Dynamic appearance of [4-14C] dehydroepiandrosterone and [7 alpha-3H] dehydroepiandrosterone sulphate metabolites in urine of normal and obese female subjects.

[4-14C]-Dehydroepiandrosterone and [7 alpha-3H]-Dehydroepiandrosterone sulphate were injected simultaneously to normal and obese female subjects. The percentage recovery of 14C and 3H radioactivies in dehydroepiandrosterone sulphate, androsterone sulphate, etiocholanolone sulphate, androsterone glucuronoside and etiocholanolone glucuronoside was determined in the day-to-day urine collections for 72 hr. Results showed a normal total 3H recovery and a poor 14C recovery in urinary conjugates of obese patients. The rate of appearance of 3H activity was not identical in the individual metabolites of normal subjects, and it was not normal in obesity. Overweight subjects exhibited an acceleration in [7 alpha-3H]-Dehydroepiandrosterone sulphate metabolism to androsterone glucuronoside. The observation regarding the rate of appearance of urinary conjugates bearing 14C isotope correlate with our previous finding in which a glandular overproduction of free dehydroepiandrosterone was found and an uptake of this steroid by the adipose tissue was suggested. Our results showed that the poor recovery of 14C radioactivity in urine of obese female subjects was not an aspecific consequence of illness.     

Estrogen metabolism as measured in blood and urine in female rhesus monkeys

In order to measure the interconversions of estrone (E1) and estradiol (E2) and their conversion to the 16 alpha-hydroxylated estrogens, 16 alpha-hydroxy estrone (16 alpha-OHE1) and estriol (E3), we infused 11 female rhesus monkeys with [3H]E2 and [14C]E1 and measured radioactivity in the blood as E1, E2 and 16 alpha-OHE1 (n = 9) and in the urine as the glucuronides of E1, E2, 16 alpha-OHE1, and E3 (n = 11). The mean conversion of E1 to E2 as measured in blood (percent of infused E1 measured in blood as E2, [rho]1.2BB) was 29.2 +/- 1.6% and as measured in the urine of the same animals, [rho]1.2BM, was 77.4 +/- 5.9%. The mean conversion of E2 to E1, [rho]2.1BB was 21.5 +/- 1.0% and as measured in urine, [rho]2.1BM was 67.7 +/- 4.6%. Thus for both estrone and estradiol only 30-35% of the interconversions occurred in pools which were in equilibrium with the blood pool of these estrogens. The remaining 65-70% occurred in a pool, probably liver, in which glucuronidation occurred immediately after conversion. The conversion ratios (the ratio of the concentration in the blood of radioactivity as 16 alpha-OHE1 to its precursor, CRPrec,16 alpha-OHE1) was 0.036 +/- 0.008 for CRE1,16 alpha-OHE1 and 0.0039 +/- 0.0010 for CRE2,16 alpha-OHE1. The percentages of administered E1 excreted in the urine as the glucuronides of E1, E2, 16 alpha-OHE1 and E3 were 20.1 +/- 1.5, 1.6 +/- 0.2, 0.96 +/- 0.20 and 0.76 +/- 0.07 respectively. The percentages of administered E2 excreted in the urine as E1, E2, 16 alpha-OHE1 and E3 were 14.4 +/- 1.0, 2.2 +/- 0.3, 0.57 +/- 0.05 and 0.68 +/- 0.11 respectively. Thus there are minor differences in the patterns of excreted metabolites of E1 and E2. Furthermore, 16 alpha-OHE1 and E3 are not major metabolites of E1 or E2 in the female rhesus monkey.     

Estimating the contribution of mutation, recombination and gene conversion in the generation of haplotypic diversity

Recombination occurs through both homologous crossing over and homologous gene conversion during meiosis. The contribution of recombination relative to mutation is expected to be dramatically reduced in inbreeding organisms. We report coalescent-based estimates of the recombination parameter (rho) relative to estimates of the mutation parameter (theta) for 18 genes from the highly self-fertilizing grass, wild barley, Hordeum vulgare ssp. spontaneum. Estimates of rho/theta are much greater than expected, with a mean rho/theta approximately 1.5, similar to estimates from outcrossing species. We also estimate rho with and without the contribution of gene conversion. Genotyping errors can mimic the effect of gene conversion, upwardly biasing estimates of the role of conversion. Thus we report a novel method for identifying genotyping errors in nucleotide sequence data sets. We show that there is evidence for gene conversion in many large nucleotide sequence data sets including our data that have been purged of all detectable sequencing errors and in data sets from Drosophila melanogaster, D. simulans, and Zea mays. In total, 13 of 27 loci show evidence of gene conversion. For these loci, gene conversion is estimated to contribute an average of twice as much as crossing over to total recombination.     

