Pool Size Measurements Research Articles

Fetal Treatment with Interleukin-1α Improves Postnatal Lung Function in Premature Lambs. ♦ 1494

Background: Interleukin-1 (IL-1) denotes two proinflammatory cytokines, IL-1α and IL-1β. Levels of IL-1 are increased in intra-amniotic infections and in other inflammatory conditions. IL-1 may affect the defence functions of the lung. Intraamniotic IL-1α was shown to up regulate the expression of surfactant proteins A and B in preterm rabbits (K. Bry, et al AJ Resp Crit Care Med, Vol. 151, A23). Objective: We measured the physiological and biochemical lung responses in preterm lambs to intra-amniotic injection of IL-1α. Methods: Ewes carrying singleton 126±1 day gestational age fetuses were randomize to receive 125 μg IL-1α (n=8) or placebo (n=9) via ultrasound guided intra-amniotic injection. Each lamb was delivered 48 h after treatment by cesarian section and mechanically ventilated for 2 h. Results: Over the 2 h ventilation period the dynamic compliance of IL-1α treated lambs was higher than controls (0.36±0.02 vs 0.28±.02 (mean ± S.E.) ml/kg/cm H2O, P<0.05). Lung volumes measured from post mortem pressure volume curves trended higher for IL-1atreated lambs than for controls (36±6 vs 24±5 ml/kg at 40 cm H2O pressure and 28±6 vs 18±4 ml/kg at 10 cm H2O). Saturated phosphatidylcholine in the lung, a measure of surfactant pool size, was higher for IL-1α treated animals than for controls (77±7 vs 54±3 μmol/kg, P<0.005). As a measure of pulmonary edema, total lung recovery of I125 albumin injected at 1.5 h of life tended to be less in IL-1α treated animals than in controls(5.2±0.8 vs 7.3±1.2%). Plasma cortisol at 2 h of age was higher in IL-1α treated lambs than in controls (3.4±0.7 vs 1.5±0.2, P<0.02), although cord blood cortisol and catecholamine levels were similar between the groups. Comparisons of IL-1α treated animals versus controls showed no differences in total white blood cells, differential percentages, or absolute neutrophils. Cardiac output and regional blood flow also were similar between the groups. Conclusion: Fetal treatment with IL-1α increased surfactant pool size, improved dynamic compliance, and was not associated with an inflammatory response, in utero cortisol response, or differences in other indicators of fetal or newborn well being.

Modeling excess sulfur deposition on wetland soils using stable sulfur isotopes

Freshwater wetlands exposed to excess S deposition can potentially store significant amounts of reduced S in soils by dissimilatory sulfate reduction. If this storage is permanent, the harmful environmental effects of S deposition and the accompanying acidity are reduced, particularly on surface waters into which wetlands drain. Total non-sulfate S in freshwater peat is divided into three fractions: reduced inorganic S, ester sulfate and carbon-bonded sulfur (CBS). Each fraction is further divided based on its origin: assimilatory via plant and microbial uptake, and dissimilatory via microbial reduction. The CBS fraction dominates in peat, so the amount of dissimilatory CBS in the soil is a direct measure of the beneficial effect of storage of reduced S. Unfortunately, there is no way to directly measure dissimilatory CBS. A model is developed which, with three assumptions, provides a method to calculate dissimilatory CBS using S pool size and stable isotope measurements. Application of the model to a wetland in the New Jersey Pinelands, U.S.A., shows that large amounts of reduced dissimilatory S are stored in the soil. As a consequence, the impact of S deposition and acidity on the surface water environment is significantly reduced.

Direct measurement of whole body thyroid hormone pool sizes and interconversion rates in fasted rats: hormone regulation implications

