Specific detection of α-amylase activity in crude plant extracts after isoelectric focusing

A new method which utilizes Procion Red MX 2B amylopectin for the detection of α-amylase in crude plant extracts is described. The substrate is specific only against α-amylase hydrolysis and β-amylase does not attack it. Paper containing Procion Red MX 2B amylopectin applied to gels after isoelectric focusing reveals α-amylase isoenzymes as white bands. When this technique is used, heat-inactivation of β-amylase is not required.     

Demonstration of a beta-casomorphin immunoreactive material in the plasma of newborn calves after milk intake

Blood was collected from newborn calves before and after their first milk intake after birth; extracts of plasma were assayed by radioimmunoassay for the presence of beta-casomorphin-7 immunoreactive materials. No beta-casomorphin immunoreactivity was found in samples collected before milk ingestion; however, in samples collected after milk ingestion a beta-casomorphin-7 immunoreactive material was detected. Chromatographic characterization showed that this material was not identical with beta-casomorphin-7 but might rather represent a precursor thereof. The material proved resistant to enzymatic attack during a 30-min incubation period at 37 degrees C in the plasma of newborn calves, whereas beta-casomorphin-7 was degraded under these conditions. A physiological significance of beta-casomorphin-7 eventually cleaved from such a precursor material at any site in the newborn mammal is suggested.     

Competitive antagonists of bradykinin

The first sequence-related competitive inhibitors of the classic kinin in vitro (rat uterus guinea pig ileum) and in vivo (rat blood pressure) assays have been developed. Replacement of the proline residue at position 7 of bradykinin (BK) with a D-phenylalanine residue is the key modification which converts BK agonists into antagonists. [D-Phe7]-BK exhibits moderate (pA2 = 5.0) inhibition of BK activity on the guinea pig ileum but possesses weak BK-like myotropic activity on the isolated rat uterus and 2-4% of BK depressor potency in the rat blood pressure assay. The additional replacement of the phenylalanine residues at positions 5 and 8 of [D-Phe7]-BK with the isosteric beta-(2-thienyl)-alanine residue produces a potent antagonist of BK activity on the uterus (pA2 = 6.4), ileum (pA2 = 6.3), and in the rat blood pressure assay. The antagonism of BK action on smooth muscle is specific for kinins (BK, kallidin, Met-Lys-BK), but neither inhibitor antagonizes the smooth muscle activity of angiotensin or substance P. Inhibition is competitive and fully reversible.     

Effect of oral contraceptives on the rat brain and pituitary opioid peptides

This study was designed to explore the hormonal regulation of CNS opioid peptide levels in female Sprague Dawley rats. Forty-eight animals were divided into 2 equal groups for acute and chronic studies. Each group was further divided into 4 subgroups, each containing 6 animals. Each rat in the control group received an inert pill (in 0.25 ml corn oil daily by gavage); the second group, 15 micrograms norethindrone (NE, a potent progestin present in the oral contraceptive Micronor); the third group, 15 micrograms NE and 1 microgram ethinyl estradiol, EE2 (present in the oral contraceptive Modicon) and the fourth group, 10 times the dose of the third group. Rats were treated either acutely for 5 days or chronically for 7 weeks. Opioid peptides were estimated by radioimmunoassay. Acute administration of 150 micrograms NE + 10 micrograms EE2 decreased the levels of methionine-enkephalin (ME), leucine-enkephalin (LE), dynorphin (DYN) and beta-endorphin like immunoreactivity (beta-EI) by about 50% in the pituitary. The same dose on chronic administration also decreased DYN, but increased the levels of ME and LE in the pituitary by 331 and 69%, respectively. In the hypothalamus, chronic administration of NE + EE2 increased the level of ME (155%) and LE (87%) as well as of DYN (97%). In the striatum, the levels of LE (33%) and DYN (115%) were elevated during chronic administration. It is concluded that the acute administration of NE + EE2, in general, reduces the levels of ME, LE, DYN and beta-EI.(ABSTRACT TRUNCATED AT 250 WORDS)     

Experimental dissociation of food intake and plasma beta-endorphin following 2-deoxy-D-glucose in rats

The present studies were undertaken to further assess the role of plasma beta-endorphin (beta-EP) in the hyperphagia induced by the glucose antimetabolite, 2-deoxy-D-glucose (2-DG). Plasma concentrations of immunoreactive beta-EP (ir-beta-EP) were measured at the end of the first hour of feeding in all animals treated with 400 mg/kg 2-DG. Previous studies had shown a consistent, positive association between 2-DG hyperphagia and plasma ir-beta-EP concentrations, but the present data revealed dissociations between hyperphagia and plasma ir-beta-EP. Dexamethasone administration blocked the 2-DG-induced rise in plasma ir-beta-EP, but had no effect on the 2-DG hyperphagia measured at 1 hour. Forced drinking of a 2% NaCl solution decreased 2-DG hyperphagia, but not the 2-DG induced rise in plasma ir-beta-EP. Thus, elevations in plasma ir-beta-EP are not necessary for the full expression of 2-DG-induced hyperphagia in dexamethasone-treated rats. Furthermore, decreased feeding responses to 2-DG could coexist with increased levels of plasma ir-beta-EP in NaCl-treated normal rats. Elevations in plasma ir-beta-EP do not appear to be the critical opiate link in 2-DG induced hyperphagia.     

