Novel and sensitive noncompetitive enzyme immunoassay for kassinin in rat plasma

A novel and sensitive noncompetitive enzyme immunoassay for kassinin is described. Kassinin was biotinylated using sulfosuccinimidyl-6-(biotinamido)hexanoate. The biotinylated kassinin was trapped on anti-kassinin IgG-coated polystyrene balls and, after washing to eliminate other biotinylated substances, was eluted with HCl. The biotinylated kassinin eluted was reacted with anti-kassinin Fab'-peroxidase conjugate and trapped onto streptavidin-coated polystyrene balls. Peroxidase activity bound to the polystyrene balls was assayed by fluorimetry. The detection limit of kassinin was 0.13 pg (0.1 fmol)/tube or 0.065 microgram/l of rat plasma, which was 750-fold or 15-fold lower than that for competitive radioimmunoassay.     

Heterologous immunoadsorption in the purification of enzymes: N-acetyl-β-glucosaminidase

Summary An immunoadsorbent column was prepared using a specific antibody toN-acetyl--glucosaminidase of human origin. Although no precipitating activity of the antisera was found with mouse or rat liver extracts, enzyme was easily eluted from the column which provided about 50fold, single-step purifications of these heterologous enzymes.     

Enzyme-linked immunosorbent assay for measuring serum IgE

Summary Serum IgE was measured by a sandwich method using polystyrene beads coated with anti-IgE -globulin and peroxidase labelled anti-IgE -globulin. The method was simple, and as sensitive as the radioimmunoassay.We are grateful for the skillful assistance of Mrs K. Kobayashi.     

Effects of female sex hormones on polyamine-oxidizing enzyme activities and polyamine concentrations in immature rat uterus and liver

17-estradiol (E₂) and progesterone (P) treatment of immature female rats (10 g/100 g body weight) respectively resulted in 1.38-fold (p<0.02) and 1.42-fold (p<0.02) increase in the uterine polyamine oxidase activity, and 2.45-fold (p<0.001) and 1.43-fold (p<0.02) increase in the uterine diamine oxidase activity, as compared to the controls. E₂ caused a 5-fold (p<0.05) and a 1.36-fold (p<0.05) increase in putrescine and spermidine concentration respectively in rat uterus. Increases of 1.7-fold (p<0.02) and 1.6-fold (p<0.05) in putrescine and spermine concentration were determined in the P-treated uterus, as compared to the controls. The spermidine/spermine ratio, which is regarded as an index of growth rate, was higher in the E₂-treated uterus and lower in the P-treated uterus than in the control uterus. No statistically significant hormonal effects were estimated in the immature liver. The data reported suggest the possibility of an involvement of polyamine-oxidizing enzymes in the modulation of polyamine concentrations in rat uterus by the female sex hormones.     

Enhancement of fluorescence of pyrene-containing lipids by polar media,detergents and phospholipids

Summary The fluorescence intensities of a medium-chain fatty acid and of several amphiphilic lipids, each containing pyrene in covalent linkage, were enhanced considerably by: 1) Dissolving in mixtures of a polar solvent (e.g. methanol, ethanol, tetrahydrofuran or dimethylsulfoxide) and water; for each individual compound, a certain ratio of solvent to water provided maximal fluorescence intensity. 2) Incorporating into micelles of reduced Triton X-100; an excess of detergent was used so that, statistically, only one molecule of lipid resided in one micelle of the Triton X-100. 3) Incorporating into liposomes of egg phosphatidylcholine; maximal fluorescence was observed using a large excess of phosphatidylcholine. When related to the fluorescence intensities in chloroform/methanol (21, by vol.) or water, the enhancement of fluorescence in the above three systems was about 2-6-fold or up to 60-fold, respectively.     

The free radical oxidation of the tetrahydrocannabinols

Summary The free radical oxidation of ¹ and ⁶-tetrahydrocannabinol has been examined by spin trapping techniques and intermediates that would lead to cannabinol have been trapped. The 1st step in the oxidation of ¹-THC involves the removal of 3-H, while for ⁶-THC, either 2-H or 5-H. Intermediates were isolated which could be pyrolysed to the diene and cannabinol.     