Food deprivation markedly reduces thyroid hormone levels in mammalian plasma, but existing data are incomplete and equivocal regards extrathyroidal hormone production and other indices of overall hormone economy. We have used a novel experiment design and analysis to directly measure the whole-body rate of conversion of T4 into T3 and several other steady-state whole organism parameters, in 4-day fasted and fed control rats. Trace amounts of 125I-labeled T3 (T3) or T4 (T*4) were infused for 7 days from osmotic minipumps implanted sc. On day 7, rats were anesthetized, bled, and killed and carcasses were frozen in liquid N2, pulverized, homogenized, and extracted. Extracts and plasma samples were chromatographed on both Sephadex and HPLC. Tracer infusion rates, whole rat tissue weights, and steady state tissue, blood, and plasma T*3, T*4, and total radioactivity concentrations provided all kinetic parameters of interest from simple steady state computations. T4 secretion (SR4) and whole body pool sizes were reduced 49-55% in fasted rats. But the most notable results were that the percent of available extrathyroidal T4 converted to T3 in fasted [41.6 +/- 7.9% (SD)] was 87% greater than that in the fed (22.3 +/- 7.69%) rats and this, in turn, generated an absolute rate of production of T3 from T4 not significantly different in fasted vs. fed controls (7.17 +/- 2.40 vs. 7.54 +/- 3.10 ng/h.100 g BW). The surprisingly high 42% conversion ratio in fasting is explained in part by larger T3 blood pools (which are not sites of T3 production from T4) relative to tissue T3 pools in fasted rats, not accounted for in earlier whole-body studies. In contrast with this finding of an increased T4 to T3 conversion ratio in fasted rats, based on whole body measurements, T3 plasma concentrations (Cp3), clearance rates (PCR3), appearance rates (PAR3 = PCR3Cp3), and more conventional indirect estimates of the T4 to T3 conversion ratio (100 PAR3/SR4) were all substantially reduced, consistent with reports in fasting humans limited to measurements of T3 and T3 turnover in plasma and interpreted as indicative of reduced whole body T4 or T3 conversion. Directly measured total T3 extrathyroidal distribution volumes, reduced 55% in the fasted group from 241 +/- 19.5 to 109 +/- 8.14 ml/100 g BW, are also of interest because fed rat values are 27-61% greater than virtually all previous estimates of this index of total body T3.

Direct measurement of whole body thyroid hormone pool sizes and interconversion rates in fasted rats: hormone regulation implications.

Food deprivation markedly reduces thyroid hormone levels in mammalian plasma, but existing data are incomplete and equivocal regards extrathyroidal hormone production and other indices of overall hormone economy. We have used a novel experiment design and analysis to directly measure the whole-body rate of conversion of T4 into T3 and several other steady-state whole organism parameters, in 4-day fasted and fed control rats. Trace amounts of 125I-labeled T3 (T3) or T4 (T*4) were infused for 7 days from osmotic minipumps implanted sc. On day 7, rats were anesthetized, bled, and killed and carcasses were frozen in liquid N2, pulverized, homogenized, and extracted. Extracts and plasma samples were chromatographed on both Sephadex and HPLC. Tracer infusion rates, whole rat tissue weights, and steady state tissue, blood, and plasma T*3, T*4, and total radioactivity concentrations provided all kinetic parameters of interest from simple steady state computations. T4 secretion (SR4) and whole body pool sizes were reduced 49-55% in fasted rats. But the most notable results were that the percent of available extrathyroidal T4 converted to T3 in fasted [41.6 +/- 7.9% (SD)] was 87% greater than that in the fed (22.3 +/- 7.69%) rats and this, in turn, generated an absolute rate of production of T3 from T4 not significantly different in fasted vs. fed controls (7.17 +/- 2.40 vs. 7.54 +/- 3.10 ng/h.100 g BW). The surprisingly high 42% conversion ratio in fasting is explained in part by larger T3 blood pools (which are not sites of T3 production from T4) relative to tissue T3 pools in fasted rats, not accounted for in earlier whole-body studies. In contrast with this finding of an increased T4 to T3 conversion ratio in fasted rats, based on whole body measurements, T3 plasma concentrations (Cp3), clearance rates (PCR3), appearance rates (PAR3 = PCR3Cp3), and more conventional indirect estimates of the T4 to T3 conversion ratio (100 PAR3/SR4) were all substantially reduced, consistent with reports in fasting humans limited to measurements of T3 and T3 turnover in plasma and interpreted as indicative of reduced whole body T4 or T3 conversion. Directly measured total T3 extrathyroidal distribution volumes, reduced 55% in the fasted group from 241 +/- 19.5 to 109 +/- 8.14 ml/100 g BW, are also of interest because fed rat values are 27-61% greater than virtually all previous estimates of this index of total body T3.(ABSTRACT TRUNCATED AT 400 WORDS)