Plasma and Urinary 11-Hydroxycorticosteroids in Differential Diagnosis of Cushing's Syndrome

Morning plasma 11-hydroxycorticoids, urinary 11-hydroxycorticoids, and urinary 17-oxogenic steroids were measured before and during a dexamethasone suppression test. This consisted in the administration by mouth of 2 mg of dexamethasone daily for 48 hours, followed by 8 mg daily for 48 hours. In addition midnight plasma 11-hydroxycorticoids were measured before the start of the test. The subjects investigated were 21 patients with Cushing''s syndrome, 27 obese female patients, 10 female patients with the Stein-Leventhal syndrome, and 8 female patients with idiopathic hirsutism.The results showed that the clearest distinction between the groups was made by measurement of the basal urinary 11-hydroxycorticoid excretion, where, in the group of patients with Cushing''s syndrome, all the levels were well above the upper limit of normal. In addition raised midnight plasma 11-hydroxycorticoid levels were of great diagnostic value. By using these results together with those of the dexamethasone suppression tests it was possible to make a firm preoperative diagnosis of pituitary-dependent Cushing''s syndrome in 90% of patients in this series.     

Isolation and partial characterization of Borrelia burgdorferi inner and outer membranes by using isopycnic centrifugation.

In order to characterize the protein composition of the outer membrane of Borrelia burgdorferi, we have isolated inner and outer membranes by using discontinuous sucrose density step gradients. Outer and inner membrane fractions isolated by this method contained less than 1 and 2%, respectively, of the total lactate dehydrogenase activity (soluble marker) in cell lysate. More importantly, the purified outer membranes contained less than 4% contamination by the C subunit of F1/F0 ATPase (inner membrane marker). Very little flagellin protein was present in the outer membrane sample. This indicated that the outer membranes were relatively free of contamination by cytoplasmic, inner membrane or flagellar components. The outer membrane fractions (rho = 1.19 g/cm3) contained 0.15 mg (dry weight) of protein per mg. Inner membrane samples (rho = 1.12 g/cm3) contained 0.60 mg (dry weight) of protein per mg. Freeze-fracture electron microscopy revealed that the outer membrane vesicles contained about 1,700 intramembranous particles per micron 2 while inner membrane densities for inner and outer membranes. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and nonequilibrium pH gel electrophoresis-SDS-PAGE analyses of inner and outer membrane samples revealed several proteins unique to the inner membrane and 20 proteins that localized specifically to the outer membrane. This analysis clearly shows that the inner and outer membranes isolated by this technique are unique structures.     

Mitochondrial membrane contact sites of yeast. Characterization of lipid components and possible involvement in intramitochondrial translocation of phospholipids

Submitochondrial membrane fractions from yeast that are enriched in inner and outer membrane contact sites were analyzed with respect to their lipid composition. Characteristic features were the significantly reduced content of phosphatidylinositol, the decreased amount of phosphatidylcholine, and the enrichment in phosphatidylethanolamine and cardiolipin. Coisolation of phosphatidylserine synthase with the outer membrane portion and enrichment of phosphatidylserine decarboxylase in the inner membrane portion of isolated contact sites provided the basis for a metabolic assay to study phosphatidylserine transfer from the outer to the inner mitochondrial membrane via contact sites. The efficient conversion to [3H]phosphatidylethanolamine of [3H]phosphatidylserine synthesized from [3H]serine in situ supports the notion that mitochondrial membrane contact sites are zones of intramitochondrial translocation of phosphatidylserine.     