Metallothionein induction by cadmium and zinc in rat secretory organs

Summary Metallothionein (MT) levels were determined in four secretory organs of the rat following administration of zinc (Zn) and cadmium (Cd). The concentrations of MT in the lacrimal, parotid and adrenal glands of untreated rats were in the range of 2.2–4.9 g/g wet weight tissue while in the pancreas it was shown to be 15.2 g/g. Injection of zinc at total doses of 16, 32 and 80 mg/kg resulted in a 1.8-, 3.2- and 5.9-fold increase in lacrimal MT content, respectively, while a 10.2- and 13.1-fold elevation was observed following treatment with 4 and 8 mg/kg of Cd, respectively. Similar findings were found in the adrenal gland. The parotid MT was elevated 5.9 and 17 times following Zn treatment at doses of 16 and 80 mg/kg respectively, whereas 4 mg/kg of Cd increased MT 14.4 times in this gland. Pancreatic MT was elevated by 39- and 40-fold after injection of Zn at doses of 16 and 32 mg/kg respectively, whereas 4 and 8 mg/kg of Cd caused a 9.8- and 17.9-fold induction, respectively. These results may indicate that secretory organs participate in metabolism of heavy metals in the mammalian body.     

Vergleichende Untersuchungen über die angeborene Grössenunterscheidung beiTilapia nilotica undHemihaplochromis multicolor (Pisces; Cichlidae)

Summary Female mouth-brooding Cichlid fishes pick up the young fry again when disturbed. The young, for their part, collect about the head of the mother fish and, contacting it, try to enter the mouth cavity. This contact behaviour is based upon an inborn readiness to react on the visual stimulus offered by the mother. Mother fishes inTilapia nilotica are much larger than those ofHemihaplochromis multicolor. By comparative experimental studies with models of various size (black balls of different diameters) it is shown that the young of the 2 species strictly prefer different sizes of the model, corresponding with the different sizes of the parent fishes.

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Herrn Prof.H. M. Peters zum 60. Geburtstag gewidmet.     

Absence of low molecular weight DNA polymerase activity from the nuclei ofAmoeba discoides

Summary Amoeba discoides nuclear protein partially purified by passage through Sephadex G-200 showed 3 high-mol.-wt DNA polymerase activities which eluted in and just following the void volume. No low-mol.-wt (45,000 daltons) DNA polymerase activity was detected. Nuclear protein layered on 5–20% sucrose gradients also showed an absence of lowmol.-wt DNA polymerase . The void volume enzyme showed deoxyribonuclease activity, but no low-mol.-wt nuclease activity was detected.     

Rapid purification of lactate dehydrogenase X from mouse testes by two steps of affinity chromatography on oxamate-sepharose

Summary At concentrations of 200 M NADH and 0.5M NaCl LDH-X is separated from the other LDH isozymes of mouse testes on oxamate-sepharose. In a second step LDH-X is bound to the same matrix at lower NADH and NaCl concentrations and the pure enzyme can subsequently be eluted.Acknowledgment. This work was supported by grants of the Deutsche Forschungsgemeinschaft awarded to the Sonderforschungsbereich 29 Embryonale Differenzierung und Entwicklung.     

Development of a new tachykinin antagonist

The mouse urinary bladder possesses a tachykinin receptor which responds to kassinin and eledoisin, but not to some other tachykinins. The action of kassinin, but not that of eledoisin, was blocked in a surmountable manner by a new tachykinin antagonist, D-Pro4, Val8, D-Trp (SP).     

