Free intracellular and protein bound amino acids in tissues as affected by a mixedβ-adrenergic agonist

The administration of metaproterenol induced an increase in gastrocnemius muscle weight without change in body growth rate or tissue protein concentrations, while epididymal fat was reduced. This effect was accompanied by an enhancement in the levels of intracellular amino acids in muscle. By contrast, liver amino acids were unaffected by treatment with the mixed -adrenergic agonist.     

Variability of β-amylase isoenzymes within a collection of inbred lines of rye (Secale cereale L.)

Summary Within one collection of 18 inbred rye lines, seven distinct isoenzymatic patterns of -amylase were found, and five of them formed a group of similar patterns shifting to distinct positions in the gel. It was proved that the shift of the pattern was due to genetic factors. In crossed immunoelectrophoresis, drastic differences were shown in the quantity of the different -amylase constituents. No antigenic differences could be demonstrated between the analyzed isoenzymes.     

A new semisynthetic ergot peptide alkaloid: DCN 203-922

Summary We report the synthesis, stereochemistry and preliminary pharmacological evaluation of DCN 203-922, a novel ergot alkaloid of the cyclol type, which contains in its peptide moiety the uncommon amino acid L-allo-isoleucine.Part of this paper was reported by this author at the Herbstversammlung der Schweizerischen Chemischen Gesellschaft, Bern, in October 1986.     

17β-Estradiol-sensitivity of cultured myometrial cells

Summary The estrogen sensitivity of cells cultured from the rat myometrium was studied by growing the cells in the absence or presence of 1 nM 17-estradiol. Following a time lag of 10 days, exposure to estrogen resulted in increased incorporation of radiothymidine by the cells. Estrogen treatment also decreased isoproterenol-dependent and GTP-dependent adenylate cyclase activity, but had no effect on basal activity. These cultured cells have been shown previously to have some properties of uterine smooth muscle. The effects estrogen has in viyro, therefore, may reflect important properties in vivo that account for the mechanism by which the sex steroid decreases the sensitivity of the myometrium to isoproterenol.     

Human and rodent type 1 11β-hydroxysteroid dehydrogenases are 7β-hydroxycholesterol dehydrogenases involved in oxysterol metabolism

Interconversion between cortisone and the glucocorticoid receptor ligand cortisol is carried out by 11-hydroxysteroid dehydrogenase (11-HSD)isozymes and constitutes a medically important example of pre-receptor control of steroid hormones. The enzyme 11-HSD type 1 (11-HSD1) catalyzes the conversion of cortisone to its active receptor-binding derivative cortisol, whereas 11-HSD type 2 performs the reverse reaction. Specific inhibitors against the type 1 enzyme lower intracellular levels of glucocorticoid hormone, with an important clinical application in insulin resistance and other metabolic disorders. We report here on the in vitro oxysterol-metabolizing properties of human and rodent 11-HSD1. The enzyme, either as full-length, membrane-attached, or as a transmembrane domain-deleted, soluble form, mediates exclusively conversion between 7-ketocholesterol and 7-hydroxycholesterol with similar k_cat values as observed with glucocorticoid hormones. Thus, human, rat, and mouse 11-HSD1 have dual enzyme activities like the recently described 7-hydroxysteroid dehydrogenase/11-hydroxysteroid dehydrogenase from hamster liver, but differ fundamentally from the latter in that 7-OH rather than 7-OH dehydrogenase constitutes the second activity. These results demonstrate an enzymatic origin of species differences in 7-oxysterol metabolism, establish the origin of endogenous 7-OH cholesterol in humans, and point to a possible involvement of 11-HSD1 in atherosclerosis.Received 30 December 2003; received after revision 16 February 2004; accepted 16 February 2004     

Role of Cys221 and Asn116 in the zinc-binding sites of the Aeromonas hydrophila metallo-β-lactamase

The CphA metallo--lactamase produced by Aeromonas hydrophila exhibits two zinc-binding sites. Maximum activity is obtained upon binding of one zinc ion, whereas binding of the second zinc ion results in a drastic decrease in the hydrolytic activity. In this study, we analyzed the role of Asn116 and Cys221, two residues of the active site. These residues were replaced by site-directed mutagenesis and the different mutants were characterized. The C221S and C221A mutants were seriously impaired in their ability to bind the first, catalytic zinc ion and were nearly completely inactive, indicating a major role for Cys221 in the binding of the catalytic metal ion. By contrast, the binding of the second zinc ion was only slightly affected, at least for the C221S mutant. Mutation of Asn116 did not lead to a drastic decrease in the hydrolytic activity, indicating that this residue does not play a key role in the catalytic mechanism. However, the substitution of Asn116 by a Cys or His residue resulted in an approximately fivefold increase in the affinity for the second, inhibitory zinc ion. Together, these data suggested that the first zinc ion is located in the binding site involving the Cys221 and that the second zinc ion binds in the binding site involving Asn116 and, presumably, His118 and His196.Received 3 March 2003; received after revision 4 August 2003; accepted 25 August 2003     

