Oxygen limitation of N2 fixation in stem-girdled and nitrate-treated soybean

The effects of increasing rhizosphere pO₂on nitrogenase activity and nodule resistance to O₂diffusion were investigated in soybean plants [Glycine max (L.) Merr. cv. Harosoy 63] in which nitrogenase (EC 1.7.99.2) activities were inhibited by (a) removal of the phloem tissue at the base of the stem (stem girdling), (b) exposure of roots to 10 mM NO₃over 5 days (NO₃-treated), or (c) partial inactivation of nitrogenase activity by an exposure of nodulated roots to 100 kPa O₂(O₂-inhibitcd). In control plants and in plants which had been treated with 100 kPa O₂, increasing rhizosphere O₂concentrations in 10 kPa increments from 20 to 70 kPa did not alter the steady-state nitrogenase activity. In contrast, in plants in which nitrogenase activities were depressed by stem girdling or by exposure to NO₃, increasing rhizosphere pO₂resulted in a recovery of 57 or 67%, respectively, of the initial, depressed rates of nitrogenase activity. This suggests that the nitrogenase activity of stem-girdled and NO₃-treated soybeans was O₂-limited. For each treatment, theoretical resistance values for O₂diffusion into nodules were estimated from measured rates of CO₂exchange, assuming a respiratory quotient of 1.1 and 0 kPa of O₂in the infected cells. At an external partial pressure of 20 kPa O₂, the stem-girdled and NO₃^--treated plants displayed resistance values which were 4 to 8.6 times higher than those in the nodules of the control plants. In control and O₂-inhibited plants, increases in pO₂from 20 to 70 kPa in 10 kPa increments resulted in a 2.5- to 3.9-fold increase in diffusion resistance to O₂, and had little effect on either respiration or nitrogenase activity. In contrast, in stem-girdled and NO₃^--treated plants, increases in external pO₂had little effect on diffusion resistance to O₂, but resulted in a 2.3- to 3.2-fold increase in nodule respiration and nitrogenase activity. These results are consistent with stem-girdling and NO₃^--inhibition treatments limiting phloem supply to nodules causing an increase in diffusion resistance to O₂at 20 kPa and an apparent insensitivity of diffusion resistance to increases in external pO₂.     

Bile acid: CoASH ligases from guinea pig and porcine liver microsomes. Purification and characterization

A procedure for the purification of the enzyme bile acid:CoA ligase from guinea pig liver microsomes was developed. Activity toward chenodeoxycholate, cholate, deoxycholate, and lithocholate co-purified suggesting that a single enzyme form catalyzes the activation of all four bile acids. Activity toward lithocholate could not be accurately assayed during the earlier stages of purification due to a protein which interfered with the assay. The purified ligase had a specific activity that was 333-fold enriched relative to the microsomal cell fraction. The purification procedure successfully removed several enzymes that could potentially interfere with assay procedures for ligase activity, i.e. ATPase, AMPase, inorganic pyrophosphatase, and bile acid-CoA thiolase. On sodium dodecyl sulfate-polyacrylamide gel electrophoresis the purified ligase gave a single band of approximately 63,000 Mr. A molecular size of 116,000 +/- 4,000 daltons was obtained by radiation inactivation analysis of the ligase in its native microsomal environment, suggesting that the functional unit of the ligase is a dimer. The purified enzyme was extensively delipidated by adsorption to alumina. The delipidated enzyme was extremely unstable but could be partially stabilized by the addition of phospholipid vesicles or detergent. However, such additions did not enhance enzymatic activity. Kinetic analysis revealed that chenodeoxycholate, cholate, deoxycholate, and lithocholate were all relatively good substrates for the purified enzyme. The trihydroxy bile acid cholate was the least efficient substrate due to its relatively low affinity for the enzyme. Bile acid:CoA ligase could also be solubilized from porcine liver microsomes and purified 180-fold by a modification of the above procedure. The final preparation contains three polypeptides as judged by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The three peptides range in size from 50,000 to 59,000, somewhat smaller than the guinea pig enzyme. The functional size of the porcine enzyme in its native microsomal environment was determined by the technique of radiation inactivation analysis to be 108,000 +/- 5,000 daltons. Thus, the functional form of the porcine enzyme also appears to be a dimer.     

