Epizooic metazoan meiobenthos associated with tubeworm and mussel aggregations from cold seeps of the northern Gulf of Mexico.

The abundance and higher taxonomic composition of epizooic metazoan meiobenthic communities associated with mussel and tubeworm aggregations of hydrocarbon seeps at Green Canyon, Atwater Valley, and Alaminos Canyon in depths between 1400 and 2800 m were studied and compared to the infaunal community of non-seep sediments nearby. Epizooic meiofaunal abundances of associated meiobenthos living in tubeworm bushes and mussel beds at seeps were extremely low (usually <100 ind. 10 cm(-2)), similar to epizooic meiofauna at deep-sea hydrothermal vents, and the communities were composed primarily of nematodes, copepods, ostracods, and halacarids. In contrast, epizooic meiobenthic abundance is lower than previous studies have reported for infauna from seep sediments. Interestingly, non-seep sediments contained higher abundances and higher taxonomic diversity than epizooic seep communities, although in situ primary production is restricted to seeps.

Determination of concentrations of sex steroids in blood plasma and semen of male dogs treated with delmadinone acetate or finasteride.

The concentrations of testosterone, 5 alpha-dihydrotestosterone, oestradiol and oestrone were determined in peripheral blood plasma and semen of male dogs. In an experimental study, three Beagles were treated once with delmadinone acetate (1 mg kg-1 body weight, i.m.) and three were submitted to oral applications of finasteride (1 mg kg-1 body weight) once a day for 3 weeks. In a clinical study, 51 dogs of different breeds were divided into four groups according to the total number of spermatozoa in ejaculates (normospermia, slight oligozoospermia, severe oligozoospermia and azoospermia). The testosterone concentrations were significantly lower in sperm-rich ejaculate fractions and prostatic secretions compared with blood plasma (P < 0.05). The lowest concentration of testosterone was found in prostatic fluid. Concentrations of 5 alpha-dihydrotestosterone were similar in blood plasma and sperm-rich fractions, and significantly lower in prostatic secretions (P < 0.05). The concentrations of oestradiol and oestrone did not differ between blood plasma and either ejaculate fraction. Significantly higher 5 alpha-dihydrotestosterone concentrations and significantly lower concentrations of oestradiol and oestrone were found in prostatic secretions from azoospermic ejaculates compared with prostatic secretions of normospermic and oligozoospermic ejaculates. Delmadinone acetate and finasteride caused reversible suppression of the secretory activity of the prostate gland. The application of delmadinone acetate led to a temporary alteration of maturation of epididymal spermatozoa.

Development of a predictive statistical model for the analgesic activity of a family of imides.

Quantitative structure-activity relationship (QSAR) studies were done with a family of cyclic imides. Promising efforts to create a QSAR model with substantial predictive power for the design of novel cyclic imides with improved analgesic activity are reported.

Structure-activity relationships for a class of inhibitors of purine nucleoside phosphorylase.

Values of inhibition constants, Ki, for a family of structurally related, competitive inhibitors of calf spleen purine nucleoside phosphorylase (PNP) have been determined employing both inosine as substrate and a manual assay and 2-amino-6-mercapto-7-methylpurine ribonucleoside (MESG) as substrate and a robot-based enzyme kinetics facility. Several of the values determined robotically were confirmed employing the same substrate and a manual assay. Surprisingly, for many of the inhibitors examined, values of Ki determined with MESG as substrate are smaller than those obtained employing inosine as substrate by a factor that varies from less than 2 to 10. Values of concentrations required for 50% inhibition of PNP, IC50, have also been determined for the same family of inhibitors employing inosine as substrate. Values of IC50ino and those for Kiino and Kimesg for subsets of the inhibitors have been employed as training sets to create quantitative structure-activity relationships (QSAR) which have substantial power to predict values of IC50 and Ki for inhibitors outside the training set. These QSAR models should be useful in guiding future medicinal chemistry efforts designed to discover inhibitors of PNP having increased potency.

Magnetic resonance imaging of the efficacy of specific inhibition of 5 alpha-reductase in canine spontaneous benign prostatic hyperplasia.

