An agglutinin with unique specificity for N-glycolyl sialic acid residues of thyroglobulin in the hemolymph of a marine crab Scylla serrata (Forskal).

A novel agglutinin with specificity for sialic acid sequence of sugars in thyroglobulin is identified in the hemolymph of Scylla serrata. The physico-chemical characteristics of its binding affinity, such as pH and temperature optima, and cationic requirements are defined. N-glycolyl neuraminic acid (NeuGc) (at 0.6 mM), in contrast to N-acetyl neuraminic acid (NeuAc) (at greater than 5.0 mM), is the potent inhibitor of hemagglutination. Bovine and porcine thyroglobulins containing NeuGc, inhibited the agglutination. NeuGc-acid glycoprotein fraction (bovine) but not NeuAc-acid glycoprotein fraction (human) inhibited the hemagglutination. The inability of other NeuGc-glycoproteins (bovine submaxillary mucin) to inhibit the agglutination suggests that the agglutinin may also recognize glycosidic linkage associated with NeuGc. The potential of the agglutinin in identifying NeuGc containing human tumor associated antigens is discussed.     

An agglutinin with unique specificity for N-glycolyl sialic acid residues of thyroglobulin in the hemolymph of a marine crabScylla serrata (Forskal)

A novel agglutinin with specificity for sialic acid sequence of sugars in thyroglobulin is identified in the hemolymph ofScylla serrata. The physico-chemical characteristics of its binding affinity, such as pH and temperature optima, and cationic requirements are defined. N-glycolyl neuraminic acid (NeuGc) (at 0.6 mM), in contrast to N-acetyl neuraminic acid (NeuAc) (at >5.0 mM), is the potent inhibitor of hemagglutination. Bovine and porcine thyroglobulins containing NeuGc, inhibited the agglutination. NeuGc-acid glycoprotein fraction (bovine) but not NeuAc-acid glycoprotein fraction (human) inhibited the hemagglutination. The inability of other NeuGc-glycoproteins (bovine submaxillary mucin) to inhibit the agglutination suggests that the agglutinin may also recognize glycosidic linkage associated with NeuGc. The potential of the agglutinin in identifying NeuGc containing human tumor associated antigens is discussed.     

Modulation of 3-hydroxy-3-methyl glutaryl CoA reductase by 2,3-diphosphoglyceric acid

Rat liver microsomal 3-hydroxy-3-methylglutaryl CoA (HMG-CoA) reductase was activated by 50% at a concentration of 0.4 mM 2,3-diphosphoglyceric acid (DPG) and by 11-fold at 10 mM DPG. DPG also prevented the inactivation of HMG-CoA reductase by ATP and Mg++. Rat liver microsomal HMG-CoA reductase prepared in the presence of 1 mM DPG was significantly more active than when prepared in the absence of DPG. Activation of the enzyme by DPG and protection of the enzyme against inhibition by ATP and Mg++ by DPG were also observed with solubilized HMG-CoA reductase.     

Modulation of 3-hydroxy-3-methyl glutaryl CoA reductase by 2,3-diphosphoglyceric acid

Summary Rat liver microsomal 3-hydroxy-3-methylgularyl CoA (HMG-CoA) reductase was activated by 50% at a concentration of 0.4 mM 2,3-diphosphoglyceric acid (DPG) and by 11-fold at 10 mM DPG. DPG also prevented the inactivation of HMG-CoA reductase by ATP and Mg⁺⁺. Rat liver microsomal HMG-CoA reductase prepared in the presence of 1 mM DPG was significantly more active than when prepared in the absence of DPG. Activation of the enzyme by DPG and protection of the enzyme against inhibition by ATP and Mg⁺⁺ by DPG were also observed with solubilized HMG-CoA reductase.This work was supported by Research Award # 697 G2-1 from the American Heart Association, Greater Los Angeles Affiliate, and by grant # 1R01 HL22672 from the National Institutes of Health. We thank M. Brun and M. Curtis for their excellent technical assistance.     

