Small amount of concanavalin A modifies radiation-induced alteration in cell-surface charge depending on its binding condition

Cell electrophoretic mobility of cultured melanoma cells or rat erythrocytes decreased with time after X-irradiation. Addition of tetravalent concanavalin A or divalent succinyl-concanavalin A before (not after) irradiation, completely blocked the mobility reduction in greater concentrations than 5 μg/l.At 5 μg/1 only 3.7 · 10³ concanavalin A molecules bound to receptors per cell, while 4.18 · 10⁷ molecules/cell bound at saturating concentrations. Preincubation with concanavalin A at 37°C was effective even when the cells were treated with α-methylmannoside immediately after irradiation. At low temperature, however, concanavalin A was not effective despite a sufficient amount of bound ¹²⁵I-labelled concanavalin A. Treatment with α-methylmannoside following the binding of concanavalin A at 37°C before irradiation inhibited the concanavalin A effect depending on temperature. The residual amount of bound lectin could not account for the temperature dependence. The amount of sialic acid (the main charged substance) was not altered by X-irradiation with or without the lectin. Divalent succinyl-concanavalin A was also effective in blocking the radiation effect on electrophoretic mobility. These results seem to suggest that binding of a very small amount of concanavalin A without causing cell agglutination or clustering of its receptors, induces some alteration in the conformation of receptor glycoprotein, which blocks the internalization of acidic sugar residues by subsequent irradiation.     

Electrokinetic properties of asparaginase sensitive or resistant mouse leukemic cells treated with L-asparaginase

The electrophoretic mobility of L5178Y cells in 0.0145 M NaCl, 4.5% sorbitol, 0.6 mM NaHCO₃, pH 7.2, at 25°C was — 1.78 μ·s^?1·V^?1·cm^?1 while that of an L-asparaginase resistant subline, L5178Y/ASN, was — 1.11 μm·s^?1·V^?1·cm^?1. Both cell lines were characterized by terminal sialic acid residues on their surfaces. Treatment of L5178Y cells for 90 min with 10 units of L-asparaginase per ml in saline decreased the electrophoretic mobility of the cells to — 1.65 μm·s^?1·V^?1·cm^?1 while treatment in Fischer's medium decreased the mobility to — 1.25 μm·s^?1·V^?1·cm^?1; neither treatment had a significant effect on the L5178Y/ASN electrophoretic mobility. The results suggest that L-asparaginase has an immediate and specific effect on synthesis of cell surface asparaginyl glycoproteins.     

Antigen-specific mouse lymphocyte stimulation by DNP-conjugated T-independent antigens studied by photobleaching recovery

Fluorescence photobleaching recovery techniques have allowed us to measure the lateral mobility of T-independent antigens bound to antigen-specific mouse B cells. The in vitro immunogenicity or tolerogenicity of antigens we have examined, DNP-polymerized flagellin (DNP-POL), and DNP-linear dextran (DNP-DEX), depend upon the antigen dose and epitope density. These factors also determine the mobility of antigen bound to B cell surfaces. For DNP-POL bound to DNP-specific cells, the observed diffusion constants D decrease monotonically with increasing antigen dose and epitope density. Values of D range from 10.4 × 10^?11 cm² sec^?1 for DNP_0.4-POL at 0.15 μg/ml to 0.8 × 10^?11cm² sec^?1for DNP_3.5-POL at 30 μg/ml. For receptor-bound DNP-DEX, D depends strongly on antigen epitope density but not observably on antigen concentration. For epitope densities of 1.2 or less, D is close to the value of 21 × 10^?11cm²sec^?1 observed for single slg receptors. By an epitope density of 4.8, D has fallen to 2.1 × 10^?11cm²sec^?1. Peak immunogenicities for DNP-POL and DNP-DEX arc observed when antigen- receptor aggregates have mobilities 14-fold and 3-fold lower, respectively, than a single slg molecule.     

Tryptophan uptake by Mycobacterium tuberculosis H37Rv--inhibition by isoniazid.