Risk factors for uterine fibroids: reduced risk associated with oral contraceptives.

Risk factors for pathologically confirmed uterine leiomyomas (fibroids) were investigated using data from the Oxford Family Planning Association study, a long term follow up study of women using various methods of contraception. For each of 535 women who had had a fibroid an individual control was selected who matched the patient on age, date of entry into the cohort, and family planning clinic at recruitment and who was alive (and still being followed up) at the date the patient underwent surgery for fibroids. Case-control analysis showed that reproductive experiences were closely linked to development of fibroids. Risk of fibroids decreased consistently with increasing number of term pregnancies; women with five term pregnancies had only a quarter of the risk of women who had had none. Risk also decreased consistently with increasing duration of oral contraceptive use; the risk of fibroids was reduced by some 31% in women who had used oral contraceptives for 10 years. Risk was strongly related to weight: women who weighed under 55 kg had a particularly low risk, and overall the risk rose roughly 21% for each 10 kg increase. Cigarette smoking was associated with a decreased risk of fibroids; smokers of 20 cigarettes a day had a risk roughly two thirds that of non-smokers. These risk factors have all previously been identified as risk factors for endometrial cancer; this strongly suggests that the underlying risk factor is "unopposed" oestrogen.     

Preliminary Evaluation of Four Oral Contraceptives Containing only Progestogens

One hundred and seventy-five women took part in a comparative clinical trial of four progestogen-only oral contraceptives and were followed for either a year or until treatment was discontinued. Megestrol acetate 0·25 mg. was found to be a very ineffective contraceptive, 21 out of 43 women becoming pregnant. One, three, and four pregnancies occurred during treatment with norethisterone acetate 0·3 mg., norgestrel 0·05 mg., and chlormadinone 0·5 mg., respectively, corresponding to pregnancy rates of 4, 9, and 12 per 100 woman-years of use.All three effective progestogens were very much less acceptable than modern low-dose combined oral contraceptives. Discontinuation of treatment for medical reasons (particularly menstrual disturbances) during the course of only one year affected 24% receiving norethisterone acetate, 38% receiving norgestrel, and 46% receiving chlormadinone.     

Controlled trial of three different antismoking interventions in general practice.

Of 6052 adult patients who consulted their doctors in six Oxfordshire general practices between October 1980 and February 1981, 2110 (35%) were smokers. The smokers were allocated to one of four study groups--a control (non-intervention) group; a group that received verbal and written antismoking advice from the general practitioner; a group that received this advice and also a demonstration of exhaled carbon monoxide; and a group that received the advice plus the offer of further help from a health visitor. After one year 72% of smokers replied to a postal follow up questionnaire: 11% of the control group claimed to have stopped smoking compared with 15% in the group that received advice alone, 17% in the exhaled carbon monoxide group, and 13% in the health visitor group. Validation of these findings by assays of urinary concentrations of cotinine showed that between 24% and 40% of subjects may have misreported their smoking habits, but there was no indication that the rate of misreporting was higher in the intervention groups than in the control group. Giving advice routinely against smoking has a useful effect, and showing an immediate, personal, and potentially harmful consequence of smoking using a CO-oximeter may improve this, particularly in lower socioeconomic groups.     

Studies of the relationship between the catalytic activity and binding of non-substrate ligands by the glutathione S-transferases.