Modulation of carcinoembryonic antigen release by HT-29 colon carcinoma line in the presence of different agents

In this study we followed the effects of various differentiating agents on the expression of carcinoembryonic antigen (CEA) released into the medium by a colon carcinoma cell line HT-29. Butyric acid 1 mM markedly increased the level of CEA (12-fold in comparison to control levels). 12-O-tetradecanoyl-phorbol-13-acetate (TPA) 50 ng/ml and 5-azacytidine 4 x 10(-6) M increased the amount of CEA, 2- and 1.5-fold respectively. On the other hand retinoic acid 10(-5) M, N methyl-formamide 1% and N,N hexamethylene bisacetamide 2.5 mM decreased CEA 2-, 4- and 3-fold respectively. Our results emphasize that various differentiating agents affect CEA levels differently. Thus changes in CEA levels appear not to be reliable as a marker of a more differentiated phenotype.     

Effects of alanine, glycine and glutamic acid on nitrogenous excretion by Amphiuma means liver in organ culture.

High concentrations (10 mM) of alanine, glycine, and glutamic acid in the culture medium had no effect on urea production in Amphiuma means liver in organ culture. Ammonia production was increased in media containing added alanine and glycine, but reduced in medium with added glutamic acid.     

Potentiation of the biological activities of daunomycin and adriamycin by ascorbic acid and dimethylsulfoxide

Summary L-Ascorbic acid (0.57 mM) and dimethylsulfoxide (14.1 mM) were found to potentiate four times the antibacterial activities of daunomycin and adriamycin in theStaphylococcus aureus test. This effect, however, could not be demonstrated against eukaryotic cells and leukemia P388 in mice. The authors thank Dr A. Maráz, Department of Microbiology, J.A. University, Szeged, and Dr Zs. Somfai, Institute of Oncology, Budapest, for collaboration in the experiments with yeast and tumor cells.     

Inhibition of human platelet lipoxygenase by cyanide

Summary Potassium cyanide inhibited the lipoxygenase activity of a human platelet cytosolic fraction in a concentration-dependent manner (ID₅₀=2 mM). The inhibition was monitored by spectrophotometry (conjugation of diene bonds at 236 nm), by chromatography (inhibition of formation of 12-hydroperoxy eicosatetraenoic acid) as well as by measuring suppression of oxygen consumption. The lipoxygenase activity of intact platelets was also inhibited by KCN as evidenced by the reduction in 12-hydroxy-eicosatetraenoic acid formation in response to thrombin.Acknowledgments. This work was supported in part by NIH grant HL-14890. D.A. was a recipient of the Pharmaceutical Manufacturers Association Advanced Pre-Doctoral Fellowship.     

Modulation of carcinoembryonic antigen release by HT-29 colon carcinoma line in the presence of different agents

Summary In this study we followed the effects of various differentiating agents on the expression of carcinoembryonic antigen (CEA) released into the medium by a colon carcinoma cell line HT-29. Butyric acid 1 mM markedly increased the level of CEA (12-fold in comparison to control levels). 12-O-tetradecanoyl-phorbol-13-acetate (TPA) 50 ng/ml and 5-azacytidine 4×10^–6M increased the amount of CEA, 2- and 1.5-fold respectively. On the other hand retinoic acid 10^–5M, N methyl-formamide 1% and N,N hexamethylene bisacetamide 2.5 mM decreased CEA 2-, 4- and 3-fold respectively. Our results emphasize that various differentiating agents affect CEA levels differently. Thus changes in CEA levels appear not to be reliable as a marker of a more differentiated phenotype.     

