High Control Values Research Articles

Improved method for recovery and quantitation of assimilated radiolabel from marine sediment bacteria

Radiotracer studies of substrate assimilation by bacteria in marine sediments are complicated by high abiotic control values, poor sampling reproducibility, and incomplete recovery and quantitation of incorporated radiolabel. An alkaline extraction approach has been developed to improve radiolabel recovery. Net acetate and glucose assimilation were 11.1–22.9 pmol ml −1 h −1 and 0.7–1.3 pmol ml −1 h −1, respectively, by microorganisms in estuarine sediments ranging from 166–233 μm median grain size and 3.1–4.1% silt and clay. The alkaline extraction method resulted in abiotic control values averaging 8.6% of experimental values, reproducible sub-sampling for radiolabel quantitation (S.E. mean −1 ranged from 0.04–0.4 for 42 sample sets), and efficient recovery of incorporated radiolabel. The procedure recovered more than twice the radiolabel recovered using acid extraction or hot water extraction with identical sediments. Alkaline extraction was significantly different from acid and hot water extraction (ANOVA, F = 40.70, p < 0.0001). Use of these methods allows detection of changes in substrate assimilation patterns in fine-grained marine sediments.

Energy-dependent reactions supported by several substrates in coupled Euglena gracilis mitochondria

The ability of glutamate + malate, 2-oxoglutarate, NADH, succinate and DL-lactate to support oxidative phosphorylation, the formation of membrane potential, swelling and Ca 2+ uptake in Euglena mitochondria was studied. Oxidative phosphorylation was supported by the five substrates assayed with the appropiate ADP/O ratios ad high respiratory control values. Oxidative phosphorylation supported by lactate was resistant to antimycin, but sensitive to 2-heptyl-4-hydroxyquinoline- N-oxide (HQNO), myxothiazol and cyanide. Respiration and membrane potential resistant to cyanide were observed with lactate and, to a lesser extent, with glutamate + malate. A similar pattern of inhibition, confirmed in thisstudy, has been described for oxidation of D-lactate in bacterial systems. Oxidation of lactate induced an increase in matrix NAD(P)H; however the retention of lactate oxidase activity in submitochondrial particles suggested that, similar to lactate oxidation in bacterial systems, NAD(P) was not an essential requirement in this reaction. Substrate oxidation induced swelling whereas ATP synthesis caused shrinkage. Ca 2+ uptake was only partially inhibited by ruthenium red and was completely abolished by an uncoupler.

The pattern and kinetics in human skin of erythema and mediators during the acute and late-phase response (LPR)

To investigate the kinetics and pattern of allergenically induced mediator release in the human skin, we have studied 24 ragweed- and grass-allergic patients with a blister-chamber technique. Chambers sealed to the skin around a denuded area, formed by unroofing a blister, were challenged with 0.5 ml of either diluent or 10 or 100 times the concentration of allergen required for 4+ early intradermal reaction. Chamber fluids were removed hourly 1 to 8 hours after antigen challenge and examined for the presence of histamine, leukotriene C 4 (LTC 4), and prostaglandin D 2 to compare inflammatory mediator levels with the clinical early response and late-phase response (LPR), as assessed by erythema around the chamber. An initial erythema developed rapidly and began to subside after 1 hour in all patients, but a late-phase local erythema and subcutaneous swelling around the chamber (i.e., >2.5 cm, the outside diameter of the chamber) developed in 13/15 challenges only when the higher concentration of antigen was used. At both allergen concentrations, histamine levels peaked sharply at the first hour (20.6 ±2.3 ng/ml) and progressively declined during the next 4 hours by 75%, but remained above control levels for at least 7 hours. Despite high control values, LTC 4 levels were significantly elevated ( p < 0.01) 4 to 6 hours after challenge. In visible reactions, maximal LPR around the chamber correlated with LTC 4 levels obtained 6 and 7 hours after challenge ( p < 0.05). Prostaglandin D 2 rose gradually in antigen-challenged chambers to a peak at 5 to 6 hours. Thus, early rises in histamine were temporally related to the immediate erythema, whereas the arachidonic acid metabolites from both cyclooxygenase and lipoxygenase pathways that appeared in the skin after allergen challenge followed kinetics that corresponded to the time course of cutaneous LPR.

Long-term effects of dietary marine omega-3 fatty acids upon plasma and cellular lipids, platelet function, and eicosanoid formation in humans.