A leading role for prostatic levels of dihydrotestosterone (DHT) in the pathogenesis of benign prostatic hyperplasia is well established, if incompletely understood. The present study provides initial confirmation that 5 alpha-reductase inhibition alone is sufficient to prevent prostatic accumulation of DHT and to produce epithelial regression in the canine prostate. In dogs treated with the specific 5 alpha-reductase inhibitor finasteride, prostatic volume decreased to one-third of the baseline volume, while the prostatic concentration of DHT fell fivefold: both were constant in placebo control dogs. Demonstration that MR imaging can serve as accurate modality to assess prostatic volume was provided by serial measurements of the canine prostate and by correlation of the last imaging measurement with the weight of the excised prostate. Significant intensity changes were observed in T2-weighted images measured post-treatment; these changes correlated with the histopathology of the prostate. These results suggest that beyond quantifying regression, multiecho T2 measurements can be useful in probing accompanying changes occurring on the cellular level.

Inhibition of rabbit lung angiotensin-converting enzyme by N alpha-[(S)-1-carboxy-3-phenylpropyl]L-alanyl-L-proline and N alpha-[(S)-1-carboxy-3-phenylpropyl]L-lysyl-L-proline.

Two novel peptide analogs, N alpha-[(S)-1-carboxy-3-phenylpropyl]L-alanyl-L-proline and the corresponding L-lysyl-L-proline derivative, have been demonstrated to be potent competitive inhibitors of purified rabbit lung angiotensin-converting enzyme: Ki = 2 and 1 X 10(-10) M, respectively, at pH 7.5, 25 degrees C, and 0.3 M chloride ion. Second-order rate constants for addition of these inhibitors to enzyme under the same conditions are in the range 1-2 X 10(6) M-1 s-1; first-order rate constants for dissociation of the EI complexes are in the range 1-4 X 10(-4) s-1. The association rate constants are similar to those measured for D-3-mercapto-2-methylpropanoyl-L-proline, captopril, but the dissociation rate constants are severalfold slower and account for the higher affinity of these inhibitors for the enzyme. The dissociation constant for the EI complex containing N alpha-[(S)-1-carboxy-3-phenylpropyl]L-alanyl-L-proline is pH-dependent, and reaches a minimum at approximately pH 6: Ki = 4 +/- 1 X 10(-11) M. The pH dependence is consistent either with a model for which the protonation state of the secondary nitrogen atom in the inhibitor determines binding affinity, or one for which ionizations on the enzyme alone influence affinity for these inhibitors. The affinity of this inhibitor for the zinc-free apoenzyme is 2 X 10(4) times less than for the zinc-free apoenzyme is 2 X 10(4) times less than that for the holoenzyme. If considered as a "collected product" inhibitor, N alpha-[(S)-1-carboxy-3-phenylpropyl]L-alanyl-L-proline appears to derive an additional factor of 375 M in its affinity for the enzyme compared to that of the two products of its hypothetical hydrolysis, a consequence of favorable entropy effects.

Purification of angiotensin-converting enzyme from rabbit lung and human plasma by affinity chromatography.

Lisinopril (N alpha-[(S)-1-carboxy-3-phenylpropyl]L-lysyl-L-proline), a potent angiotensin-converting enzyme inhibitor, is an exceptionally selective affinity chromatography ligand for this enzyme. Affinity chromatography furnishes electrophoretically homogeneous enzyme directly from crude homogenates of rabbit lung tissue, a 1,000-fold purification; also, it affords a 100,000-fold enrichment of the more rare human plasma enzyme in a single step. The affinity of angiotensin-converting enzyme for the Sepharose-spacer-lisinopril matrix (Ki matrix = 1 X 10(-5) M) is weak compared to its affinity for free lisinopril (Ki = 1 X 10(-10) M). The capacity of the affinity column is described quantitatively as a function of Ki matrix, lisinopril, and enzyme concentrations. The recovery of bound enzyme is low in chromatography of crude tissue samples (10-40%), although it approaches a reversible process (70-100%) with pure enzyme. The holoenzyme is converted to Zn2+-free apoenzyme to effect removal of lisinopril. In this process, the rate constant for spontaneous dissociation of Zn2+ from free enzyme is 1 X 10(-2) s-1 (t 1/2 = 1 min), which places a lower limit of 3 X 10(-10) M on the dissociation constant of Zn2+ at neutral pH from angiotensin-converting enzyme. The exceptional selectivity of lisinopril as an affinity chromatography ligand for angiotensin-converting enzyme suggests it is among the most specific inhibitors designed for any enzyme.