Mutations affecting phenol oxidase activity inDrosophila: quicksilver andtyrosinase-1

The complex enzyme phenol oxidase plays a major role in sclerotization and melanization of cuticle in insects. Production of active enzyme from the inactive proenzyme involves at least six protein components in Drosophila. We examine here the biochemical phenotype of two loci that affect phenol oxidase activity--quicksilver (qs; 1-39.5) and tyrosinase-1 (tyr-1; 2-54.5). Three mutations isolated by different procedures in three different laboratories are alleles at the quicksilver locus. The effects of these mutations have been monitored by means of enzyme assays in vitro and in polyacrylamide gels and by measurement of catecholamine pool sizes. The activity of all three active enzyme components (A1, A2, and A3) is reduced in qs mutants. The activated enzyme of one qs allele is thermolabile, while its activator is normal. Deletion and genetic mapping place tyr-1 near purple (pr; 2-54.5). Enzyme activity is reduced to 10% of normal but is not thermolabile and the activator is normal. The activity of all three A components is reduced. The diphenol oxidase activity in double mutant combinations shows that these mutations and Dox-A2 (Pentz et al., 1986) affect this enzyme in different ways.

Synthesis and metabolism of uracil-containing deoxyribonucleic acid in Escherichia coli.

Significant amounts of uracil were found in the deoxyribonucleic acids (DNAs) of Escherichia coli mutants deficient in both uracil-DNA glycosylase (ung) and deoxyuridine 5'-triphosphate nucleotidohydrolase (dut) activities, whereas little uracil was found in the DNAs of wild-type cells and cells deficient in only one of these two activities. The amounts of uracil found in the DNAs of dut ung mutants were directly related to the growth temperature of the cultures, apparently because the deoxyuridine 5'-triphosphate nucleotidohydrolase synthesized by dut mutants was temperature sensitive. The dut mutant used failed to grow exponentially, became filamentous at temperatures above 25 degrees C, and exhibited a hyperrec phenotype; however, the ung mutation suppressed all of these effects. Although the dut ung mutants grew exponentially at all temperatures, their growth rates were always slower than the growth rate of the wild type. Since pool size measurements indicated that both deoxyuridine triphosphate and deoxythymidine triphosphate pools were markedly elevated in dut mutants, the reduced growth rate of dut ung cells apparently was due to the actual presence of uracil in the DNA, rather than to a deficiency of deoxyuridine triphosphate and deoxyribosylthymine triphosphate for DNA synthesis. The presence of uracil in E. coli donor DNA also markedly reduced the recombination frequency when the recipient cells were ung+, indicating that DNA repair commenced before the entering DNA could be replicated.

The generation of memory B-cell subpopulations capable of proliferation and expansion of the pool: Effect of time and antigen

The ability of antigen to induce proliferation of memory B lymphocytes, thus perpetuating and expanding the memory cell pool, has been examined using an antibody-forming cell (AFC) assay. This method provided confirmation of previous studies in which serum antibody titer was used as a relative measure of pool size and demonstrated directly that the number of antibody-forming cells is increased. Memory cell subpopulations were prepared by lg velocity sedimentation from recently immunized donors (2 weeks) and tested for their ability to proliferate, thus expanding the memory cell pool. Both large and small immature DNP-specific memory cells displayed antigen-dependent and antigen-independent proliferation while mature cells (8 weeks postpriming) were capable only of antigen-dependent proliferation. Chicken γ-globulin (CGG) specific memory cells were also evaluated in this system and were found to differ from DNP-specific cells in several ways. (A) DNP-specific AFC were found to be concentrated in the spleen while CGG-specific AFC were found predominantly in the bone marrow early after transfer and in the spleen upon delayed challenge. (B) The rate of maturation of CGG-specific memory cells capable of antigen-driven proliferation and pool expansion was delayed in comparison to DNP-specific memory cells. The relationship of these functionally defined subsets to previously described memory cell subpopulations is discussed.