Prolonged decrease in cardiac volumes after maximal upright bicycle exercise

Sequential exercise-gated cardiac blood pool scintigrams provide a noninvasive technique for evaluating the effect of therapeutic interventions on cardiac volumes and function only if both exercise periods are equivalent in the absence of an intervention. To assess whether they are indeed equivalent, 14 healthy subjects underwent gated blood pool scintigraphy during two maximal upright exercise periods separated by 60 min without changing position. Although resting cardiac output and blood pressure returned to base-line values 60 min after the first exercise period, mean resting heart rate was markedly higher (89.4 +/- 2.7 vs. 66.5 +/- 2.5 beats/min, P less than 0.001) and upright cardiac volumes lower [39.1 +/- 4.9 vs. 56.3 +/- 6.0 ml, P less than 0.001, for end-systolic volume (ESV) and 112.6 +/- 8.0 vs. 144.9 +/- 9.0 ml, P less than 0.001, for end-diastolic volume (EDV)] than before the first exercise period. These differences persisted during low levels of the subsequent exercise but not at high and maximum work loads. Cardiac volumes and heart rate 60 min after an identical exercise protocol in a second group of 22 subjects who received propranolol, 0.15 mg/kg iv, after their initial exercise, however, were the same as those preexercise. Thus higher sympathetic tone may be responsible for the persistently higher heart rate and decreased cardiac volumes after exercise, and the assumption that cardiac volumes and function are similar during two closely spaced sequential exercise studies is not always valid.     

Brown trout Salmo trutta released to support recreational fishing in a Norwegian fjord

One- and two-year-old hatchery reared juveniles of seven freshwater resident and anadromous populations of Scandinavian brown trout were released in the outer and inner part of the Oslofjord and in the River Akerselva, flowing through the city of Oslo. Recapture rates were highest (mean 20.3%) for river released fish and lowest for those released in the outer (16.8%) and inner (12.1%) fjord. In general, recapture rate increased with fish size at release (r=0.76). When released in fresh water, most of the recaptures were from fresh water and when released in the fjord, most recaptures were from the fjord. In general, freshwater resident stocks showed a higher degree of freshwater residency than anadromous stocks. However, mean migratory distance was longer for freshwater resident than anadromous fish. Trout moved longer distances at sea when released in the outer than in the inner fjord. Specific growth rate and size at recapture varied among release sites and stocks; they were highest for fish released in the outer fjord and lowest for river-released trout. There was no consistent difference in sea growth between freshwater resident and anadromous stocks. Estimated total yield was highest for fish released in the outer fjord, whereas there was no significant difference in yields between trout released in the river and the inner Oslofjord.     

Ketone body kinetics in humans: the effects of insulin-dependent diabetes, obesity, and starvation

The kinetics of acetoacetate (A) and beta-hydroxybutyrate (B) have been studied following the injection as a pulse or continued infusion of [3-14C]acetoacetate (A*) or [14C]beta-hydroxybutyrate (B*) into six newly diagnosed, untreated, ketotic diabetic patients, ten obese subjects in the postabsorptive state, and the ten obese subjects after 1-2 weeks starvation (50 cal per day). Employing a compartmental model of acetoacetate and beta-hydroxybutyrate kinetics developed using CONSAM for normal subjects, the rate coefficients (Lij), rates of release of newly synthesized acetoacetate and beta-hydroxybutyrate into the blood (UA, UB), and fractional removal of each compound (FCRA and FCRB) were calculated. Ketone body release into blood (UA + UB) in diabetic subjects was threefold higher than normal (mean +/- SD, 208 +/- 118 versus 81 +/- 66 mumol min-1 m-2) and in obese subjects the rate increased on starvation from 171 +/- 70 to 569 +/- 286 mumol min-1 m-2. In each case most of the increase was in beta-hydroxybutyrate. The major change in diabetes and on starvation of the obese subjects was in the rate coefficient for removal of ketone bodies. Normally 0.168 +/- 0.109 min-1, it was 0.055 +/- 0.040 min-1 in the diabetic patients and fell from 0.066 +/- 0.040 to 0.027 +/- 0.019 min-1 in the obese subjects on starvation. In normal subjects, FCRA was similar to FCRB (0.226 +/- 0.142 versus 0.188 +/- 0.124 min-1). However, in diabetics, FCRA was 0.074 +/- 0.044 and FCRB was 0.050 +/- 0.034 min-1 and both were lower than normal. On starvation of obese subjects, FCRA fell from 0.199 +/- 0.047 to 0.089 +/- 0.035 min-1, whereas FCRB fell from 0.141 +/- 0.040 to 0.033 +/- 0.012 min-1. Therefore, the removal of beta-hydroxybutyrate was impaired more than that of acetoacetate in all patients. Our results confirm previous observations that ketosis is associated with high rates of ketogenesis and a decrease in fractional clearance. In addition, we found that in diabetes, obesity, and in obese subjects following starvation, most of the increased synthesis was in beta-hydroxybutyrate and that the clearance of beta-hydroxybutyrate decreased more than that of acetoacetate.     

