Correlation of conformational changes in the acrosome stabilizing factor (ASF) with its biological activity

The rabbit Acrosome Stabilizing Factor (ASF) is a glycoprotein synthesized in the corpus epididymis that reversibly decapacitates sperm. The effects of altering the conformation of ASF were evaluated by using a competitive enzyme-linked immunoabsorption assay (ELISA) with monoclonal antibodies that recognized either sequential or conformational determinants and/or an in vivo decapacitation assay. Heat denaturation (80 degrees C for 30 min) of affinity-purified ASF resulted in destruction of its native conformation concurrent with its loss of biological activity. Acid pH treatment of ASF also resulted in a conformational change in ASF, which caused a shift from the dimeric form (MW = 260,000) to the monomeric form (MW = 130,000). This manipulation allowed the biological activity of the monomeric form of ASF to be assayed separately from the dimer. The monomer was found to be biologically inactive. Proteolysis with trypsin or Staphylococcus-V8 treatment resulted in loss of the native conformation of the molecule, but Staphylococcus-V8 did not destroy the sequential determinant recognized in this analysis. This work indicates that conformation of the ASF macromolecule is important for its biological activity, and also provides a rapid means to evaluate potential decapacitation activity of purified ASF.     

Androgenic regulation of luteinizing hormone secretion: relationship to androgen binding in sheep pituitary

Castrated ram lambs (wethers) were investigated for sensitivity to androgen feedback and to determine whether this feedback inhibition of luteinizing hormone (LH) was associated with changes in pituitary androgen receptors. Administration of Silastic capsules containing either dihydrotestosterone or testosterone was found to produce dose-dependent inhibitory effects on serum LH levels in wethers. Physiological dosages of these androgens (i.e., those that produce serum levels of dihydrotestosterone [0.24 ng/ml] or testosterone [2.1 ng/ml] similar to those of intact rams) resulted in differential inhibition of serum LH and LH content of the anterior pituitary. Whereas the inhibitory effect of dihydrotestosterone on pituitary LH content was much more dramatic than that seen with testosterone, the high dosage of testosterone also produced a substantial decrease in pituitary LH content. Responses of the pituitary to changes in serum androgen were compared to responses of the seminal vesicle, which served as a control androgen target organ. Androgen levels were positively correlated with seminal vesicle weights, but pituitary weights were unaffected by castration and/or androgen replacement. Treatments with dihydrotestosterone were associated with decreased cytosol androgen binding activity (i.e., receptors) in pituitary and seminal vesicle, suggesting that both of these tissues were sites of androgen action. Although testosterone inhibited serum LH levels, pituitary cytosol androgen receptors were not affected by changes in serum testosterone. We conclude from these data that dihydrotestosterone is a physiological regulator of pituitary LH secretion in the ram and that further study is needed to investigate the complex actions of testosterone and its metabolites on pituitary function.     

Oxytocin response to conditioned and nonconditioned stimuli in lactating ewes

We have measured oxytocin release during lactation in the ewe in response to normal tactile sucking stimuli as well as exteroceptive stimuli emanating from the lamb. Four puerperal ewes that had indwelling catheter inserted in the femoral artery while still pregnant were used. Each nursed a single lamb. Each was studied 2 or 3 times between Days 1 and 15 of lactation during a 2.5-h experimental period that was preceded by a 2-h separation from the lamb in the early morning. Samples were taken before and after the lamb was brought within sight and sound of the ewe but without contact, and then 0.5, 1, 5, 10, 15, 30, and 60 min after suckling began. When another suckling episode intervened, the same sampling schedule was immediately restarted. Suckling occurred in an intermittent fashion; 3 to 4 episodes of 2.9 +/- 2.0 (SD)-min duration took place with variable intervals during the 2.5-h experimental period. Exteroceptive stimuli emanating from the lamb caused plasma oxytocin to rise significantly from basal levels of 10.0 +/- 4.5 to 21.8 +/- 5.7 pg/ml (mean +/- SE, n 10, p less than 0.05). This rise was not seen on Day 1 and in only half of the ewes on Day 2, but thereafter the rise occurred in every instance. A further rise in plasma oxytocin was observed in almost all instances (86%) at suckling. Peak levels were usually observed within 1 min. They were quite variable, ranging from 15 pg/ml to 287 pg/ml, and not related to the milk yield, but were significantly greater than spontaneous pulses observed in nonlactating puerperal sheep.(ABSTRACT TRUNCATED AT 250 WORDS)     

Structure and dynamics of one-dimensional ionic solutions in biological transmembrane channels.

The structure and dynamics of solvated alkali metal cations in transmembrane channels are treated using the molecular dynamics simulation technique. The simulations are based on a modified Fischer-Brickmann model (Fischer, W., and J. Brickmann, 1983, Biophys. Chem., 18:323-337) for gramicidin A-type channels. The trajectories of all particles in the channel as well as two-dimensional pair correlation functions are analyzed. It is found from the analysis of the stationary simulation state that one-dimensional solvation complexes are formed and that the number of water molecules in the channel varies for different alkali metal cations.     

On the small-sample performance of Efron's and of Gill's version of the product limit estimator under nonproportional hazards

Various proposals have been made to extend the product limit estimator to survival times beyond the largest observation in case that observation is censored. Two extreme extensions are examined with respect to bias and mean squared error (MSE). Their quality depends considerably on the "censoring constellation." The MSE of one extension appears to be robust against a wide variety of nonproportionalities of the hazard rates of the distributions of lifelength and time to censoring.     