The effect of amino acids on the temperature sensitive phenotype of the mammalian leucyl‐tRNA synthetase mutant ts Hl and its revertants

The temperature sensitive leucyl-tRNA synthetase mutant tsHl and two revertants have been compared to the parental Chinese hamster ovary cells with respect to the effects of amino acid concentrations in the medium on growth. Elevating the leucine concentration 30- or 100-fold allowed tsHl to grow exponentially at 38.5 degrees C, normally the nonpermissive temperature. Partial revertants that had recovered some enzyme activity required smaller supplements for growth. Measurements of the leucine pools indicated that they respond directly to the extracellular leucine concentration and may mediate the effect. Use of combinations of amino acids confirmed that isoleucine has a similar though weaker effect on tsHl and identified an even weaker protection by valine. The triple combination of leucine, isoleucine and valine was a much more efficient medium supplement and three times normal concentrations of these amino acids supported growth of tsHl at 38.5 degrees C. It is postulated that they are acting at their respective aminoacyl-tRNA synthetases to help stabilize a complex which also contains the mutant leucyl-tRNA synthetase. The pool size measurements also showed that the leucine pools of tsHl and a revertant increased 2-fold more in a response to increased temperature than those of WT. It is suggested that this is a regulatory response to low leucyl-tRNA synthetase activity and is important in determining growth phenotypes.

Deoxyribonucleoside triphosphate pools and differential thymidine sensitivities of cultured mouse lymphoma and myeloma cells

The effects of various concentrations of thymidine on DNA synthesis and deoxyribonucleoside triphosphate contents of a highly thymidine-sensitive cultured mouse lymphoma cell line (WEHI-7) and a relatively resistant mouse myeloma cell line (HPC-108) have been studied by 32P-labelling techniques. DNA synthesis in the myeloma cells was inhibited by thymidine at concentrations of 10 −3 M or greater, while DNA synthesis in the lymphoma cells was inhibited by concentrations 30-fold lower, consistent with the 25-fold difference between the two cell lines in sensitivity to growth inhibition by thymidine. Thymidine caused marked elevation of the dTTP and dGTP pools, slight elevation or no change in the dATP pool and a marked decrease in the dCTP pool in cells of both lines. The greater resistance of HPC-108 cells to thymidine inhibition was related to the finding that they normally contained a much higher concentration of dCTP than did the WEHI-7 cells. Pool size measurements on thymidine-treated (10 −4 M) cells of an additional seven sensitive lymphoma and six relatively resistant myeloma cell lines indicated that in all 15 lines studied, with one exception, a critical concentration of dCTP of about 32 nmol per ml of cell volume was required for the maintenance of normal rates of DNA synthesis. The dCTP content found normally in the lymphoma cells was only a little above this concentration. Amongst the myeloma lines, three contained similarly low levels of dCTP, but were more resistant to thymidine inhibition probably because of their inefficient production of dTTP from thymidine. Cells of the other four myeloma lines (including

Metabolism of potential precursors of chenodeoxycholic acid in cerebrotendinous xanthomatosis (CTX).

In patients with cerebrotendinous xanthomatosis (CTX), diminished cholic acid production is associated with incomplete oxidation of the cholesterol side chain and the excretion of C(25)-hydroxy bile alcohols. The aims of this investigation were 1) to provide quantitative information on the pool size and production rate of chenodeoxycholic acid by the isotope dilution technique; and 2) to investigate the possible existence of a block in chenodeoxycholic acid synthesis and explain the absence of chenodeoxycholic acid precursors in CTX. After the injection of [24-(14)C]chenodeoxycholic acid, measurements of chenodeoxycholic acid pool size and production rate in a CTX subject were, respectively, 1/20 and 1/6 as great as controls. Further, three potential precursors of chenodeoxycholic acid, namely [G-(3)H]7alpha-hydroxy-4-cholesten-3-one, [G-(3)H]5beta-cholestane-3alpha,7alpha,25-triol, and [G-(3)H]5beta-cholestane-3alpha,7alpha,26-triol, were administered to the CTX and control subjects and the specific activity curves of [G-(3)H]cholic acid and [G-(3)H]chenodeoxycholic acid were constructed and compared. In the control subjects, the two bile acids decayed exponentially, but in the CTX patient maximum specific activities were abnormally delayed, indicating the hindered transformation of precursor into bile acid. These results show that chenodeoxycholic acid synthesis is small in CTX and that the conversion of 7alpha-hydroxy-4-cholesten-3-one, 5beta-cholestane-3alpha,7alpha,25-triol, and 5beta-cholestane-3alpha,7alpha,26-triol to both chenodeoxycholic acid and cholic acid were similarly impaired.

