a prominent 36-kDa species are specifically recognized by antibodies against the C terminus of the Sst2 protein. Purification and chemical sequencing of the 36-kDa species revealed cleavage sites after Ser-414 and Ser-416, just preceding the region of RGS homology. Expression of a mutationally truncated form of the protein (C-Sst2) could not restore function to an sst2Delta mutant strain. In contrast, co-expression of C-Sst2 with the N-terminal domain (N-Sst2) partially restored the ability to regulate the growth arrest response but not the transcription induction response. Whereas the full-length protein was localized to the microsomal and plasma membrane fractions, the N-Sst2 species was predominantly in the microsomal fraction, and C-Sst2 was in the soluble fraction. Mutations that block proteasome or vacuolar protease function, or mutations in the cleavage site Ser residues of Sst2, did not alter processing. However, Sst2 processing did require expression of other components of the pheromone response pathway, including the receptor and the G protein. These results indicate that Sst2 is proteolytically processed, that this event is regulated by the signaling pathway, and that processing can profoundly alter the function and subcellular localization of the protein.

Tissue concentrations of clindamycin after multiple oral doses in normal cats.

Eighteen normal cats were randomly allocated into two blocks with three treatment groups and dosed orally with clindamycin aqueous solution for 10 days at a dosage rate of 5.5 mg/kg twice daily (Group 1), 11 mg/kg twice daily (Group 2), or 22 mg/kg once daily (Group 3). At the end of dosing, all cats were killed and tissues were taken for clindamycin concentration analysis. Clindamycin was extracted from tissues using solid-phase extraction columns followed by microbiological assay of clindamycin using a cylinder plate assay using M. luteus. Recovery from each tissue was determined by inoculating known concentrations of clindamycin into drug-naive tissues and comparing the observed concentration from the expected concentration. Confirmation that the bioassay detected clindamycin and not N-desmethylclindamycin, its active metabolite, was done using gas-chromatography-mass-spectrometry. Concentrations were highest in the lung, with tissue:serum ratios greater than 3 in all groups. Concentrations were higher in Group 3 than Group 1 (P less than 0.05). Only liver concentrations in Group 3 were statistically higher than in Group 2, although all tissues except bone marrow and CSF had numerically higher concentrations in Group 3 than Group 2. The tissue:serum ratio was greater than 1 in all tissues studied except bone, cerebrospinal fluid, brain, and skeletal muscle.

The lack of a growth-promoting effect of orally administered bovine somatotropin in the rat body-weight-gain bioassay.

Bovine somatotropin (bSt) was given either orally or subcutaneously to groups of female hypophysectomized rats daily for 9 days. Ten rats per dose group were given oral dosages of 0 (buffered-water vehicle control), 40, 400, 2000, and 4000 micrograms of bSt per day. Similar groups of ten rats each received subcutaneous doses of 0 (buffered-water vehicle control), 15, 30, and 60 micrograms of bSt per day. Rats were weighed daily to observe their body-weight gain, which is a measure of the biological activity of bSt in the hypophysectomized rat. At study termination, serum of treated rats was monitored for the presence of bSt and antibody to bSt. Bovine somatotropin was detected in the serum of the subcutaneously treated rats, but not in those rats treated orally. Of 18 rats treated subcutaneously with bSt, 14 developed antibodies to bSt, whereas of 38 rats treated orally with bSt, 11 developed antibodies. Subcutaneously treated rats grew in a dose-related manner as expected in this assay. Orally administered bSt failed to elicit a growth response at any dose in this sensitive bioassay system. The data suggest that neither bSt nor growth-promoting fragments of bSt are absorbed after oral administration of doses up to 40,000 micrograms/kg/day in the hypophysectomized rat.

Amine nitration and nitrosation by gaseous nitrogen dioxide.

Reaction of N-methylaniline in solution with ca. 100,000 ppm gaseous NO2 leads to significant amounts of ring- and N-nitro products, in addition to N-methyl-N-nitrosoaniline. Reaction of heterocyclic amines in solution with 5-1000 ppm gaseous NO2 gives significant amounts of N-nitrosamines and much higher amounts of N-nitramines than those obtained from concentrated gaseous NO2. The results are interpreted as evidence that N-nitrosation proceeds via an unsymmetrical ON-ONO2 dimer, whereas N-nitration may involve a free-radical process involving NO2, itself.