The effect of various sub-inhibitory concentrations of isoniazid on tryptophan uptake by $\text{Mycobacterium tuberculosis}$ H₃₇Rv grown $\text{in vitro}$ and $\text{in vivo}$ was studied. Uptake, measured after 3 minutes of drug exposure was inhibited mildly by 0.1 μg/ml and 0.2 μg/ml concentration and completely by 0.3 μg/ml. However, with the minimal inhibitory concentration (MIC)⁷ of 0.5 μg/ml, not only inhibition but also a strong efflux of the preformed tryptophan pool were observed. The results are discussed in the light of the theory that isoniazid interferes with the cell wall mycolate synthesis.     

The relationship of protein synthesis to cell division and oral development in synchronized Tetrahymena pyriformis GL-C: An analysis employing cycloheximide

The effects of cycloheximide on synchronized Tetrahymena pyriformis strain GL-C were investigated at concentrations ranging from 0.01 to 10 μg/ml. The initial inhibition of protein synthesis was nearly total (>85%) at 1 μg/ml and above, partial (50–80%) at 0.2 to 0.05 μg/ml, and slight (<30%) at 0.02 μg/ml. Eventual recovery of protein synthesis to a rate approaching that of the controls took place at concentrations of 1 μg/ml and less. When the drug was added before a “transition point” at 55 minutes after the end of the synchronizing treatment (EST), cell division was blocked by 10 μg/ml, and delayed at concentrations of 1 μg/ml or less. The duration of delay was related to the degree of initial inhibition, and to the time required for recovery of protein synthesis; it also depended on the time after EST at which the drug was added. At a given concentration, maximum division delay was observed just prior to the “transition point;” this maximum delay was correlated with resorption of differentiating oral primordia, followed by the appearance of new primordia. The lesser delays observed at earlier times were correlated with temporary blockage of development of primordia in the “stomatogenic field” stage. Resumption of oral primordium development was, in both cases, temporally correlated with a substantial recovery of protein synthesis. After the “transition point,” cell division, and completion of oral development, was delayed slightly at the lower concentrations, and more substantially at 1 and 10 μg/ml, with some division-arrest at the latter concentration. Except for the recovery phenomenon, the developmental responses elicited by cycloheximide were similar to those observed earlier with puromycin. The bearing of these findings on the mechanism of synchronization in Tetrahymena is considered in the Discussion.     

Electrophoretic detection of reversible chlorpromazine · HCl binding at the human erythrocyte surface

The binding of chlorpromazine · HCl at the human erythrocyte surface has been detected through its effect on cellular electrophoretic mobility. Incubation of erythrocytes (approx. 5 · 10⁶/ml) in 23 μM chlorpromazine · HCl resulted in a reduction of negative electrophoretic mobility from the control value of $\text{?1.11 ± 0.01}$ (μm · s^?1)/(V · cm^?1) to $\text{?1.00 ± 0.02}$ (μm · s^?1)/(V · cm^?1) (pH 7.2, ionic strength 0.155). This mobility change was completely reversed when chlorpromazine · HCl was removed by centrifugal washing. Increasing the drug concentration to 70μM did not affect the mobility change, indicating saturation of the electrophoretically detectable drug binding sites over chlorpromazine · HCl concentration range studied here. The effect of the 23 μM chlorpromazine · HCl on electrophoretic mobility was also measured in isotonic media of reduced ionic strength. The drug-induced reduction in negative surface charge density was found to be independent of ionic strength over the range 0.155 (Debye length, 0.8 nm) to 0.00310 (Debye length, 5.7 nm).Fixation of erythrocytes with glutaraldehyde affected neither the normal electrophoretic mobility of discocytes nor the reduced electrophoretic mobility of chlorpromazine · HCl-induced stomatocytes. When these stomatocytes were first fixed with glutaraldehyde, then washed free of chlorpromazine · HCl, they retained the stomatocyte form while regaining a normal control electrophoretic mobility. Conversely, when discocytes fixed in that form were treated with chlorpromazine · HCl, they showed the same mobility change as did fixed or unfixed stomatocytes. The drug-induced mobility change is therefore independent of the shape change, but reflects a contribution to cellular surface charge density from the membrane-bound chlorpromazine · HCl molecules. From the charge reduction, it is estimated that about 10⁶ chlorpromazine · HCl molecules are bound at the electrokinetic cell surface and occupy approximately 0.4% of the total surface area.     

