Identification of residues in the cysteine-rich domain of Raf-1 that control Ras binding and Raf-1 activity

We have identified mutations in Raf-1 that increase binding to Ras. The mutations were identified making use of three mutant forms of Ras that have reduced Raf-1 binding (Winkler, D. G., Johnson, J. C., Cooper, J. A., and Vojtek, A. B. (1997) J. Biol. Chem. 272, 24402-24409). One mutation in Raf-1, N64L, suppresses the Ras mutant R41Q but not other Ras mutants, suggesting that this mutation structurally complements the Ras R41Q mutation. Missense substitutions of residues 143 and 144 in the Raf-1 cysteine-rich domain were isolated multiple times. These Raf-1 mutants, R143Q, R143W, and K144E, were general suppressors of three different Ras mutants and had increased interaction with non-mutant Ras. Each was slightly activated relative to wild-type Raf-1 in a transformation assay. In addition, two mutants, R143W and K144E, were active when tested for induction of germinal vesicle breakdown in Xenopus oocytes. Interestingly, all three cysteine-rich domain mutations reduced the ability of the Raf-1 N-terminal regulatory region to inhibit Xenopus oocyte germinal vesicle breakdown induced by the C-terminal catalytic region of Raf-1. We propose that a direct or indirect regulatory interaction between the N- and C-terminal regions of Raf-1 is reduced by the R143W, R143Q, and K144E mutations, thereby increasing access to the Ras-binding regions of Raf-1 and increasing Raf-1 activity.     

Differential regulation by estrogens of growth and prolactin synthesis in pituitary cells suggests that only a small pool of estrogen receptors is required for growth

PR1 cells are a prolactin (PRL)-secreting cell line derived from a pituitary lactotroph tumor found in 17beta-estradiol-treated Fischer 344 rats. We examined the effect of estrogen on cell proliferation and PRL synthesis under various culture conditions. Estrogen, at extremely low concentrations, induces cell proliferation in this cell line, whereas antiestrogen inhibits proliferation. Interestingly, the proliferation response is much more sensitive than the PRL response because 0.01 pM estradiol or diethylstilbestrol induces half-maximal growth induction [ approximately 0.1% estrogen receptor (ER) occupancy is required], whereas 0.01 nM concentration is required for half-maximal PRL induction ( approximately 50% ER occupancy is required). The proliferation response is not as sensitive to antiestrogen as the PRL response, because 10 nM concentration of the pure antiestrogen ICI 182,780 could not inhibit 1 nM estradiol- or diethylstilbestrol-induced proliferation. The same concentration of ICI 182,780 decreased PRL secretion to 1% of estradiol- or diethylstilbestrol-induced prolactin secretion suggesting a possible dichotomy of ER control of proliferation and PRL synthesis. The Kd of ER binding in these cells is about 3 x 10(-11) M. These results with the PR1 cells extend previous studies in other estrogen- regulated systems and suggest that only a small pool of ER is required for cell proliferation in contrast with the regulation of expression of specific genes. They also raise questions as to how a dimeric receptor functions when only one ligand site is occupied or when both an estrogen and an antiestrogen occupy one dimer.     

Human spinal arachnoid villi and granulations

A commercially available recording of Northwestern University Auditory Test No. 6 (N.U. No. 6) produced by Auditec of St. Louis was evaluated in a series of four studies. Interlist equivalence of this version was initially investigated, and then normative intelligibility functions were developed using listerners with normal hearing. Intelligibility functions for the original version of N.U. No. 6. prepared by Northwestern University, were then derived, concurrently with functions of the Auditec version, using (1) a group of listeners with normal hearing; and (2) a group with sensorineural hearing loss. The results demonstrated good interlist equivalence for the Auditec version of N.U. No. 6. The comparative intelligibility functions for the Auditec and Northwestern versions of N.U. No 6 furthermore indicated a difference between the two recordings, but it was concluded that for clinical purposes the two versions may be considered equivalent.     

Hemodynamic responses to stellate ganglion stimulation in mongrels and greyhounds

A comparison of the effects of left stellate ganglion stimulation (SGS) on central and aortic hemodynamics has been made in chloralose-anesthetized mongrel (M), and greyhound (GH) dogs. Measurements of aortic pressure and flow, and left ventricular pressure were made during stimulation of the decentralized left SG at different frequencies from 0 to 20 Hz. The increases in aortic pressure and flow with SGS were larger in the GH, especially for low frequencies of stimulation. Stroke volume was increased with SGS in the GH at all stimulation rates, whereas in the M it was unchanged. A greater decrease in left ventricular end-diastolic pressure with SGS was found in the GH. These results suggest that differences exist in both the intrinsic and extrinsic control of cardiac output in the greyhound dog compared to the mongrel. These differences may be in part responsible for the elevated arterial blood pressure in the greyhound compared to the mongrel.     

A simple program which processes liquid scintillation counting data from samples containing both 125I and 3H under conditions of varying quench

A human study of the effects of topical nystatin (Mycostatin) therapy of oral candidiasis showed that effects of treatment were limited to the time in which the drug was used. Two weeks of therapy resulted in significant reduction in number of organisms and marked improvement in signs and symptoms of candidiasis. The condition recurred rapidly following cessation of treatment. No change in specific anticandida antibody in saliva or in adherence of Candida albicans to mucosal epithelium (in vitro) was seen with treatment.