A competitive protein binding method for the measurement of unconjugated and sulfate-conjugated dehydroepiandrosterone in peripheral plasma

Dehydroepiandrosterone (D) can be readily measured in plasma by means of competitive protein binding analysis as its derivative, 5-androstenediol (5-Adiol; androst-5-en-3β, 17β-diol). Ether extraction of 1–5 ml plasma and thin layer chromatography are performed as previously described for testosterone and androstenedione with modifications permitting radiochromatogram scanning. The 17β-hydroxyl derivative of D, 5-Adiol, is then formed enzymatically. A glass fiber Chromatographie step separates 5-Adiol from the 17β-hydroxy derivatives of closely related 17-ketosteroids. Assay is then performed using “testosterone binding globulin” and 5-Adiol standards. The method possesses high accuracy, sensitivity, precision and specificity. Application of the method to the measurement of plasma D-sulfate (DS) after solvolysis yields results in agreement with analysis by gas-liquid chromatography and Zimmermann reaction. Less than 0.18% of DS-7a-³H contributes to D-7a-³H. D and DS values obtained in normal subjects correspond to those obtained by established, more complex methods.     

3-Methylhistidine turnover in the whole body, and the contribution of skeletal muscle and intestine to urinary 3-methylhistidine excretion in the adult rat.

The tissue origin of 3-methylhistidine (N tau-methylhistidine) was investigated in adult female rats. The decay of labelling of urinary 3-methylhistidine was compared with the labelling of protein-bound 3-methylhistidine in skeletal muscle and intestine after the injection of [methyl-14C]methionine. The decay curve for urinary 3-methylhistidine was much steeper than that in muscle or intestine, falling to values lower than those in either tissue after 30 days. The lack of decay of labelling in muscle during the first 30 days is shown to result from the persistence of label in the precursor S-adenosylmethionine. The relative labelling of urinary, skeletal-muscle and intestinal 3-methylhistidine cannot be explained in terms of skeletal muscle accounting for a major proportion of urinary 3-methylhistidine. Measurements were also made of the steady-state synthesis rate of protein-bound 3-methylhistidine in intestinal smooth muscle in vivo in adult female rats. This involved measurement of the overall rate of protein synthesis and measurement of the relative rates of synthesis of 3-methylhistidine and of mixed protein. The synthesis rate of 3-methylhistidine was 29.1%/day, compared with the overall rate of 77.1%/day for mixed, non-mucosal intestinal protein. Measurement of the amount of 3-methylhistidine in skeletal muscle (0.632 +/- 0.024 mumol/g) and in the whole body (0.332 +/- 0.013 mumol/g) indicate that, although the muscle pool is 86% of the total, because of its slow turnover rate of 1.1-1.6%/day, it only accounts for 38-52% of the observed excretion. Measurements of the mass of the intestine (9.95 g/250 g body wt.) and protein-bound 3-methylhistidine content (0.160 mumol/g of tissue) indicate a pool size of 1.59 mumol/250 micrograms rat. Thus 463 nmol of the urinary excretion/day would originate from the intestine, 22% of the total. The tissue source of the remaining urinary excretion is not identified, but other non-muscle sources constituting about 10% of the whole-body pool could account for this with turnover rates of only 6%/day, a much lower value than the turnover rate in the intestine.     

Physiologic mechanisms for reduced apolipoprotein A-I concentrations associated with low levels of high density lipoprotein cholesterol in patients with normal plasma lipids.