(−)-Epigallocatechin gallate,a polyphenolic tea antioxidant,inhibits peroxynitritemediated formation of 8-oxodeoxyguanosine and 3-nitrotyrosine

Reaction with peroxynitrite at pH 7.4 and 37°C was found to increase the 8-oxodeoxyguanosine levels in calf thymus DNA 35-38-fold. This oxidation of deoxyguanosine, as well as the peroxynitrite-mediated nitration of tyrosine to 3-nitrotyrosine, was significantly inhibited by ascorbic acid, glutathione and (–)-epigallocatechin gallate, a polyphenolic antioxidant present in tea. For 50% inhibition of the oxidation of deoxyguanosine to 8-oxodeoxyguanosine, 1.1, 7.6 of 0.25 mM ascorbate, glutathione or (–)-epigallocatechin gallate, respectively, was required. For 50% inhibition of tyrosine nitration, the respective concentrations were 1.4, 4.6 or 0.11 mM. Thus, (–)-epigallocatechin gallate is a significantly better inhibitor of both reactions than either ascorbate or glutathione. Reaction of (–)-epigallocatechin gallate with peroxynitrite alone resulted in the formation of a number of products. Ultraviolet spectra of two of these suggest that the tea polyphenol and/or its oxidation products are nitrated by peroxynitrite.     

Probing the penicillin sidechain selectivity of recombinant deacetoxycephalosporin C synthase

Deacetoxycephalosporin C synthase from Streptomyces clavuligerus catalyses the conversion of the five-membered penicillin ring to the unsaturated six-membered cephem ring of deacetoxycephalosporin C. The effects on enzyme activity of the penicillin substrate sidechain and various cofactors were investigated using a continuous spectrophotometric assay. The conversion of penicillin G to phenylacetyl-7-aminodeacetoxycephalo sporanic acid (G-7-ADCA) was confirmed, and further details of the reaction were elucidated. The conversion of ampicillin to cephalexin was faster than that of acetyl-6-APA to acetyl-7-ADCA kcat = 0.120 +/- 0.001 s(-1) versus 0.035 +/- 0.001 s(-1), but they had similar Km values: 4.86 +/- 0.12 and 3.28 +/- 0.26 mM, respectively. Amoxycillin and penicillin V were also converted at low levels. Conversion was not detected for penicillanate, 6-aminopenicillanate, carbenicillin, temocillin, ticarcillin or benzylpenicilloic acid, suggesting that the enzyme has a relatively strict selectivity for the sidechain of the penicillin substrate.     

Effect of (+)-catechin on renal and intestinal transport.

The ability of dog renal cortex slices to accumulate beta-methyl-glucoside or glycine is enhanced by the flavonoid (+)-catechin at a concentration of 3.5 mM. This stimulatory effect is apparently due to a decreased rate of efflux of either substrate. On the other hand, the uptake of p-amino-hippuric acid and N1-methyl-nicotinamide is inhibited by (+)-catechin. The drug at the same concentration is without action on amino-acid transport by guinea-pig intestine in vitro.     

Assessment of the scavenging action of reduced glutathione, (+)-cyanidanol-3 and ethanol by the chemiluminescent response of the xanthine oxidase reaction

The free radical scavenging capacity of reduced glutathione (GSH), (+)-cyanidanol-3 and ethanol was assessed by their interference with the maximal chemiluminescent response produced by the xanthine oxidase reaction. GSH and (+)-cyanidanol-3 induce a progressive inhibition of chemiluminescence when increasing amounts are added to the reaction mixture. GSH and (+)-cyanidanol-3 added together at low concentrations (1 and 0.05 mM respectively) exhibit an additive effect. The addition of ethanol presents a biphasic effect. It inhibits chemiluminescence at low concentrations (10-50 mM) while at higher concentrations (75-500 mM) this effect is reversed. Estimation of the concentrations required to produce half of the maximal inhibition of chemiluminescence by these agents revealed that ethanol is less effective than GSH and (+)-cyanidanol-3 as a free radical scavenger in the system used.     

The oxygen-linked hydrogen ion binding (the Haldane coefficient) of bovine hemoglobin.

