Synthesis of immobilized flavin derivatives and their use in purification of chicken egg-white ovoflavoprotein.

Affinity adsorbents for flavoproteins were prepared by the covalent attachment of polyacrylamide and agarose to flavin derivatives linked through position N(3) of the flavin nucleus. 3-Carboxymethyl-FMN covalently linked to aminoalkyl substituted agarose was successfully used for the separation and purification of the apo form of the ovoflavoprotein from chicken egg white. High yields and high purities were achieved by two different isolation procedures employing the affinity adsorbent.     

Morphological and biochemical changes in supersensitive smooth muscle.

The effect of postganglionic denervation on the incidence of nexal contacts in the smooth muscle of the rat vas deferens was investigated. The chronically denervated tissue exhibited twice as many nexuses as control. This increase in the incidence of cell contacts may contribute to the supersensitivity and/or the increase in maximum response of the denervated vas deferens. The effects of denervation, decentralization, and pretreatment with reserpine on the concentration of adenosine triphosphate (ATP) in vasa deferentia of rats and guinea pigs were also investigated. One day after denervation there was a substantial decrease in the endogenous norepinephrine and ATP concentrations. The norepinephrine concentration remained low (less than 10% of control) throughout subsequent days (up to 14 days) whereas the ATP concentration, after the first postoperative day, rose significantly. The rise in ATP concentration was temporally correlated with the development of postjunctional supersensitivity. Decentralization and pretreatment with reserpine both resulted in a significant increase in ATP concentration which preceded by 2 to 3 days a significant increase in sensitivity of the vas deferens. It appears that a change in the tissue concentration of ATP may be one of the initial events that occurs following interruption of the neural contact to the smooth muscle of the vas deferens.     

Purification and regulatory properties of chicken heart prostaglandin E 9-ketoreductase.

Prostaglandin E 9-ketoreductase was purified from chicken heart by ammonium sulfate fractionation, and DEAE-Sephadex, hydroxylapatite and phosphocellulose chromatography. Two peaks of activity were resolved during the phosphocellulose chromatographic step. Both peaks were stimulated by a substance that was not bound to the phosphocellulose column. This stimulatory substance was destroyed by treatment with phosphodiesterase and 0.1 M NaOH. It was heat-stable (100 degrees, 2 min), nondialyzable, and resistant to treatment with pronase, ribonuclease, and deoxyribonuclease; but it was dialyzable after heating or digestion with pronase. Sodium pyrophosphate also enhanced the activities of the prostaglandin E 9-ketoreductases as did angiotensin I; but not angiotensin II. In the presence of 3':5'-cyclic AMP, AMP, or several other ribonucleotides, the enhancing effects of the natural stimulatory substance, sodium pyrophosphate or angiotensin I were blocked, but these ribonucleotides themselves had little effect on the enzymes activity. The substrate specificities of the two prostaglandin E 9-ketoreductases were also studied. Both the 9-keto group and the 15-keto group of 15-ketoprostaglandin F2 alpha could be converted to the corresponding hydroxyl group; the 15-keto group was reduced faster than the 9-keto group. Prostaglandin D2, a prostaglandin with a 9-hydroxyl and an 11-keto group, could not be converted to prostaglandin F2 alpha nor could cyclohexanone be converted to cyclohexanol by the prostaglandin E 9-ketoreductase.     

Purification and properties of rabbit liver phosphorylase phosphatase.

A procedure for the purification of rabbit liver phosphorylase phosphatase is described. The specific activity of the preparation is 2,100 units/mg of protein, representing a 25,000-fold purification. During the initial steps of the purification a large activation of enzyme activity was observed. The molecular weight of the purified enzyme was estimated by Sephadex G-75 chromatography to be 35,000, and by sucrose density ultracentrifugation to be 34,000 (2.9 S). On Na dodecyl-SO4 polyacrylamide disc gel electrophoresis a single component with a molecular weight of 34,000 was observed. The pH optimum is 6.9 to 7.4, and the Km for rabbit muscle phosphorylase alpha is 2 muM. The same procedure is also applicable to the extensive purification of phosphorylase phosphatase from rabbit muscle.     

The relation between sporulation and the induction of antibiotic synthesis and of amino acid uptake in Bacillus brevis

The induction and localization of tyrocidine-synthesizing enzymes is shown to be parallel, during growth of Bacillus brevis (ATCC 8185, American Type Culture Collection, Rockville, Md.), with the induction of uptake of constitutive amino acids and of components of pantetheine, a coenzyme of tyrocidine synthesis. Antibiotic synthesis appears at the end of logarithmic growth when the first soluble enzymes may be obtained from homogenates. During this period, binding proteins for metabolite uptake were isolated by intensive sonication which, when studied by chromatography, were identified by the appearance of low molecular weight fractions binding the radioactively marked metabolites; their induction was prevented by addition of rifampicin. The major purpose of this study was a comparison of antibiotic production and sporulation, the progress of which was followed by electron microscopy. The onset of tyrocidine synthesis and metabolite uptake coincided with the appearance of septum formation indicating that sporulation had progressed to stage II. With the progress of spore encapsulation, the tyrocidine production migrated from the soluble fraction into the forespore, terminating with the separation of forespores from the sporangium membrane. The resulting concentration of antibiotic in the forespore may indicate its function in sporulation, the nature of which, however, was not explored.     