Low plasma concentrations of high density lipoprotein (HDL) cholesterol and apolipoprotein A-I (apoA-I) are major risk factors for coronary heart disease (CHD). Low HDL levels are common in patients with hypertriglyceridemia, but they also occur in those with normal plasma lipids; the latter include obese patients and cigarette smokers, though other patients with low HDL levels are neither obese nor smokers. The present study was designed to define metabolic causes of low apoA-I levels in normal-weight, normolipidemic patients. ApoA-I tracer studies were carried out in two groups of normolipidemic patients having low HDL levels to determine input rates and residence times for ApoA-I; these patients included 11 nonobese nonsmokers and 11 nonobese cigarette smokers. Their results were compared to those of 20 normal-weight, normolipidemic controls with normal HDL levels and 12 obese nonsmokers also having low HDL. In all three groups manifesting low HDL-cholesterol and low apoA-I levels, residence times for plasma apoA-I were reduced by approximately 30%, compared to control subjects with normal HDL levels. In contrast, average input rates for apoA-I were similar among the three low-HDL patients and control subjects. No differences in apoA-I kinetics were observed among any of the three groups with low apoA-I concentrations. Within each of the four groups of the study, however, input rates for apoA-I were highly correlated with plasma concentrations of apoA-I. Thus, for individuals with normal levels of plasma lipids, both residence times and input rates for apoA-I appeared to be important determinants of apoA-I levels. Residence times for apoA-I were reduced in almost all patients with low apoA-I levels, regardless of concomitant factors, whereas input rates were highly variable among individuals.     

Decreased frequency of activation of replicon clusters in Down syndrome lymphocytes

Human blood lymphocytes from two normal and seven Down syndrome (DS) subjects were examined to determine rates of synthesis of individual replicon and adjacent clusters of replicons, using DNA fiber autoradiography. Lymphocytes in 6 of 7 DS patients were shown to have significantly slower synthesis of simultaneously active adjacent replicon clusters compared to normal controls. Rates of synthesis of individual replicons were the same in lymphocytes from all the subjects investigated. These results demonstrate differences in respect to the structural organization of clusters of replicons between DS and normal lymphocytes. A possible relation of the above phenomenon to the chromosomal radiosensitivity in DS cells is discussed.     

Relevance of drug uptake,cellular distribution and cell membrane fluidity to the enhanced sensitivity of Down's syndrome fibroblasts to anticancer antibiotic-mitoxantrone

Sensitivity of human fibroblasts derived from Down's syndrome (DS) individuals (S-240, T-158, T-74, T-164) and normal donors (S-126, WA-1) to anticancer antibiotic-mitoxantrone (1,4-dihydroxy-5,8-bis((2-((2-hydroxy-ethyl)amino)ethyl)amino)-9,10-anthracenedione dihydrochloride; MIT) and its relationship to the transport rate, cellular distribution and interaction with cell membrane were studied. The survival assay showed that MIT was more toxic to trisomic fibroblast lines than to normal cells. Studies of transport kinetics indicated that the amount of drug taken up and extruded by DS cells was diminished, compared to control cells. In contrast, the cellular level of MIT associated with DNA was greater in trisomic than in normal cells. The fluorescence anisotropy measurements of TMA-DPH and 12-AS demonstrated that the fluidity of the polar region of the outer lipid monolayer of DS cell membrane was decreased in comparison with normal cells. MIT treatment decreased fluidity of the inner hydrophobic region of plasma membrane, but only slightly influenced the fluidity of the outer surface of the cell membrane. Finally, we concluded that lowered membrane fluidity, diminished amount of MIT extruded by cells and the enhanced level of the drug associated with DNA could be responsible for the enhanced sensitivity of DS fibroblasts to the MIT treatment.     