The design and properties of N-carboxyalkyldipeptide inhibitors of angiotensin-converting enzyme.

Angiotensin-converting enzyme inhibitors promise to make important therapeutic contributions to the control of hypertension and congestive heart failure. The nonapeptide teprotide was the first of these inhibitors to be tested clinically. It was followed by orally active inhibitors, captopril in 1977 and enalapril in 1980. The latter is representative of a new design for the inhibition of metallopeptidases and is the subject of this review. The best of the N-carboxyalkyldipeptide inhibitors inhibits angiotensin-converting enzyme with a Ki of 7.6 X 10(-11) M. This compound is the most potent competitive inhibitor of a metallopeptidase yet to have been reported. The basis of this high potency is beginning to be understood and in part is considered to involve precisely arranged multiple interactions within the enzyme active site. X-ray crystallography of a thermolysin-inhibitor complex has been achieved. Assuming that similar interactions within the active site of angiotensin-converting enzyme are mechanistically probable, the authors hypothesize the binding of enalaprilat to converting enzyme as shown in Figure 24. Such interactions are consistent with kinetic studies (Section V) with the understanding that binding to the enzyme is not sensitive to the inhibitor's state of NH protonation. The reason for this surprising conclusion has not been established. Perhaps counterbalancing factors are involved in the energetics of binding or there may be compensating adjustments made in the enzyme which permit NH protonated and nonprotonated inhibitor to bind equally well. Figure 24 also summarizes present understanding of the conformation of enalaprilat when bound to angiotensin-converting enzyme. From studies on conformationally defined analogs of enalaprilat, it seems likely that the Ala-Pro segment of enalaprilat binds in a conformation that is close to a minimum energy conformer. This situation no doubt contributes to the potency of enalaprilat, since little binding energy would be needed to induce conformational changes in this part-structure of enalaprilat when it is bound to the enzyme. The phenethyl group of enalaprilat is believed to be near the alpha-hydrogen of the L-Ala residue in the enzyme-inhibitor complex. However, the synthesis of conformationally restricted analogs to establish this point has not yet been reached. The N-carboxyalkylpeptide design was developed from Wolfenden's collected product inhibitors of carboxypeptidase-A. Whether or not N-carboxyalkyldipeptides should be classified as collected product or transition state inhibitors is unclear.(ABSTRACT TRUNCATED AT 400 WORDS)

Effects of exogenous growth hormone and diethylstilbestrol on growth and carcass composition of growing lambs.

An 8-wk growth trial was conducted to assess the effects of ovine growth hormone (oGH; 7 mg/d, sc) on growth performance and carcass composition of normal, growing wether lambs. Diethylstilbestrol (DES; .1 mg/d, sc) and control lambs were included for comparisons. Plasma oGH levels at 8 wk were 1.9, 5.5 (P less than .05) and 138.1 ng/ml (P less than .001) for controls, DES and oGH lambs, respectively. Diethylstilbestrol did not increase plasma oGH until the fourth week. The oGH improved feed conversion 7.4% (FC; P less than .05), but did not alter average daily gain (ADG) or feed intake (ADF). Diethylstilbestrol increased ADG 15.3% (P less than .05) and improved FC 16.1% (P less than .01), with no effect on ADF. The primary effect of oGH on carcass composition was to decrease the quantity of fat 8.9% (P less than .05). In addition, oGH may have increased protein 6.5% (P less than .10) and moisture 4.0% (not significant). Diethylstilbestrol increased the quantity of carcass protein 10% (P less than .01) and moisture 8.7% (P less than .05), with no effect on fat. In these studies, the primary effect of exogenous oGH on normal, growing lambs was to reduce carcass fat, which may account for the observed improvement in FC. Diethylstilbestrol, at 1/70th of the oGH dose, was superior to oGH for improving FC (P less than .05) and ADG (P less than .10). Improvements in body weight of the lambs given DES were observed 2 wk before an increase in plasma oGH. In addition, DES, unlike exogenous oGH, did not alter the quantity of carcass fat. These observations do not support the concept that the mode of action of DES is through increased GH secretion.

Molecular and catalytic properties of rabbit testicular dipeptidyl carboxypeptidase.