Selective Methylation: an Incorrect Hypothesis

“Selective methylation,” a hypothesis proposed to explain the discrepancy found in the degree of methyl deficiency of transfer ribonucleic acid, cannot be explained on the basis of some biological phenomenon.     

Regulation of gonadotrophin secretion in rams from birth to sexual maturity. I. Plasma LH, FSH and testosterone levels.

Plasma LH, FSH and testosterone concentrations were measured by radioimmunoassays in male crossbred Merino/Corriedale sheep from birth to 45 weeks of age. FSH levels were 11 and 22 ng/ml at birth, increased to peak levels (mean value of 47 ng/ml) at 5 weeks and fluctuated between 25 and 35 ng/ml for the next 40 weeks. Similarly, LH (less than 0-5 ng/ml) and testosterone (less than 38 ng/100 ml) levels were low at birth and were significantly elevated by 5 weeks of age. LH values varied betwen 0-9 and 3-0 ng/ml for the next 30 weeks and then a secondary rise occurred reaching levels of 2-4 ng/ml by the 41st week after birth. Concentrations of LH subsequently fell to levels observed in adult rams. Testosterone levels rose gradually between the 5th and the 25th week, and then increased rapidly to values of 270-517 ng/100 ml by the 41st week after birth, a time coincident with the peak LH levels. Histological examination of testicular biopsies demonstrated that Sertoli cell maturation occurred 17-21 weeks after birth and was followed by activation of spermatogenesis leading to the presence of spermatozoa in the seminiferous epithelium by 39-42 weeks of age.     

Social grooming between squirrel monkeys and uakaris in a seminatural environment

Young squirrel monkeys (Saimiri sciureus) were reported grooming an adult female uakari (Cacajao calvus rubicundus) on four different occasions. Furthermore, the uakari was noted grooming two squirrel monkeys in separate instances. These observations took place in a seminatural rainforest (The Monkey Jungle; Goulds, Florida, U.S.A.) where provisions are provided. Some possible hypostheses tendered to account for this unusual behavior included (a) the unaverted interaction of food-seeking and fur-cleaning behavior, and (b) the compatibility of play-curiosity activities by squirrel monkeys with the uakaris' need for social contact.     

In vivo metabolism of 3H-testosterone in adult male rats: effects of estrogen administration.

The metabolism of testosterone (T) was studied in normal adult male rats using a constant infusion of trace amounts of the 3H-steroid into a tail vein for 3 h in order to attain a state of equilibrium. Samples of plasma, liver, kidney, prostate, seminal vesicles and muscle were analysed for 3H-testosterone, 3H-5alpha-dihydrotestosterone (5alphaDHT) and 3H-5alpha-androstanediol (Adiol). When compared to the 3H-T level in plasma there were high levels of 3H-T in kidney and of 3H-5alphaDHT in prostate and seminal vesicles. Intraperitoneal estradiol valerate administration (100 mug/day) for 4 days decreased and 3H-5alphaDHT levels in the prostate and seminal vesicles. The estrogen administration increased the T metabolic clearance rate from 17.5 1/24 h/100 g body wt to 22.6 1/24 h/100 g body wt.     

Oncogenic KRAS G12C: Kinetic and redox characterization of covalent inhibition

The recent development of mutant-selective inhibitors for the oncogenic KRAS^G12C allele has generated considerable excitement. These inhibitors covalently engage the mutant C12 thiol located within the phosphoryl binding loop of RAS, locking the KRAS^G12C protein in an inactive state. While clinical trials of these inhibitors have been promising, mechanistic questions regarding the reactivity of this thiol remain. Here, we show by NMR and an independent biochemical assay that the pK_a of the C12 thiol is depressed (pK_a ∼7.6), consistent with susceptibility to chemical ligation. Using a validated fluorescent KRAS^Y137W variant amenable to stopped-flow spectroscopy, we characterized the kinetics of KRAS^G12C fluorescence changes upon addition of ARS-853 or AMG 510, noting that at low temperatures, ARS-853 addition elicited both a rapid first phase of fluorescence change (attributed to binding, K_d = 36.0 ± 0.7 μM) and a second, slower pH-dependent phase, taken to represent covalent ligation. Consistent with the lower pK_a of the C12 thiol, we found that reversible and irreversible oxidation of KRAS^G12C occurred readily both in vitro and in the cellular environment, preventing the covalent binding of ARS-853. Moreover, we found that oxidation of the KRAS^G12C Cys12 to a sulfinate altered RAS conformation and dynamics to be more similar to KRAS^G12D in comparison to the unmodified protein, as assessed by molecular dynamics simulations. Taken together, these findings provide insight for future KRAS^G12C drug discovery efforts, and identify the occurrence of G12C oxidation with currently unknown biological ramifications.