Uric acid kinetic studies in the immediate post-myocardial-infarction period

The etiology of hyperuricemia following myocardial infarction was investigated by uric acid kinetic studies carried out on seven male patients following myocardial infarction and on two control subjects. The patients selected had uncomplicated myocardial infarction and were maintained on a low-purine diet. Measurements of uric acid pool size and turnover rates using 2-C 14 uric acid were made, commencing on days 2–5 following myocardial infarction. Initial concentration of serum uric acid ranged from 2.9 to 9.8 mg 100 ml . Uric acid pool size was elevated in six of seven patients. Five had a pool size of from 36.9 to 79.6 mg/kg, while the single gouty subject demonstrated 104 mg/kg compared with 12.6 and 16.8 mg/kg for the control subjects. Turnover rates were also increased, ranging from 1036 to 2772 mg/day (controls, 612 and 872 mg/day). Twenty-four-hour urine uric acid excretions ranged from 358 to 623 mg 24 hr . Serum lactic acid concentration was normal (1.03 ± 0.17 μ M/ml), and endogenous creatinine clearance in all cases was 77.9 ml/min or greater. These data suggest that following myocardial infarction there is an expansion of the uric acid pool with an increased uric acid turnover rate. Only the patient with a previous history of gout had uric acid excretion outside the normal range.

Flux of free fatty acids among host tissues, ascites fluid, and Ehrlich ascites carcinoma cells

The role of plasma free fatty acids (FFA) in the transport of fatty acids from host tissues to Ehrlich ascites carcinoma in mice was studied. [9,10-(3)H] Palmitate complexed to mouse serum (albumin) was injected either intraperitoneally or intravenously into unanesthetized tumor-bearing mice. The incorporation of radioactivity into tumor extracellular fluid FFA, tumor cell FFA, neutral lipid, phospholipid, water-soluble material in cells and fluid, plasma FFA, host carcass total lipid fatty acids, and water-soluble (i.e., nonlipid) material was measured. In addition, the quantity of fatty acid in each of the above lipid fractions was determined. The data were analyzed by multicompartmental analysis (SAAM) using a digital computer, and fractional rate constants of FA movement within and out of the host-tumor system were calculated. These rate constants and pool size measurements were used to estimate the corresponding fluxes. Although FFA in the tumor's extracellular fluid were replaced rapidly, almost none of the newly formed fluid FFA was derived from plasma FFA. Moreover, the transfer of FFA from the tumor extracellular fluid FFA to plasma FFA was virtually negligible. We suggest that the net amount of FFA required to replace the fluid FFA utilized for tumor energy and growth may be derived from direct transfer of FFA from host tissues to the ascitic fluid and that plasma FFA is not an intermediate in this transport process. The transport of FFA from the host to tumor cell lipids through the tumor extracellular fluid was about 26-fold greater than that required to account for net lipid accumulation during growth.

The study of bile salt kinetics in the experimental animal using a new technique

Six female baboons (Papio) have undergone cholecystopexy to facilitate bile sampling from the gallbladder for experimental studies. The animals have had several measurements of bile salt pool size and kinetics utilizing 14C-labeled primary bile acids, cholic and chenodeoxycholic acids. The data presented confirm that the gallbladder functions normally following cholecystopexy and suggest that cholic and chenodeoxycholic acids may vary independently in their kinetic activity.