The isolation and characterization of 2-carboxyethyl adducts following in vitro reaction of acrylic acid with calf thymus DNA and bioassay of acrylic acid in female Hsd:(ICR)Br mice

Reaction of acrylic acid (AA) at pH 7.0 and 37 degrees C for 40 days with 2'-deoxyadenosine (dAdo), 2'-deoxycytidine (dCyd), 2'-deoxyguanosine (dGuo) and thymidine (dThd) resulted in the formation of 2-carboxyethyl (CE) adducts via Michael addition. The alkylated 2'-deoxynucleoside adducts isolated (percent yield after 40 days) were 1-CE-dAdo (5%), N6-CE-dAdo (11%) (via Dimroth rearrangement of 1-CE-dAdo), 3-CE-dCyd (7.5%), 7-CE-Gua (4%), 7,9-bis-CE-Gua (0.9%) (formed by reaction of AA with depurinated 7-CE-Gua during the course of the reaction) and 3-CE-dThd (0.5%). The products isolated following in vitro reaction of AA with calf thymus DNA at pH 7.0 and 37 degrees C for 40 days were (nmol/mg DNA) 1-CE-Ade (9.9), N6-CE-Ade (8.2), 7-CE-Gua (7.2) and 3-CE-Thy (1.9). Compound 3-CE-Cyt was not detected. Thus the adducts formed following in vitro reaction of AA with DNA are identical to those formed by in vitro reaction of the carcinogen beta-propiolactone (BPL) with DNA as reported in an earlier paper. Structures were assigned on the basis of identical UV spectra, Rf values on paper chromatograms and Rt values on HPLC as marker compounds prepared from reactions of BPL with 2'-deoxynucleosides and 2'-deoxynucleotides-5'-monophosphoric acids. AA was assayed for carcinogenic activity by s.c. injection (20 mumol, once a week for 52 weeks) in female Hsd: (ICR)Br mice. Two mice with sarcomas at the site of application were observed out of 30 mice. Malignancies were not observed in solvent and no-treatment controls. The bioassay results reported in this paper and elsewhere in the same strain of mice suggest that AA is a weak carcinogen in female Hsd:(ICR)Br mice.     

Gallbladder pressure, compliance, and hysteresis during cyclic volume change

The gallbladder has both storage and contractile properties, but is pressure-volume characteristics have not been fully described. We studied gallbladder pressure, compliance, stress relaxation, and the work performed during infusion-withdrawal of fixed volumes of bile at increments of base-line volume under halothane anesthesia in 13 dogs. Two cannulae were inserted in the gallbladder fundus: one for cyclic infusion-withdrawal of bile, and one for pressure monitoring. Following ligation of the cystic duct, the gallbladder was fully aspirated and then filled to successive predetermined base-line volumes (15, 20, 25, 30, or 35 mL). At each base-line volume, 5 mL of bile was infused and withdrawn at 2.47 mL/min. Studies were performed under basal conditions and with cholecystokinin octapeptide (CCK) infusion (10 or 30 ng.kg-1.h-1iv). Pressure was measured at base-line, after infusion, and after withdrawal. Compliance during infusion (delta V/delta P) was calculated for each cycle. Stress relaxation was defined as the difference between base-line pressure and any reduction in pressure after withdrawal. Hysteresis, the difference between work of infusion and work of withdrawal, was calculated. The results were such that base-line, end-infusion, and end-withdrawal pressures; work of infusion, work of withdrawal, and hysteresis; and stress relaxation all increased significantly with increases in the predetermined base-line volumes (p less than 0.001). Compliance decreased significantly (p less than 0.001) with increasing base-line volume. CCK at 10 ng.kg-1.h-1iv had no effect, but infusion of CCK at 30 ng.kg-1.h-1 significantly (p less than 0.05) increased pressure and work, decreased compliance, and increased stress relaxation compared with controls.(ABSTRACT TRUNCATED AT 250 WORDS)     

Ethanol vapours above lacrimal fluid in the rabbit

Ethanol was administered intravenously to rabbits. The concentration of ethanol, determined by gas chromatographic analysis, in lacrimal fluid was shown to reflect the concentration in plasma. The vapour above lacrimal fluid was analyzed in situ by the use of a small resistivity sensor that measures ethanol vapours. After a dose of approximately 750 mg/kg, the metabolic rates of ethanol determined by gas chromatographic analysis of plasma (226 +/- 13 mg.kg-1.h-1) and by eye ethanol vapour analysis (210 +/- 8 mg.kg-1.h-1) were virtually identical. The data suggest that ethanol eye vapour analysis may be an attractive, noninvasive method for the determination of ethanol in animals.     

On the shoulders of giants: the discovery of atrial natriuretic factor

Investigations culminating at the beginning of this century clearly established that the cardiac muscle cell (cardiocytes) is differentiated for excitation, conduction, and contraction. All of the physiology and pathophysiology of the heart was developed subsequently based on this concept. However, morphological investigations in the mid 1950s suggested a secretory function for mammalian atrial cardiocytes. These cells contain storage granules, the specific atrial granules, which resemble granules found in polypeptide hormone-producing cells. The development of techniques for the study of these granules using a combined biochemical-morphological approach during the 1970s defined their general chemical nature and their behaviour under different experimental conditions. Because the number of atrial granules change dramatically following upsets of water and electrolyte balance, atrial muscle extracts were tested for effects on kidney function. In 1981, it was reported that atrial extracts contain a natriuretic factor (ANF) capable of inducing massive diuresis, increases in hematocrit, and lowering of blood pressure. It was demonstrated soon thereafter that ANF is stored within specific atrial granules. More recent work has defined ANF as a polypeptide hormone that appears to modulate or antagonize the renin-angiotensin-aldosterone system. Current work attempts to define the physiological and pathophysiological role for ANF as well as possible therapeutic uses.