Asymmetric synthesis followed by transmembrane movement of phosphatidylethanolamine in rat liver endoplasmic reticulum

Studies with phospholipase C have indicated that two-thirds of the phosphatidylethanolamine of rat liver endoplasmic reticulum is located in the inner leaflet of the membrane bilayer. Phosphatidyl[¹⁴C]ethanolamine is synthesised in microsomes incubated with CDP[¹⁴C]ethanolamine. Using phospholipase C as a probe we have observed that the labelled phospholipid is initially (1–2 min) concentrated in the ‘outer leaflet’ of the membrane bilayer. The specific activity of this pool of phosphatidylethanolamine was 3.5 times that of the inner leaflet. If, however, the microsomes were opened with 0.4% taurocholate or the French pressure cell to make both sides of the bilayer available to phospholipase C, the phosphatidylethanolamine behaves as a single pool for hydrolysis. On longer incubation, up to 30 min, with CDP[¹⁴C]ethanolamine the specific activity of the outer leaflet phosphatidylethanolamine becomes close to that of the inner leaflet. In chase experiments, in which microsomal phosphatidylethanolamine was labelled by incubation with CDP[¹⁴C]ethanolamine for 1 min, the reaction stopped by addition of calcium, and the microsomes isolated by centrifugation and reincubated, labelled phosphatidylethanolamine was transferred from the ‘outer leaflet’ to the ‘inner leaflet’, so that both were equally labelled. These observations suggest that phosphatidylethanolamine is synthesised at the cytoplasmic leaflet of the endoplasmic reticulum and subsequently transferred across the membrane to the cisternal leaflet of the bilayer. Transmembrane movement is apparently temperature-dependent and independent of continued synthesis of phosphatidylethanolamine.     

The metabolism in man of (3H)-5alpha-16-androsten-3alpha-ol and of (3H)-5alpha-16-androsten-3-one

The in vitro metabolism in man of two 16-androstene steroids, 5alpha-16-androsten-3-one and 5alpha-16-androsten-3alpha-ol, has been studied using 3H-labelled tracers. 4 healthy subjects (2 of each sex) were chosen, and each labelled steroid was administered, by a single injection, to 1 man and 1 woman. Disappearance of (3H)-3alpha-androstenol in the subjects receiving this compound followed a curve which indicated a two-pool distribution in both cases; metabolic clearance rates for these subjects were found to be 3,790 1/24 h in the man and 3,120 1/24 h in the woman. Blood production rates calculated for the 3alpha-androstenol-treated subjects were 875 microgram/24 h in the man and 1,780 microgram/24 h in the woman. Recovery of 3H in the urine of all 4 subjects was very low, between 28 and 42%. Conversion of the injected precursors to urinary 3alpha-androstenol was 13.5 and 12.7% in the 2 men and 6.1 and 5.9% in the 2 women. The male subjects were found to have a lower 24-hour urinary 3alpha-androstenol output (570 and 387 microgram/24 h) than the average for men of their age. The urinary 3alpha-androstenol output in the women was 225 and 276 microgram/24 h, and was within the normal range for women. The urine production rates of 3alpha-androstenol were 2,470 and 4,090 microgram/24 h in the male and female subjects, respectively; the difference between the blood and urine production rates of this compound are thought to indicate the direct secretion of conjugates. Urine production rates of 5alpha-androstenone (measured as 3alpha-androstenol) were 2,370 and 4,340 microgram/24 h in the male and female subjects, respectively.     

Direct measurement of apolipoprotein B synthesis in human very low density lipoprotein using stable isotopes and mass spectrometry

Stable isotope methodology has been adapted to the study of lipoprotein turnover in human subjects. Using endogenous [15N]glycine labeling and gas-liquid chromatographic-mass spectrometric analysis, synthesis of apolipoprotein B in very low density lipoprotein (VLDL) was measured directly in five normal and two hyperlipidemic subjects. An isotopic precursor steady state was achieved during the studies by utilizing a priming dose and constant infusion containing [15N]glycine. Measurement of the plateau in 15N enrichment in the urinary hippurate produced during each study was used to estimate the 15N enrichment of the hepatic glycine precursor pool. The range of values for the fractional synthetic rate of VLDL apoB in the normal subjects obtained by this method was 5.9 to 11.5 day-1, with a mean of 9.2 +/- 2.4 (SD). This value agrees with the results of previous investigations which have utilized other methods. The method was also tested in two hypertriglyceridemic subjects and gave fractional synthetic rates of VLDL apoB that were significantly lower than in normals (1.5 and 2.8 day-1). This stable isotope method allows calculation of the fractional synthetic rate of VLDL apoB by maintaining an isotopic steady state throughout the study. It makes possible repeated studies in the same individual since no risk of exposure to radioisotopes is involved.     