Action of Heparin on Platelet Electrophoretic Mobility

THE aggregating agents adenosine diphosphate (ADP) and noradrenaline induce a biphasic change in human platelet electrophoretic mobility¹. Small concentrations (0·005 mg ml.^?1 and 0·05 mg ml.^?1) increase platelet mobility after incubation for 10 min; high concentrations (0·05 mg/ml.) induce a decrease in mobility. The same change also occurs in pig² and rabbit³ platelet mobilities. Hampton and Mitchell⁴ showed that the addition of 1–50 U of heparin to plasma has no effect on the biphasic mobility of human platelets. Nevertheless, measurements of the electrophoretic mobility of pig platelets after intravenous infusion with heparin have led to in vitro studies of both pig and human platelets, which I shall now describe.     

The effect of superoxide dismutase and catalase on metabolism of 3H-arachidonic acid by washed platelets

Previous experiments have suggested that superoxide dismutase (SOD) and catalase (CAT) may inhibit prostaglandin synthesis. The purpose of this study was to determine if these free radical scavengers can alter the metabolism of free arachidonic acid (AA) by the cyclooxygenasse and lipoxygenase enzyme systems in platelets. In control experiments washed platelets were incubated with ³H-AA for 5 minutes, extracted and the products separated by reverse phase high pressure liquid chromatography (HPLC). In normal intact platelets 13.5 ± 0.6% of the radioactivity was found in TxB₂, 16.3 ± 1.4% in HHT, 61.3 ± 1.1% in 12-HETE and 9.0 ± 1.0% was unconverted AA. Pre-incubating the platelets for 1 minute with 10 μg/ml SOD or CAT or 10 μg/ml SOD plus 10 μg/ml CAT did not inhibit AA conversion or alter the percent product distribution. Similarly, SOD and CAT had no effect on AA metabolism in broken cells. However, as expected, pretreating platelets with indomethaoin blocked TxB₂,and HHT formation (P <.0001). We conclude that SOD and CAT do not inhibit cyclooxygenase or lipoxygenase metabolism of free AA in platelets.     

The effect of 2-deoxyglucose on guinea pig polymorphonuclear leukocyte phagocytosis

The effects of 2-deoxyglucose (DOG), an inhibitor of glycolysis, on guinea pig polymorphonuclear leukocytes (PMN) obtained from peritoneal exudates was examined. ATP levels in PMN were reduced by 40% by one hour following an incubation with 2-deoxyglucose. When complement (C3) coated ¹⁴C-staphylococcus aureus, C3 coated lipopolysaccharide-paraffin oil droplets (LPSPO), ¹⁴C-pneumococcus opsonized with IgG, or albumin coated paraffin oil droplets opsonized with IgG were added to cell suspensions containing DOG, the phagocytizing rate was 1,310 ± 55 cpm/5 x 10⁶ cells/15 minutes, 6 ± 2 μg paraffin oil (PO)/10⁷ cells/minute, 2,250 ± 175 cpm/1 x 10⁶ cells/20 minutes or 0.037 ± 0.01 mg PO/10⁷ cells/minute compared to control values of 5,970 ± 275 cpm/5 x 10⁶ cells/15 minutes, 35 ± μg PO/10⁷ cells/15 minutes, 4,510 ± 200 cpm/1 x 10⁶ cells/20 minutes and 0.067 ± 0.01 mg PO/10⁷ cells/minute. In parallel studies the phagocytic index for latex was 0.74 ± 0.28 in DOG compared to control of 2.36 ± 1.13 and the phagocytic rate of albumin coated paraffin oil droplets was 0.029 ± 0.01 mg PO/10⁷ PMN/minute in DOG compared to control of 0.048 mg PO/10⁷ cells/minute. When ATP levels were maintained by the simultaneous addition of 5 mM glucose or pyruvate to media containing DOG, latex ingestion was improved to 1.15 ± 0.3 with glucose and 1.59 ± 0.64 with pyruvate and albumin coated particles to 0.045 ± 0.01 mg PO/10⁷ PMN/minute with pyruvate. There was no improvement in the uptake of either the C3 dependent particles or IgG coated Pneumococci in media containing DOG and glucose and/or pyruvate. Following the removal of DOG from the extracellular medium and the addition of pyruvate or glucose, phagocytosis of C3 dependent LPS-PO was restored to normal values. Neither the binding of C3 or IgG coated particles to the PMN nor the lateral movement of glycoprotein utilizing concanavalin A capping was affected by DOG. Thus, the presence of DOG in the PMN containing adequate amounts of ATP will selectively and reversibly inhibit those surface events required for phagocytosis of C3 and IgG bound particles but not latex particles or albumin particles which non-specifically bind to PMN.     