We studied the incorporation and metabolism of eicosapentanoic (EPA) and docosahexaenoic acid in six human volunteers who supplemented their normal Western diet for 5 mo daily with 10-40 ml of cod liver oil, rich in omega-3 polyunsaturated fatty acids. EPA and docosahexaenoic acid were incorporated into the total phospholipids of plasma, platelets, and erythrocytes in a dose- and time-dependent manner. During omega-3 fatty acid ingestion serum triacylglycerols were lowered and platelet aggregation upon low doses of collagen was reduced. Concomitantly, formation and excretion of prostanoids showed a characteristic change. As measured in serum from whole clotted blood, thromboxane A3 was formed in small amounts, whereas thromboxane A2 formation was reduced to 50% of control values. Excretion of the main urinary thromboxane A metabolites was unaltered in subjects with low basal excretion rates, but decreased markedly in two subjects with high control values. As determined from the main urinary metabolite, prostaglandin I3 was formed from EPA at rates up to 50% of unaltered prostaglandin I2 formation. The biochemical and functional changes observed lasted for the entire supplementation period of 5 mo and were reversible within 12 wk after cessation of cod liver oil intake. Favorable changes induced by long-chain omega-3 fatty acids include a dose-related and sustained shift of the prostaglandin I/thromboxane A balance to a more antiaggregatory and vasodilatory state.

Critical Parameters in the Isolation of Mitochondria from Candida utilis

The successive steps in the isolation of mitochondria from chemostat-grown Candida utilis have systematically been investigated for their effects on organelle integrity. Growth rate had a profound effect on the susceptibility of carbon-limited cells towards Zymolyase, whereas the nature of the carbon source had no effect. Stabilization of spheroplasts with at least 2M-sorbitol was required to prevent premature lysis. This was concluded from the amounts of glucose-6-phosphate dehydrogenase liberated during Zymolyase treatments. The influence of the method for disruption of spheroplasts on the quality of the mitochondria was analysed with particular emphasis on respiratory control values and the distribution of marker enzymes among the cell fractions. Disruption by osmotic shock resulted in mitochondria without respiratory control and a high degree of solubilization of NADH and NADPH dehydrogenase activities. Only a gradual decrease of the osmotic value of the medium, preferably by dialysis against a hypotonic buffer, in combination with mechanical disruption with a Potter-Elvehjem homogenizer yielded mitochondria with high respiratory control values and a high retention of NADH dehydrogenase in the organelle. It is concluded that, for the quality of mitochondrial preparations from yeasts, the distribution of NADH dehydrogenase among the cell fractions is a more reliable measure than that of the usual marker enzymes.

Oxidation of NADH and NADPH by Mitochondria from the Yeast Candida utilis

Mitochondria were isolated from Candida utilis CBS 621 grown in carbon-limited continuous cultures on glucose, gluconate, xylose, ethanol or acetate as the carbon source and ammonia or nitrate as the nitrogen source. In all cases mitochondria were isolated which could oxidize exogenous NADH and NADPH via a cyanide- and antimycin A-sensitive but rotenone-insensitive respiratory chain. Oxidation of NADH and NADPH was coupled to energy conservation as evidenced by high respiratory control values. Different respiratory control values of mitochondria with NADH and NADPH as well as variations in the ratio of NADH and NADPH oxidase activities indicate that separate systems exist for the oxidation of exogenous redox equivalents by mitochondria of C. utilis. Variation of the NADPH requirement for biomass formation by applying differnt growth conditions did not result in significant changes in NADPH oxidase activities of mitochondria. It is concluded that in C. utilis NADPH can be used in dissimilatory processes for the generation of ATP.

Problems with radiolabelling egg albumin for the farr ammonium sulphate technique for antibody estimation

High concentrations of passive haemagglutinating antibodies were raised in sheep immunised with egg albumin (Ea) in Freund's adjuvant. Further attempts to study the antigen binding capacities and avidities of these antisera by the Farr ammonium sulphate technique were hindered by unsuccessful attempts to label Ea with 125Iodine by either the chloramine T or sodium nitrite-HCl methods. This problem was attributed to the unavailability of tyrosine in the native protein. This was solved by labelling Ea with the Bolton-Hunter reagent which does not require the presence of tyrosine. However labelled Ea binds non-specifically to normal serum to give high control values of circa 40% binding. Despite this it was possible to estimate antibody concentrations and avidities of lamb sera.

Angiotensin II effects upon the glomerular microcirculation and ultrafiltration coefficient of the rat.