Resin-core and buried-bag estimates of nitrogen transformations in Costa Rican lowland rainforests

We compared the resin-core and buried-bag incubation methods for estimating nitrogen (N) transformation rates using the ¹⁵N pool dilution technique in alluvial soils of an early successional forest (ESF) and an old-growth forest (OGF) at the La Selva Biological Station in Costa Rica. Soil cores (38×100-mm) from both forests were incubated in situ for 7 days. The two methods gave generally similar estimates of net N mineralization rates for the two forests. Estimates of ammonium production by the resin-core method were higher than those by the buried-bag method in ESF, but did not differ significantly in OGF (p<0.05). Estimates of nitrate production by the two methods did not differ significantly. Nitrate averaged 74% and 81% of the total inorganic N production in ESF and OGF, respectively. Net N mineralization in ESF (6.6 mmol m^-2d^-1) did not differ significantly from that in OGF (5.0 mmol m^-2d^-1). Fluxes of ammonium and nitrate were high for both forests, but the OGF tended to have higher gross mineralization and nitrification rates than ESF. Approximately 60% of the gross nitrate production and less than 30% of the ammonium were immobilized by microorganisms.     

Purified outer membranes of Serpulina hyodysenteriae contain cholesterol.

We have isolated outer and inner membranes of Serpulina hyodysenteriae by using discontinuous sucrose density gradients. The outer and inner membrane fractions contained less than 1 and 2%, respectively, of the total NADH oxidase activity (soluble marker) in the cell lysate. Various membrane markers including lipooligosaccharide (LOS), the 16-kDa outer membrane lipoprotein (SmpA), and the C subunit of the F1F0 ATPase indicated that the lowest-density membrane fraction contained outer membranes while the high-density membrane fraction contained inner membranes and that both are essentially free of contamination by the periplasmic flagella, a major contaminant of membranes isolated by other techniques. The outer membrane fractions (rho = 1.10 g/cm3) contained 0.25 mg of protein/mg (dry weight), while the inner membrane samples (rho = 1.16 g/cm3) contained significantly more protein (0.55 mg of protein/mg [dry weight]). Lipid analysis revealed that the purified outer membranes contained cholesterol as a major component of the membrane lipids. Treatment of intact S. hyodysenteriae with different concentrations of digitonin, a steroid glycoside that interacts with cholesterol, indicated that the outer membrane could be selectively removed at concentrations as low as 0.125%.     

Delayed secretory pathway contributions to VLDL-triglycerides from plasma NEFA, diet, and de novo lipogenesis in humans

Newly synthesized triglyceride (TG) may exit the liver immediately as VLDL-TG or be stored and secreted after a delay. We quantified the contributions from plasma NEFA, diet, and de novo lipogenesis (DNL) to VLDL-TG via immediate and delayed pathways in five lean, normolipidemic subjects; six obese, hypertriglyceridemic (HPTG) nondiabetics; and six obese, HPTG diabetics. Intravenous [(2)H(31)]palmitate and [1-(13)C(1)] acetate and oral [(2)H(35)]stearate were administered for 30 h preceding an overnight fast. [1,2,3,4-(13)C(4)]palmitate was infused during the subsequent 12 h fast. Contributions from plasma NEFA via the immediate pathway were 64 +/- 15, 33 +/- 6, and 58 +/- 2% in control, HPTG, and diabetic HPTG, respectively. Delayed pool fractional contributions were as follows: dietary FA, 2.0 +/- 0.9, 2.5 +/- 1, and 12 +/- 2%; DNL, 3 +/- 0.3, 14 +/- 3, and 13 +/- 4%; delayed NEFA, 15 +/- 4, 20 +/- 4, and 30 +/- 3%. VLDL-TG production rates and absolute input rates from the delayed pool were significantly higher in HPTG and diabetic HPTG than in controls. In conclusion, we provide direct kinetic evidence for a hepatic TG storage pool in humans and document its metabolic sources. The turnover time and sources of this pool differ in diabetic HPTG and nondiabetic HPTG, with potential therapeutic implications.