Effects of β-bungarotoxin and taipoxin on contractions of canine airways caused by nerve stimulation

β-Bungarotoxin and taipoxin are snake venoms that have been reported to induce neuromuscular blockade exclusively in somatic motor neurons by a presynaptic mechanism. Taipoxin (1 μg/ml), but not β-bungarotoxin (1 μg/ml), depressed the contractile response of canine airway smooth muscle to electrical stimulation. Taipoxin (1 μg/ml) also slightly depressed the contractile activity of canine airways to two concentrations (5 × 10^?6M and 10^?5M) of exogenously administered acetylcholine. We conclude that taipoxin, but not β-bungarotoxin, induces a weak neuromuscular blockade in the parasympathetic fibers innervating canine airways. The sites of this inhibition are believed to be both presynaptic and postsynaptic.     

Comparative effects of adenine analogs upon metabolic cooperation between Chinese hamster cells with different levels of adenine phosphoribosyltransferase activity

Colony formation by variant Chinese hamster cells highly resistant to adenine analogs and deficient in adenine phosphoribosyltransferase (APRT) activity was measured after co-cultivation with APRT⁺, CHO-K1 cells in medium containing one of three different adenine analogs. Depending upon the density of APRT⁺ cells and the specific adenine analog, large differences in the recovery of APRT^? colonies were observed. The particular adenine analog and APRT⁺ cell density were more significant factors in the recovery of APRT^? colonies than the concentration of the analog or the level of APRT activity. The number of wild-type cells (CHO-K1) required to inhibit formation of APRT^? colonies by 50% (mean lethal density; MLD₅₀) with 65 μg/ml 8-aza-adenine (AzA) as the selective drug was 8.0 × 10⁵ cells/100 mm dish (1.5 × 10⁴/cm²). With 100 μg/ml 2,6-diaminopurine (DAP) the MLD₅₀ for CHO-K1 was 4.0 × 10⁵ cells/100 mm dish (7.3 × 10³/cm²). The MLD₅₀ for CHO-K1 when the DAP concentration was decreased to 50 μg/ml was only slightly higher, 5 × 10⁵ cells/100 mm dish (9.1 × 10³/cm²). The most toxic effect was observed with 2-fluoroadenine (FA). The MLD₅₀ for CHO-K1 in 2 μg/ml FA was 4.5 × 10⁴ cells/100 mm dish (8.2 × 10²/cm²), a cell density which permits minimal direct contact between APRT⁺ and APRT^? cells. The toxic effects of FA on individually resistant, APRT^? cells were found to be mediated by metabolites released into the medium by dying APRT⁺ cells. This metabolite toxicity to APRT^? cells was also demonstrated in mixtures with cells having only 8% of wild-type APRT activity. The MLD₅₀ for these APRT⁺ (8%) cells in 2 μg/ml FA was 7.5 × 10⁴ cells/100 dish (1.4 × 10³/cm²), a small difference from the MLD₅₀ for cells with wild-type levels of APRT activity. The differences in the recovery of APRT^? colonies from mixtures with APRT⁺ cells in these three adenine analogs are critical to the design of procedures for the selection of APRT^? cells from populations of APRT⁺ cells and emphasize the importance of establishing the parameters of metabolic cooperation, not only in terms of cell density but also with regard to the particular selective agent, in any experiment designed to determine precise mutation rates or to test putative mutagens upon mammalian cells in culture.     