The effects of both synthetic and biologically produced angiotensin II (AII) upon the process of glolerular filtration were examined in the plasma-expanded (2.5% body wt) Munich-Wistar rat, by micropuncture evaluation of pressures, nephron plasma flow (rpf) and filtration rate (sngfr). Plasma expansion was chosen as a control condition because (a) response to AII was uniform and predictable, (b) endogenous generation of AII was presumably suppressed, and (c) the high control values for rpf permitted accurate determination of values for the glomerular permeability coefficient (LpA) before and during AII infusion. With subpressor quantities of synthetic Asn-1, Val-5 AII (less than 5 ng/100 g body wt/min), sngfr fell from 47.7 in the control group to 39.8 nl/min/g kidney (P less than 0.005). The rpf fell to 60% of control values (P less than 0.001). Measurement of glomerular capillary (PG) and Bowman's space (Pt) hydrostatic pressures in surface glomeruli with a servo-nulling device permitted evaluation of the hydrostatic pressure gradient (deltaP = PG - Pi). DeltaP increased from 38.1 +/- 1.2 in control to 45.9 +/- 1.3 mm Hg after Asn-1, Val-5 AII and essentially neutralized the effect of decreased rpf in sngfr. The sngfr then fell as a result of a decreased in LpA from 0.063 +/- 0.008 in control to 0.028 +/- 0.004 nl/s/g kidney/mm Hg after Asn-1, Val-5 AII (P less than 0.02). Lower doses of Asp-1, Ile-5 AII (less than 3 ng/100 g body wt/min) had no effect on sngfr, rpf, deltaP, and afferent and efferent vascular resistance, but significantly elevated systemic blood pressure, suggesting peripheral effects on smooth muscle at this low dose. LpA was 0.044 +/- 0.007 nl/s/g kidney/mm Hg after low-dose Asp-1, Ile-5 AII, and 0.063 +/- 0.008 in the control group (0.02 greater than P greater than 0.1). Higher, equally pressor doses of native AII (5 ng/100 g body wt/min) produced effects almost identical to similar quantites of synthetic Asn-1, Val-5 AII upon rpf, deltaP, sngfr, and renal vascular resistance. LpA again fell to 0.026 +/- 0.004 nl/s/g kidney/mn Hg, a value almost identical to that after the synthetic AII. Paired studies with Asp-1, Ile-5 AII also demonstrated a consistent reduction in LpA.

Hemodynamic and electrocardiographic effects of disopyramide in patients with ventricular arrhythmia.

Antiarrhythmic and hemodynamic effects of i.v. disopyramide phosphate (1.7 mg/kg b.wt. over 2 min) have been studied in nine patients, several in various degrees of cardiac decompensation, with sinus rhythm and persistent ventricular ectopic beats (VEBs). In one case with primary cardiomyopathy, with greater than 30 VEBs/min, disopyramide (DE) abolished the arrhythmia for 30 min, but precipitated brief dysponea. Other side-effects were tolerable and mainly attributable to anticholinergic effects of the drug. DE either abolished or significantly reduced the arrhythmia in all cases. For 30 min, only one patient showed VEBs, and in three patients no VEBs were seen for three hours. Changes in cardiac output and pulmonary artery (PAP) and central aortic pressures were measured in eight patients. Negative inotropic effects were indicated in seven by an increased diastolic PAP/stroke volume ratio and in seven by a decreased central aortic (dp/dt)max. Patients with high control values for diastolic PAP showed marked reductions in cardiac output, stroke volume and stroke work. In predicting myocardial depressant effects of DE, the control values for diastolic PAP seemed to be superior to central venous pressure, cardiac index and systolic time intervals. Mean arterial pressure measured 5 and 10 min after drug administration showed no significant change, indicating that vasoconstrictor reflexes were well preserved, and a pressure level significantly above the control value was reached from the 20th min. It is concluded that DE is potent in suppressing VEBs but exerts negative inotropic effects that may be of clinical importance. The optimal antiarrhythmic dose is probably lower than that used in the present study.

Direct Demonstration and Quantitation of Aal, Aa2 and Aa3 Allotypic Specificities on Fd-Fragments of Rabbit Immunoglobulin G

Summary Fd-fragments were prepared from Fab-fragments obtained by papain digestion of rabbit immunoglobulin-G (IgG). In addition, a 5 S piece was obtained by cyanogen bromide treatment of IgG and an Fd derived from the 5 S piece was prepared. Polyalanylation was carried out in order to solubilize the Fd-fragments. Some masking of a-locus-allotypic specificity was demonstrated when IgG and 5 S piece were polyalanylated. 125I-labeled fragments were allowed to react with either anti-a1, -a2 or -a3 antisera and supernatant portions were precipitated with appropriate anti-allotype antisera specific only for the antibody portion of the soluble complexes. In spite of some probable masking due to polyalanylation, the majority of polyalanylated Fdfragments (generally more than 60%) had demonstrable determinants associated with a-locus-allotypic specificity. Fab-fragment (a2), from which noncovalently linked glycopeptides were removed on Sephadex in 1 M propionic acid, retained a2 allotypic specificity. Fd-fragments derived from CNBr treated IgG, especially those with short polyalanine chains, gave high control values with normal rabbit sera, suggesting either a nonspecific or a specific affinity for immunoglobulins in normal rabbit sera.

Improved Method for the Determination of Ethylenebisdithiocarbamate Residues in Plants, Fruits, and Vegetables

Abstract A modified method for the determination of dithiocarbamate fungicide residues on crops is presented. A large representative subsample of the frozen crop is blended in ice-cold deaerated water and an aliquot of the homogenate is added to the analytical apparatus containing hot 5 0% sulfuric acid. Dithiocarbamates are decomposed to evolve CS2 which is removed by a continuous gentle air-sweep from the digestion flask. Variations in technique allow the analysis of dithiocarbamate fungicide residues in several ranges, 1-10, 10-200, and 200-1000 /ig maneb. Recoveries from a wide variety of crops averaged 70 to 103%. Certain crop types present low recoveries and/or high apparent control values, but modifications in the analytical procedure are successful in solving these problems.