The dimeric enzyme glutathione S-transferase B is composed of two dissimilar subunits, referred to as Ya and Yc. Transferase B (YaYc) and two other transferases that are homodimers of the individual Ya and Yc subunits were purified from rat liver. Inhibition of these three enzymes by Indocyanine Green, biliverdin and several bile acids was investigated at different values of pH (range 6.0-8.0). Indocyanine Green, biliverdin and chenodeoxycholate were found to be effective inhibitors of transferases YaYc and YcYc at low (pH 6.0) but not high (pH 8.0) values of pH. Between these extremes of pH intermediate degrees of inhibition were observed. Cholate and taurochenodeoxycholate, however, were ineffective inhibitors of transferase YcYc at all values of pH. The observed differences in bile acids appeared to be due, in part, to differences in their state of ionization. In contrast with the above results, transferase YaYa was inhibited by at least 80% by the non-substrate ligands at all values of pH. These effects of pH on the three transferases could not be accounted for by pH-induced changes in the enzyme's affinity for the inhibitor. Thus those glutathione S-transferases that contain the Yc subunit are able to act simultaneously as both enzymes and binding proteins. In addition to enzyme structure, the state of ionization of the non-substrate ligands may also influence whether the transferases can perform both functions simultaneously.     

Evidence for multiple enzymes in the dolichol utilizing pathway of glycoprotein biosynthesis

A comparison has been made of the enzymes catalyzing the transfer of manose, glucose and N-acetylglucosamine from, respectively, GDPmannose, UDP-glucose and UDP-N-acetylglucosamine to endogenous dolichol phosphate (Dol-P) in liver Golgi membranes. Evidence is presented which suggests that all three reactions utilize the same pool of Dol-P. The transfer of mannose from GDP-Man to Dol-P is not inhibited by 0.1 mM UDP or UMP; 0.1 mM GDP did block the accumulation of mannose in Dol-P-Man. The net transfer of glucose and N-acetylglucosamine to Dol-P is prevented by 0.1 mM UDP but not 0.1 mM GDP. UDPglucose inhibits the reverse of the glucose transfer reaction but not reverse of the N-acetylglucosamine or mannose transfer reaction. On the basis of this, and other data, it is concluded that the three sugar transfer reactions utilize separate enzymes.     

The co-purification and common identity of cholyl CoA:glycine- and cholyl CoA:taurine-N-acyltransferase activities from bovine liver.

A procedure for the purification of cholyl CoA:glycine and taurine N-acyltransferase activities from the soluble cell fraction of bovine liver is described. The procedure results is an 900-fold enrichment relative to the soluble cell fraction. The final preparation gives a single band on sodium dodecyl sulfate-polyacrylamide gel electrophoresis, Mr = 50,900, and runs as a single peak, Mr = 47,000, on gel filtration. The preparation is approximately 80% pure as judged by isoelectric focusing and focuses at a pH of 6.6. The glycine and taurine conjugating activities co-purified and did not separate to any extent in any of the chromatographic steps employed, including a gradient elution from an affinity column and an isoelectric focusing column. Also, kinetic analysis revealed that glycine and taurine appear to compete for a common active site. The two activities had identical temperature-denaturation curves and were equivalently stabilized against temperature denaturation by taurocholate. This data provides strong evidence for a common enzyme for both glycine and taurine conjugation in bovine liver. A preliminary kinetic characterization of the enzyme revealed non-Michaelis-Menten kinetics.     

Membrane-bound estrone as substrate for microsomal UDP-glucuronyltransferase.

Nonpolar substrates of microsomal UDP-glucuronyl-transferase partition between the hydrophobic phase of the microsomal membrane and the bulk aqueous phase in a suspension of microsomes in water. Partitioning of estrone into the membranes was measured in the studies presented and was extensive. Comparison of the rate of conjugation of estrone and the rate of its release from microsomes into the bulk aqueous phase showed that the pool of estrone within the membrane is the substate for UDP-glucuronyl-transferase. The rate of conjugation of estrone was 6-fold greater than the rate of release of estrone from the membrane into the aqueous phase. Several additional experiments showed that the rate of glucuronidation of estrone did not depend on the amount of estrone in the bulk aqueous phase. It is concluded that the microsomal membrane serves to concentrate nonpolar substrates of UDP-glucuronly-transferase. The phospholipid region of the microsomal membrane also may be a co-factor of UDP-glucuronyltransferase in the sense that binding of estrone to the membrane restricts its orientation in a manner that facilitates catalysis.