Stability of rapidly labelled messenger ribonucleic acid in Bacillus amyloliquefaciens during the phases of minimum and maximum extracellular enzyme formation.

The stability of rapidly labelled hybridizable messenger RNA in both exponential and post-exponential phase cells of Bacillus amyloliquefaciens was measured in terms of the rate of loss of its radioactivity. In the exponential phase, where 96% of the mRNA was specific for cell proteins and only 4% was exoprotein mRNA, the label was lost exponentially from the rapidly labelled hybridizable mRNA fraction with a half-life of six minutes at 30 °C. The antibiotic rifampicin, at a concentration of 10 μg/ml, had no effect on the characteristics of decay of this exponential-phase mRNA. In the post-exponential phase, where there were equal amounts of cell protein and exoprotein-specific mRNA, rapidly labelled hybridizable mRNA decayed exponentially in the presence of rifampicin (10 μg/ml), with a half-life of six minutes at 30 °C. In the absence of rifampicin the characteristics of decay were more complex. The evidence available suggested that this was due to the superimposition of a component attributable to reincorporation of degradation products of radioactive RNA on the characteristic exponential decay pattern of the post-exponential mRNA.Measurement of the stability of active mRNA, by studying the loss of ability to incorporate l-[¹⁴C]leucine into protein in the presence of rifampicin (10 μg/ml), gave half-lives of 4.5 minutes and six minutes, respectively, for exponential and post-exponential material.     

Effect of cycloheximide on protein and DNA synthesis and on the yields of chromosomal aberrations induced by DEB and ENU in vicia faba

Incubation of root tips in cycloheximide (CHM) at concentrations of 0.3–50 μg/ml inhibits the incorporation of [¹⁴C]leucine by 40–100% within 2 h. A depression in the incorporation of [³C]thymidine was observed after a 2-h incubation in CHM solution at 1 μg/ml.In root tips exposed for 2 h to CHM at 1 μ/ml the mitotic activity of cells was severely depressed within 15 h of recovery. Metaphases appearing after 20 h carried infrequent aberrations of the chromatid type. CHM at this concentration had no effect on the yield of aberrations induced by the alkylating agents diepoxybutane (DEB) and N-ethyl-N-nitrosourea (ENU) when applied as post-treatment.     

Ultrafiltration-light absorption spectrophotometric determination of silicon in blood

An ultrafiltration-light absorption spectrometric method for soluble molybdate-reactive silicon was assessed and applied to bovine and ovine blood plasma and sera, giving precise analytical results. Interfering protein above molecular weight 10,000–25,000 was removed by ultrafiltration, and silicon in ultrafiltrates was quantitated by measuring light absorption at 810 nm of the 1,2,4-aminonaphthol sulfonic acid/ascorbic acid-reduced silicomolybdate. Chemical interferences on the color-forming reaction of remaining blood components were tested by measuring recoveries of silicon added to real blood plasma samples and to synthetic blood plasma solutions, the latter containing typical levels of the major ions Na⁺, K⁺, Ca²⁺, HCO₃^?, and Cl^?, together with varying quantities of the potential interferants (amount per analytical reaction): phosphate (0–0.5 mg P), ferric ion (0–3 mg), fluoride (0–1.25 mg), vanadate (0–0.5 mg V), arsenate (0–10 μg As), and germanate (0–0.5 μg Ge). The mean recovery of added 0.8–9 μg silicon/g of bovine and ovine plasma was 97.7% (SE = 1.0, n = 17); the mean recovery of 1 and 5 μg silicon from synthetic blood plasma solutions with interferant levels up to 50-fold that in normal plasma was 99.2% (SE = 0.3, n = 47). Silicon concentrations found in bovine and ovine blood plasma and sera were typically around 7 μg/ml with procedural reagent blanks consistently low at a mean of 0.12 μg/test (SD = 0.011, n = 20). The silicon level in Center for Disease Control bovine serum (reference specimen Lot R-2274) was found to be (mean ± SE, n = 10) 1.147 ± 0.013 μg/g or 1.172 ± 0.013 μg/ml (25°C). The method detectivity (detection limit) was estimated at 0.03 μg.     