The Haldane coefficient (the amount of the oxygen-linked hydrogen ion binding of hemoglobin) was determined in bovine erythrolysate (Hb concentration equals 13.5 mM) by means of the differential titration method with varying PCO2 from 0 to 74 mm Hg and pH from 6.0 to 8.5 at 37 degrees C. The maximum value of the coefficient was found to be 0.49 mM per mM Hb at PCO2 equals 0 and pH 7.20. With increasing of PCO2, the coefficient became smaller in all ranges of pH studied. The coefficient under the conditions of pH 7.20 and PCO2 equals 45 mm Hg that are normally prevailing in the interior of bovine erythrocytes was 0.31.     

Effect of indole-3-acetic acid on the kinetics of horseradish peroxidase catalyzed scopoletin oxidation

Summary The kinetics of horseradish peroxidase catalyzed scopoletin oxidation were observed to be sigmoidal. The apparent K_ms for scopoletin is 0.9 mM. Inhibition of scopoletin oxidation by indole-3-acetic acid (IAA) appears to be non competitive. Non competitive inhibition by IAA suggests a more significant role of the peroxidase protein matrix in regulating the activity of the heme moiety.     

Hormone content of mouse pancreatic islets subjected to different in vivo and in vitro functional demands

Summary Mice treated for 4 days with tolbutamide displayed decreased serum glucose values with a concomitant decrease of their islet insulin content. Mouse islets cultured for 1 week at a low (3 mM) or a high (28 mM) glucose concentration contained less insulin than non-cultured islets and islets cultured at a medium (11 mM) glucose concentration. All groups of cultured islets contained more glucagon than non-cultured islets. The somatostatin content of high- and medium-glucose cultured islets was higher than that of freshly isolated islets.The skilled technical assistance of Astrid Nordin, Ewa Forsbeck and Eva Törnelius is gratefully acknowledged. Financial support was received from the Swedish Medical Research Council (12X-109; 12X-2297), the Nordic Insulin Fund and the Swedish Diabetes Association.     

Characterization of the (Na+-K+)-ATPase from endometrial plasma membranes of immature mammals]

The (Na+-K+)-ATPase in plasma membrane from Mammiferous endometrium is characterized by the Mg/ATP ratio equal to one, and by a distinct affinity for Na+ (1.3 mM) and K+ (2 mM). The activity is maximum for pH 7.4-7.5 in presence of Mg++ 2mM and ATP 2 mM, Na+ 140 mM and K+ 10 mM.     

Degradation of pheromone biosynthesis-activating neuropeptide (PBAN) by hemolymph enzymes of the tobacco hornworm,Manduca sexta,and the corn earworm,Helicoverpa zea

The tritium-labeled bis-norleucine analog ofHelicoverpa zea pheromone biosynthesis-activating neuropeptide ([³H]NLPBAN) was incubated in vitro with hemolymph fromManduca sexta orH. zea adult females. The incubations resulted in the formation of several tritium-labeled degradation products. At a [³H]NLPBAN concentration of 0.9 μM the degradation proceeded at a very slow but physiologically plausible rate (2–10 fmol/min/μl hemolymph). The primary [³H]NLPBAN degradation reaction inM. sexta hemolymph was not inhibited by 20 μM leupeptin, 0.1 mM amastatin, 1 mM EDTA, 1 mM EGTA, 1 mM 1,10-phenanthroline, or 2 mM 4-(2-aminoethyl)benzenesulfonyl fluoride; but secondary reactions may have been affected, as some of the inhibitors changed the radio-HPLC profile of the degradation products. It is concluded that hemolymph ofM. sexta andH. zea contains peptidase(s) capable of inactivating circulating PBAN.     

Effects of Ca2+ and Mg2+ on motility of sea urchin spermatozoa

Summary Swimming speed of sea urchin spermatozoa, measured by a light scattering technique, did not change with 0-20 mM Ca²⁺ in the medium. The speed was maximum at the normal concentration of Mg²⁺ (49 mM) in sea water.Supported by grants-in-aid from the Ministry of Education, Science and Culture, Japan, and a grant from the Ford Foundation.