In vitro translation of human pheochromocytoma messenger RNAs: characterization of tyrosine-hydroxylase and dopamine-beta-hydroxylase

mRNAs extracted from human pheochromocytoma were translated in vitro in a lysate of a rabbit reticulocytes. Two enzymes of the biosynthetic pathway of the catecholamines, tyrosine-hydroxylase (TH) and dopamine-beta-hydroxylase (DBH), were characterized as translation products after immunoprecipitation by specific antisera and electrophoretic analysis. The precursor of TH is a polypeptide having a molecular mass of 62,000 identical to that found for the mature protein. The molecular mass of the precursor of DBH 73,000 while that of the mature form is 79,000. TH and DBH have been translated from mRNAs having sedimentation coefficients of 22S and 25S, respectively.     

The binding of monosaccharides to wheat germ agglutinin: fluorescence and NMR investigations

The synthesis of N-acetyl- and N-trifluoroacetyl-glucosaminides was reported. The interaction of these compounds with wheat germ agglutinin, a plant lectin specific for N-acetyl-glucosamine and sialic acid, was investigated by two complementary approaches: 1H and 19F NMR, and fluorescence spectroscopy. This last technique relies on the existence of a competitive equilibrium involving the protein, the ligand and O-(methylumbelliferyl)-N-acetyl-glucosaminide, a fluorescent saccharide. The binding constants and the chemical shifts in the complex were determined and were related to the protein structure.     

Studies on the C-24 configurations of Δ7-sterols in theseeds of Cucurbita maxima

Uncertainties surrounding the structures of the Δ⁷-sterols in the seeds of Cucurbita maxima have been resolved. Seven components were found by TLC, GLC, HPLC, mass spectrometry and ¹H NMR. They were 24β-ethyl-5α-cholesta-7,22,25(27)-trien-3β-ol, 24β-ethyl-5α-cholesta-7,25(27)-dien-3gb-ol, avenasterol, spinasterol, 24-dihydrospinasterol, 24ζ-methyllathosterol and 25(27)-dehydrofungisterol. The ¹H NMR spectra indicated that the sterols with an ethyl substituent at C-24 occurred in the absence of their C-24 epimers. This seems to be the first instance of the detection of 25(27)-dehydrofungisterol in a higher plant.     

NADPH-generating system: Influence on microsomal mono-oxygenase stability during incubation for the liver-microsomal assay with rat and mouse S9 fractions

Activity levels of 7-ethoxycoumarin O-deethylase (ED), aminopyrine N-demethylase (APD), p-nitroanisoleO-demethylase (p-NAD) and glucose-6-phosphate dehydrogenase (G-6-PDH) were determined in incubation mixtures for the liver-microsomal assay (LMA) at time 0 and after 1 and 2 h incubation under conditions for mutagenic assay. The experiments were performed with S9 liver fractions from mice (induced with Na-phenobarbital and β-naphthoflavone) and rats (induced with Aroclor 1254) with and without G-6-PDH in the incubation mixtures.

In the absence of G-6-PDH the activities were significantly lower at time 0 in the mouse. The pattern of stability, however, was similar for the activities, with an increase of stability after 1 and 2 h of pre-incubation (an exception for p-NAD).

Only ED activity showed a similar behaviour in the rat. No differences were present for APD and p-NAD activities at time 0 in the rat, but the enzyme stabilities were significantly decreased after 2 h of incubation (about 15% and 10% for APD and p-NAD respectively) in the absence of G-6-PDH.

At time 0, the amounts of G-6-PDH differed between mouse and rat fractions; however, during the incubations for LMA they decreased by about 57% and 53% for the two species, respectively. In addition to the above biochemical results, the presence of exogenous G-6-PDH in the incubations for the mutagenic assay, significantly increased the mitotic gene conversion and mitotic crossing-over of dimethylnitrosamine (DMN) and AR2MNFN (a nitroimidazo[2,1-b]thiazole) in the D₇ strain of Saccharomyces cerevisiae.