Dissociation constants of glucan phosphorylases of rabbit tissues studied by polyacrylamide gel disc electrophoresis

Using polyacrylamide gel disc electrophoresis, a simple and sensitive stain method for glucan phosphorylase (EC 2.4.1.1) was developed. With this method 0.3–1.5 μg or 1–5 units of phosphorylase could be demonstrated as a sharp band within a few hours. Mobility of phosphorylase fraction was retarded in gels containing glycogen. From the change of mobility as a function of glycogen concentrations, the dissociation constants of phosphorylases of rabbit skeletal muscle, liver, and brain with rabbit liver glycogen was calculated. They were 6.1 × 10⁻⁴, 22 × 10⁻⁴, and 13 × 10⁻⁴m, respectively. From the electrophoretic mobility, rabbit tissue phosphorylases could be classified into two: those of brain and kidney, and those of skeletal muscle and liver. When the electrophoresis gel, however, contained glycogen in a considerable concentration, their mobilities were retarded, and the retardation was more marked with those of skeletal muscle and brain than with those of liver and kidney. Hence, all four tissue phosphorylases could be distinguished only by the disc gel containing glycogen.     

Radioprotection of bone marrow stem cells by superoxide dismutase.

In mouse bone marrow cells the radioactivity from intravenously injected ¹²⁵I-superoxide dismutase reached its maximum level 1 h later. The 1 h interval was used while examining the effect of the unlabelled enzyme on the proliferative capacity of X-irradiated hematopoietic stem cells, as measured by their ability to form colonies in host spleens. When administered to donor mice in the amount of 35 μg/g body weight, the enzyme protected the cells, exposed to 350 rads, by a factor of 2.16 ±0.18. At doses of 15, 70, and 100 μg/g, this factor was reduced to 1.44 ± 0.18, 1.15 ± 0.15, and 0.7 ± 0.1, respectively. The value of 2.16 at 35 μg/g was increased to 2.5 by giving an additional therapeutic dose of the same size 1 h after the X-irradiation.     

Incorporation of 14C-Tyrosine into Soluble Ribonucleic Acid from Baker’s Yeast

The incorporation of ¹⁴C-tyrosine into S-RNA catalyzed by a partially purified tyrosine activating enzyme from baker’s yeast was observed. The maximum incorporation was shown in the presence of 5 μmoles of ATP, 10 μmoles of MgCl₂ and 10~100 μmoles of KCl in the reaction mixture of total volume of 1ml, at pH 7.8 when 1.2 mg of S-RNA, 0.1 μmole of ¹⁴C-tyrosine and 400 μg of enzyme protein were used. Beyond the concentration of ATP, MgCl₂ and KCl described above, the tendency of inhibition was observed. The incorporation was strongly inhibited by pCMB and reactivated by cysteine. Manganese and calcium ions were effective as substitutes for magnesium. S-RNA used was prepared from whole baker’s yeast cell with phenol, but S-RNA obtained from the supernatant of the ground yeast had lost its incorporating activity.     

Identification and determination of pirlimycin residue in bovine milk and liver by high-performance liquid chromatography-thermospray mass spectrometry