Rabbit testicular dipeptidyl carboxypeptidase activity was purified by a procedure exploiting its affinity for N-alpha-[1-(S)-carboxy-3-phenylpropyl]-L-lysyl-L-proline. The molecular, catalytic, and immunological properties of the testicular enzyme are presented and compared with the corresponding properties of pulmonary angiotensin-converting enzyme. Although catalytically similar and immunologically related to pulmonary dipeptidyl carboxypeptidase, the testicular enzyme has a molecular weight (100,000) which is lower by a factor of about one-third and differs in its NH2 and COOH termini. Furthermore, we present evidence that the testicular enzyme is not a post-translation product of the pulmonary type enzyme. These data suggest that testicular and pulmonary dipeptidyl carboxypeptidase are two distinct proteins which are catalytically similar and immunologically closely related.

Carbon-13 nuclear magnetic resonance studies of cholesterol-egg yolk phosphatidylcholine vesicles.

Proton-decoupled natural-abundance (13)C nuclear magnetic resonance spectra at 63 KG were obtained for isolated single-bilayer egg yolk phosphatidylcholine-cholesterol vesicles containing a variable phospholipid/cholesterol ratio. Numerous well-resolved singly carbon resonances of phospholipid and cholesterol carbons were observed. Carbon resonances from different parts of the phospholipid show markedly different behavior as a function of cholesterol content of the vesicles. The line widths of resonances for carbon atoms in the head-group region and the sn-3 carbon of the phospholipid glycerol backbone are relatively independent of cholesterol content. In contrast, resonances from the sn-1 and sn-2 carbon atoms of the glycerol backbone and the envelopes containing the olefinic and aliphatic carbon resonances of the fatty acyl chains of the phospholipids broaden markedly with increasing content of cholesterol. The most prominent cholesterol ring resonance is that for C6. This is, in part, due to its location in a clear window of the spectrum, where it is unobscured by interfering phospholipid resonances. However, resonances for cholesterol ring carbons C9 and C14, 17, which should also appear in clear regions of the spectrum, are not observable. It is suggested that these resonances are broadened by dipolar interactions with neighboring protons. Anisotropic rotation of the cholesterol molecule about its long axis is suggested to be the major mechanism responsible for decreased dipolar interactions for the C6 carbon, while retaining the large dipolar coupling of the C9 and C14, 17 carbons with their neighboring protons. The temperature dependence of spectra of single-bilayer phosphatidylcholine vesicles containing epicholesterol (alpha-cholesterol) is different from that of corresponding cholesterol-containing vesicles. Resonances from the C9 and C14, 17 carbons of epicholesterol in EYPC vesicles are detectable at 35 degrees C whereas the corresponding resonances from beta-cholesterol are not. These data suggest that in vesicles the rotation of cholesterol is more anisotropic than that of epicholesterol and that the stereochemistry of the C3 hydroxyl group of cholesterol is at least partly responsible for the highly anisotropic rotation of the steroid ring within the bilayer.

Kinetic alpha-deuterium isotope effects for Escherichia coli purine nucleoside phosphorylase-catalyzed phosphorolysis of adenosine and inosine.

Kinetic alpha-deuterium isotope effects have been measured for the purine nucleoside phosphorylase-catalyzed phosphorolysis of adenosine and inosine by a competitive double label technique at saturating concentrations of the second substrate, phosphate. Under these conditions the observed isotope effect, kH/kD, is on the second order rate constant, Vmax/Km, for reaction of nucleoside with the Michaelis complex of enzyme and phosphate. For adenosine, at neutral pH, the isotope effect is unity. For inosine, kH/kD was determined as a function of pH (the numbers in parentheses are the ratios of Vmax at that pH to Vmax at pH 7.3): 1.10 at pH 5.0 (0.19); 1.10 at pH 6.1 (0.72); 1.01 at pH 7.3 (1.00); 1.16 at pH 8.4 (0.22); and 1.18 at pH 9.4 (0.04). These values suggest a mechanism for purine nucleoside phosphorylase involving a change in rate-limiting step from one at pH values near neutrality for which cleavage of the nucleoside C-N bond is not rate limiting to a step at extremes of pH with a transition state having considerable oxocarbonium ion-like character.

Lipoprotein-X: carbon-13 nuclear magnetic resonance studies on native, reconstituted, and model systems.