Increased Bacterial Degradation of Bile Acids in Cholecystectomized Patients

Bile acid metabolism was studied in 10 patients previously cholecystectomized for cholesterol cholelithiasis, in 12 healthy subjects, and in 13 patients with cholesterol cholelithiasis (disease control subjects). The rate of bacterial deconjugation and 7-dehydroxylation was measured by using [2, 4-3H ]cholyl- [1-14C]glycine; measurements of synthesis and pool size were made simultaneously. Total bile acid pool size and biliary bile acid composition were determined by gas-liquid chromatography. Compared with healthy subjects, patients with cholecystectomy showed significantly increased deconjugation and dehydroxylation of the labeled cholylglycine. However, the pool of cholylglycine was considerably smaller, so that the absolute rate of deconjugation was similar. The pool of deoxycholylglycine was twice as large as that in the healthy control and the disease control subjects. In addition, ketohydroxy bile acids (resulting from bacterial dehydrogenation) composed 7 to 15% of the bile acid pool. The 7-keto and 12-keto derivatives of cholic acid were identified by mass spectrometry. The total bile acid pool in cholecystectomized patients did not differ from that in healthy control subjects but was larger than that in the disease control subjects. The size of the total bile acid pool in cholecystectomized patients correlated highly with the size of the pool of secondary bile acids, suggesting that its apparent return to a normal size in these patients after cholecystectomy could be explained in part by the increased input of secondary bile acids from the intestine.

Analysis of precursor pool labeling kinetics without pool size measurements: A computer-assisted approach

A general method is described which permits the correction of macromolecular radioactive labeling data for precursor pool specific activity variation during a labeling period. Measurement of the precursor pool size is made unnecessary. The method involves: 1. (1) Determination of radioactivity in the precursor pool at various times during an extended labeling period. 2. (2) Derivation of a theoretical equation describing the kinetics of labeling of the precursor pool. 3. (3) Computer-assisted fitting of the theoretical equation to the observed labeling data. 4. (4) Use of the parameters obtained from the curve-fitting to calculate the time course of saturation of the precursor pool with exogenous radioactive precursor . The derived saturation curves may be used to correct macromolecular labeling data, on a relative basis, for precursor pool saturation. The method may also be extended to provide absolute specific activity estimates. The application of the method is shown for the UTP and S-adenosyl methionine pools of cultured lymphocytes.

The economy of cholesterol in man: drug effects.

A partial list of the factors controlling the movement and storage of cholesterol in man must include: intake; absorption; lipoprotein synthesis and transport capacity; tissue equilibria with plasma cholesterol; endogenous synthesis; conversion to bile acids; pool sizes; excretion rates and pathways. All of these can now be defined with directness and exactitude except for measurement of pool sizes and of tissue-plasma equilibria, utilizing sterol balance and isotope kinetic techniques. Although pool sizes can be estimated by isotope kinetics, as yet we have no independent means by which to verify these estimates.

Age effects on the variation of RNA synthesis in the nurse cells in drosophila

1. (1)The RNA synthetized during Drosophila ovogenesis is mainly ribosomal. Its total amount per egg results from the rate of r RNA synthesis by the nurse cells and the time required for egg formation. The RNA content per egg does not signifantly decrease with female age; the duration of maturation is 5 days in young individuals and 2·5 days in old ones. These facts would suggest a rate of synthesis twice as high in old individuals. 2. 2. Precursors pool size measurements, autoradiography and histophotometry studies of polyploid nurse nuclei labelled with tritiated uridine confirm this hypothesis. 3. 3. The relationship between polyploidy and rate of RNA synthesis is not linear but shows peaks of activity for some specific DNA contents corresponding to phases of DNA synthesis. 4. 4. Small nuclei from early developing cysts are considerably more active in old individuals but the difference is much smaller when large nuclei from later stages are concerned. Three mechanisms are examined to explain these differences: unequal r DNA redundancy, uneven precursor ribosomal RNA synthesis, altered rate of precursor processing.

Availability and Metabolism of Various Substrates in Ruminants. I. Absorption and Metabolism of Acetate

Summary Two experiments were conducted in which K-acetate-1-C 14 was either injected or infused intravenously into a mature dairy steer to obtain measurements of pool size, turnover rate, and turnover time of blood acetate. Estimates were made of the amount of acetate absorbed and metabolized to carbon dioxide following feeding. It was shown that a single injection of K-acetate-1-C 14 is not applicable to the measurement of acetate pool size or turnover rate because of its apparent rapid metabolism before complete mixing with the unlabeled acetate in the pool is effected. However, the turnover time of the acetate pool was calculated to be 83min. The amount of acetate entering the pool after the consumption of 4.3lb. of grain was estimated to be 67.24g. per hour. Of this amount, 50% was oxidized to CO 2 and 50% was metabolized via other routes. The acetate oxidized accounted for 50% of the expired carbon dioxide.