Purification and characterization of aβ-glucosidase (linamarase) from the haemolymph ofZygaena trifolii Esper, 1783 (Insecta,Lepidoptera)

Summary A -glucosidase (linamarase) was purified 52-fold with a recovery of 27% from the haemolymph of the larvae ofZygaena trifolii, ESPER, 1783 (Lepidoptera, Zygaenidae). The final enzyme preparation was found to be nearly homogeneous on both disc polyacrylamide gel electrophoresis and SDS-polyacrylamide gel electrophoresis. The molecular weight of the enzyme was determined to be about 130 kDa; it consisted of two subunits of about 66 kDa. The enzyme showed an optimum between pH 4.5 and 5 with linamarin and a broad optimum between pH 3.5 and 6.5 for p-nitrophenyl--D-glucoside; the temperature optimum was 40°C. The -glucosidase showed a high specificity for its endogenous substrates linamarin and lotaustralin. Among the other natural and artificial substrates tested, only prunasin and p-nitrophenyl--D-glucoside were hydrolyzed by the enzyme, whereas linustatin, salicin, cellobiose and trehalose were not. The enzyme is strongly inhibited by -glucosylpiperidine.     

Development of a new tachykinin antagonist

Summary The mouse urinary bladder possesses a tachykinin receptor which responds to kassinin and eledoisin, but not to some other tachykinins. The action of kassinin, but not that of eledoisin, was blocked in a surmountable manner by a new tachykinin antagonist, D-Pro⁴, Val⁸, D-Trp^7,9,10 (SP)^4–11.Acknowledgments. We thank Mrs Danielle Solomos for technical help. This study was financed by a grant from the Swiss National Science Foundation (3.800-80.2).     

Methyl cellosolve-induced sensitization of mice to bacterial endotoxin

Summary Treatment of mice with a sublethal dose of methyl cellosolve renders the animals 64-fold more sensitive to the toxic effects of bacterial endotoxin.Acknowledgments. I would like to thank Mr Larry Davis for his excellent technical assistance in these studies.This work was supported by the Naval Medical Research and Development Command, Research Work Unit No. MF585240013.1026. The opinions and assertions contained herein are the private ones of the writer and are not to be construed as official or reflecting the views of the Navy Department or the naval service at large. The experiments conducted herein were conducted according to the principles set forth in the Guide for the Care and Use of Laboratory Animals, Institute of Laboratory Resources, National Research Council.     

Cholesterol feeding to rats does not modulate the expression of binding sites for HDL on liver membranes

Summary The binding of HDL, Apo-E-free, was studied in rats fed a cholesterol rich diet for 2, 4 and 7 days. Plasma cholesterol increased up to 16-fold (from 55 to 900 mg/dl); liver cholesterol was also raised, from 0.5 to 16 mg/g of tissue. The HDL binding to membrane preparations was not affected while the binding of VLDL was reduced to about 50% of the controls. These data show, therefore, that liver binding sites for HDL are refractory to regulation by dietary cholesterol.     

Thrombospondin co-localises with TGFβ and IGF-I in the extracellular matrix of human osteoblast-like cells and is modulated by 17β estradiol

Thrombospondin (TSP) is a multifunctional glycoprotein which is synthesised by several cell types including osteoblasts, and incorporated into the extracellular matrix (ECM) of these cells. The function and regulation of TSP in bone is not clear. In this study, using a long term culture model of human osteoblast-like cells, we examined the distribution of TSP in the ECM and its modulation by added estradiol. In this model the osteoblast-like cells form a regular multilayer which continues to increase in depth up to 50 days post confluence. In the ECM of these cultures and in 19-week fetal bone, the bone markers osteocalcin and alkaline phosphatase were diffusely distributed in the matrix. In contrast, labelling for TSP was concentrated, confined to the banded collagen and its immediately adjacent ECM. This pattern of labelling resembled that of the growth factors transforming growth factor-I (TGF), and insulin-like growth factor-I (IGF-I), with which TSP label co-localised. Labelling intensities were comparable between fetal bone and the in vitro material for TSP, TGF and IGF-I. TSP label was present by 10 days post confluence, reached a maximum by 20 days, and declined slowly thereafter, a time course which was similar to that of IGF-I. Incubation of osteoblast-like cell cultures with 17 estradiol resulted in an increase in multilayer depth and a maximal 3-fold increase in TSP labeling at 30 days as well as approximately 2-fold increases for TGF and IGF-I. The dose-response relationship for these responses to estradiol treatment was biphasic with maximal increases at 10^–10 M–10^–11 M of added estradiol. Treatment with 17 estradiol produced labelling intensities that were not significantly different from controls. Studies with other cell types have suggested that TSP may be involved in modulation of growth factor activity. The similarities between TSP, TGF and IGF-I, in terms of their distribution and regulation by 17 estradiol treatment, may indicate a role for TSP in modulating bone cell proliferation and function through interaction with local growth factors.     