Lipoprotein-X (LP-X), a lipoprotein isolated from human cholestatic plasma by ethanol--acetate precipitation and zonal ultracentrifugation, has been studied by 13C NMR at 67.9 MHz. Spectra of LP-X and its three subfractions are markedly different from those of normal human high-density lipoprotein3 (HDL3) or low-density lipoprotein (LDL). Spectra of LP-X are characterized by the presence of unusually broad resonance lines, especially those attributable to C6 of unesterified cholesterol (160--260 Hz) and to C beta of phospholipid glyceride (240--290 Hz). In contrast, the CH2O, CH2N, and N(CH3)3 choline resonances have line widths comparable to those of normal LDL and HDL3. For the subfraction LP-X1, spin--lattice relaxation times (T1) of the fatty acyl olefin resonances at 129.8 and 128.0 ppm and of the unesterified cholesterol C6 at 120.1 ppm were measured to be 675, 766, and 162 ms, respectively. These times are comparable to those measured for the corresponding resonances in single bilayer vesicles whose lipid composition approximates that of LP-X. The three LP-X subfractions isolated by zonal ultracentrifugation gave spectra which are identical, within experimental error, as judged qualitatively from their appearance and quantitatively from the line widths of selected resonances. In addition, 13C NMR spectra of sonicated total LP-X lipids are similar to spectra of the intact native lipoprotein. This study suggests (a) that motions of lipids in LP-X as probed by 13C NMR are similar to the motions of lipids found in model vesicular systems, (b) that the motions of the cholesterol rings and phospholipid fatty acyl chains are significantly more restricted in LP-X than in HDL3 and LDL, and (c) that the motions of the phosphoryl moieties in all three systems are similar.

Lipoprotein-X: proton and phosphorus-31 nuclear magnetic resonance studies on native, reconstituted, and model systems.

LP-X, a lipoprotein present in the low-density range (d 1.006--1.063 g/mL) of cholestatic human plasma, has been studied with its normal counterpart (LDL) by 1H and 31P nuclear magnetic resonance. The 220-MHz 1H spectrum of LP-X contains four major lines: the choline CH2N and N+(CH3)3 resonances and the cholesteryl--acyl CH2 and CH3 envelopes. The widths of these four lines at 37 degrees C are approximately 24, 10, 124, and 48 Hz, respectively. The latter two line widths are much greater than the corresponding ones of LDL (28 and 20 Hz), suggesting the much more restricted motion of acyl chains and/or cholesteryl rings in LP-X. This difference persists over the temperature range 15--52.5 degrees C. The microscopic fluidity of LP-X and LDL was compared by titration with 2,2,6,6-tetramethylpiperidinyl-1-oxy (Tempo), a paramagnetic amphiphile which distributes between the bulk aqueous phase and the fluid lipid phase of lipoproteins. Tempo is much less effective in broadening the 1H resonances of LP-X than of LDL, indicating the lower permeability/fluidity of the former. The 40.5-MHz 31P spectrum of LP-X consists of a single resonance whose line width is approximately 20 Hz and whose spin--lattice relaxation time is 2.23 +/- 0.15 s. Titration of LP-X with Pr3+ ions splits this resonance into two lines, one remaining at the chemical shift of the original resonance and the other paramagnetically shifting downfield. The ratio of integrated areas for these two lines was 1:1.72. Titration of phosphatidylcholine--cholesterol vesicles alone, vesicles containing apolipoprotein-C and albumin, or vesicles containing apolipoprotein-X gave results similar to those obtained with native LP-X, suggesting the presence of a single bilayer structure in all of these systems.

Alteration of the fatty acid composition of Escherichia coli by growth in the presence of normal alcohols.

The addition of normal alcohols in the series n-butanol to n-octanol to cultures of Escherichia coli ML308 grown on defined or lipid-free medium (at 17, 27, and 37 degrees C) caused an alteration in the fatty acid composition of this organism: the ratio of saturated to unsaturated fatty acids increased. Changes in the relative quantities of individual fatty acid species elicited by increasing concentrations of these alcohols were as follows: (i) myristic acid remained constant: (ii) palmitic acid increased; and (iii) the combined amount of palmitoleic plus cis-methylene hexadecanoic acids changed in a way which was reflected inversely by changes in the amount of cis-vaccenic acid. Comparable changes were not observed when cells were grown in the presence of n-nonanol and n-decanol in the concentration range tested. The changes observed upon addition of normal alcohols (n-butanol to n-octanol) paralleled, in part, the alterations in fatty acid composition observed when growth temperature was increased.