Determinative and confirmatory methods of analysis for pirlimycin (I) residue in bovine milk and liver have been developed based on HPLC-thermospray (TSP) MS. Milk sample preparation consisted of precipitating the milk proteins with acidified acetonitrile followed by a solvent partitioning with a mixture of n-butyl chloride and hexane, extraction of I from the aqueous phase into methylene chloride (MC), and solid-phase extraction clean-up. For liver, samples (2 g) were extracted with 0.25% trifluoroacetic acid in acetonitrile. The aqueous component was released from the organic solvent with n-butyl chloride. The aqueous solution was reduced in volume by evaporation, basified with ammonium hydroxide, then extracted with MC. The MC was evaporated to dryness and the dried residue reconstituted in 2.0 ml of 0.1 M ammonium acetate for analysis. A chromatographically resolved stereoisomer of I with TSP-MS response characteristics identical to I was used as an internal standard (I.S.) for quantitative analysis based on the ratio of peak areas of I to I.S. in the protonated molecular-ion chromatogram at m/z 411.2.The method for milk was validated by the analysis of control milk samples spiked with I at concentrations from 0.05 to 0.8 μg/ml. The overall recovery of pirlimycin across this concentration range was 95.4% ± 8.7%. The limit of quantitation (LOQ) and limit of confirmation (LOC) of the method were validated to be 0.05 μg/ml and 0.10 μg/ml, respectively.The method for liver was validated by the analysis of control liver samples spiked with I at concentrations ranging from 0.025 to 1.0 μg/g. The overall recovery of pirlimycin was 97.6% ± 5.1% in this concentration range. The validated limit of quantitation (LOQ) and limit of confirmation (LOC) of the method were 0.025 μg/g and 0.10 μg/g, respectively.Four diagnostic ions for I were monitored for confirmation: the pseudo-molecular ions (M + H)⁺ at m/z 411.2 (³⁵Cl) and m/z 413.2 (³⁷Cl), and fragment ions at m/z 375.2 and 158.1. Confirmatory criteria were defined for these assays.     

Leb-active glycolipid in human plasma: Measurement by radioimmunoassay

A radioimmunoassay that measures Le^b-active glycolipids in human plasma has been developed using antiserum from a goat immunized with a Le^b blood group hapten, lacto-N-difucohexaose I, conjugated to polylysine. Binding by the antiserum of lacto-N-difucohexaose I conjugated to ¹²⁵I-labeled bovine serum albumin is specifically inhibited by Le^b-active ceramide hexasaccharide. Plasma levels of the glycolipid are quantitated by comparing the inhibitory activity of plasma with that of the purified Le^b-active glycolipid. Plasma samples from 35 blood group O Le(a ? b +) individuals contain Le^b-active ceramide hexasaccharide at an average concentration of 0.9 μg/ml (range: 0.2 to 2.5 μg/ml); no Le^b-active glycolipid (less than 0.02 μg/ml) could be detected in plasma from blood group O Le(a + b?) or O Le(a? b?) individuals. Plasma from A₁ Le(a ? b+) individuals contains less Le^b-active glycolipid than plasma from A₂ Le(a? b+) individuals: its level in 19 samples of A, Le(a? b+) plasma averages 0.2 μg/ml (range: 0.1 to 0.45 μg/ml), and its level in 9 samples of A₂ Le(a? b+) plasma averages 1.1 μg/ml (range 0.8 to 1.3 μg/ml). About one-third of the total Le^b-active glycolipid in whole blood is associated with erythrocytes and the rest is found in plasma.     

Effect of melittin-induced membrane alterations on rat heart adenylate cyclase activity

Melittin, a surface-active, 26-amino acid polypeptide from bee venom, has been reported to alter a variety of membrane properties including stability, permeability, and fluidity, the latter having been shown to be altered in a biphasic manner. Melittin induced a biphasic alteration of rat heart microsomal adenylate cyclase activity, stimulating it at low concentration (<30 μg/ml) and inhibiting it at higher concentrations (100 μg/ml or higher). Melittin potentiated sodium fluoride and 5′-guanylylimidodiphosphate activation of adenylate cyclase below 40 μg/ml but it inhibited at high concentrations, except in the presence of high concentrations of 5′-guanylylimidodiphosphate (10^?4m). Basal and fluoride-activated adenylate cyclase exhibited no significant change in the K_m for ATP in the presence of melittin at <40 μg/ml, but the V was elevated. Potentiation by melittin of adenylate cyclase was observed at all fluoride, 5′-guanylylimidodiphosphate, and magnesium concentrations tested. The observed effects of melittin on rat heart adenylate cyclase are consistent with it acting by altering the properties of membrane lipids with which the enzyme is associated.