The lymphatic route. 1) Albumin and hyaluronidase modify the normal distribution of interferon in lymph and plasma

Summary When human recombinant interferon-₂ diluted in saline was injected s.c. into rabbits, the total amount recovered in thoracic lymph was less than 0.4%. Recoveries increased from 2- to 8-fold if interferon was injected in 4% albumin or with hyaluronidase, respectively. Albumin added to interferon acts as an interstitial fluid expander, thus favoring interferon absorption through lymphatics rather than blood capillaries. This strategy may increase the therapeutic index of interferon.This work was supported by Ministero Pubblica Istruzione and Progetto Finalizzato Oncologia, contract No. 84.00461.44, CNR, Roma.     

Enhanced heparan sulfate proteoglycan-mediated uptake of cell-penetrating peptide-modified liposomes

Protein transduction domains (PTDs) are used to enhance cellular uptake of drugs, proteins, polynucleotides or liposomes. In this study, functionalized Antennapedia (Antp, aa 43–-58) and HIV Tat (aa 47–57) peptides were coupled to small unilamellar liposomes via thiol-maleimide linkage. Modified liposomes showed higher uptake into a panel of cell lines including tumor and dendritic cells than unmodified control liposomes. Liposome uptake was time and concentration dependent as analyzed by flow cytometry and live-cell microscopy. At least 100 PTD molecules per small unilamellar liposome (100 ± 30 nm) were necessary for efficient translocation into cells. Cellular uptake of PTD-modified liposomes was 15- to 25-fold increased compared to unmodified liposomes and was inhibited by preincubation of liposomes with heparin. Glycosaminoglycan-deficient CHO cells showed dramatically reduced cell association of PTD-modified liposomes, confirming the important role of heparan sulfate proteoglycans in PTD-mediated uptake. Antp-liposomes used as carriers of the cytotoxic drug N⁴-octadecyl-1--D-arabinofuranosylcytosine-(5- 5)-3-C-ethinylcytidine showed a reduction of the IC₅₀ by 70% on B16F1 melanoma cells compared with unmodified liposomes. PTD-functionalized liposomes, particularly Antp-liposomes, represent an interesting novel carrier system for enhanced cell-specific delivery of a large variety of liposome-entrapped molecules.Received 16 April 2004; received after revision 13 May 2004; accepted 25 May 2004     

Purification of mouse liver thymidylate synthetase by affinity chromatography using 10-methyl-5,8-dideazafolate as the affinant

Summary Thymidylate synthetase (methylenetetrahydrofolate: 2-deoxyuridine-5-monophosphate C-methyltransferase; EC 2.1.1.45) from neonatal mouse liver has been purified 714-fold by affinity chromatography on aminohexylsepharose bound 10-methyl-5,8-dideazafolate.Supported by grant CA22754, awarded by the National Cancer Institute, DHEW.     

Discovery of novel avermectins with unprecedented insecticidal activity

Summary A new class of insecticidal and antiparasitic agents, 4-amino-4-deoxy avermectins, has been developed by chemical modification of avermectin B₁. The most effective of these compounds are 1500-fold more potent than avermectin B₁ (abamectin) against the beet armywormSpodoptera exigua and show similar potency against other lepidopteran larvae.