Inhibition of cadmium teratogenesis by a mercaptoacrylic acid (MFA)

Summary The teratogenic effect of cadmium can be diminished by a number of mechanisms including zinc and pretreatment with cadmium and mercury. In this study, the oral administration of -mercapto--(2-furyl)-acrylic acid (MFA) protects against cadmium-induced malformations and embryonic death. This protection is probably mediated by the chelation of the cadmium ion rather than metallothioneim (MT) synthesis.This project has been financed in part with Federal funds from the U.S. Environmental Protection Agency under grant number R811078. The contents do not necessarily reflect the views and policies of the Environmental Protection Agency nor does mention of trade names or commercial products constitute endorsement of recommendation for use.We thank Dr W. Layton for the statistical analyses of the data and G. McClain for the synthesis of MFA.     

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.     

Enhancement of fibronectin expression by herbimycin A

Summary Herbimycin A specifically inrreased the level of fibronectin mRNA in Rous sarcoma virus-infected rat kidney cells, and the time course of fibronectin expression was found to be closely related to that of morphological change induced by herbimycin A.Acknowledgments. The authors wish to thank Dr Y. Uehara, National Institute of Health, Tokyo, and Dr K. Hayashi, National Cancer Center Research Institute, Tokyo, for valuable suggestions. This work was supported in part by grants from the Ministry of Education, Science and Culture and the Ministry of Health and Welfare of Japan.     

Reduction of high affinity glutamate uptake in rat hippocampus by two polyamine-like toxins isolated from the venom of the predatory waspphilanthus triangulum F

Summary Two components of the venom of the predatory waspPhilanthus triangulum F. significantly reduce — to a greater or less extent — the high affinity uptake of glutamate in rat hippocampus. A concentration of 10 M -PTX caused a reduction of 74%, while the other component, -PTX, at the same concentration, caused a reduction of 18%. Hence the effect of -PTX on high affinity glutamate uptake in the hippocampus is comparable with its effect on high affinity glutamate uptake in insect neuromuscular junctions. Contrary to our previous findings that -PTX has no effect on high affinity glutamate uptake in insect glutamatergic terminal axons, however, -PTX significantly reduces high affinity glutamate uptake in the hippocampus, albeit less effectively than -PTX.     

Characterization of the third component of pig complement and its utilization in a C3b receptor study

Summary The third component of the pig complement system (C3) was isolated in hemolytically active form and characterized. The C3 component is a -globulin with the molecular weight of 191,000 and is composed of 2 non-identical polypeptide chains of M_t 112,000 and 74,000. The isolated C3 can be used for the detection of the C3b receptor on the membranes of heterologous peritoneal macrophages.     

Butyrate-induced reactivation of the fetal globin genes: A molecular treatment for theβ-hemoglobinophaties

The inherited -hemoglobinopathies (sickle cell disease and thalassemia) are the result of a mutation in the adult () globin gene. The fetal globin chain, encoded by the globin genes, can substitute for the mutated or defective globin chain, but expression of the globin gene is developmentally inactivated prior to birth. Reinducing expression of the normal fetal globin genes is a preferred method of ameliorating sickle cell disease and the thalassemias. Stimulation of as little as 4–8% fetal globin synthesis in the bone marrow can produce >20% fetal hemoglobin in the peripheral circulation, due to enhanced survival of red blood cells containing both sickle and fetal hemoglobin, compared to those containing sickle hemoglobin alone. Butyric acid and butyrate derivatives are generally safe compounds which induce fetal hemoglobin production by stimulating the promoter of the fetal globin genes. An initial trial with the parent compound, delivered as Arginine Butyrate, has demonstrated rapid stimulation of fetal globin expression to levels that have been shown to ameliorate these conditions. Phase 1 trials of an oral butyrate derivative with a long plasma half-life have just begun. These agents now provide a specific new apporach for ameliorating these classic molecular disorders and merit further investigation in larger patient populations.     

Hb F in sickle cell anemia

We have reviewed the methodology for an accurate quantitation of Hb F in the blood of patients with sickle cell anemia, values observed in hundreds of patients of different (racial or ethnic) backgrounds and with differences in severity of the disease, and the various factors that affect the level of Hb F. The latter include sex, age, genetic background or chromosomal haplotypes, variations in the sequences of the locus control region(s) 5 to the -globin gene, and the presence of an chain deficiency or -thalassemia. Finally, a few remarks about agents effective in increasing the in vivo Hb F synthesis are also included.     

Localisation of acetylcholinesterase in rat myotubes in the presence of β-endorphin and β-endorphin-(1-27)

Summary In rat embryo skeletal myotubes, acetylcholinesterase is present, as multiple forms, and can be detected in deposits at the cell surface. Myotubes cultured in the presence of -endorphin, exhibit an increased predominance of the globular (precursor) forms of the enzyme, which are largely restricted to intracellular sites associated with nuclei. In the presence of -endorphin-(1-27), the relative proportions of the different forms of acetylcholinesterase is similar to that seen in the controls, but the enzyme is intracellular and has a characteristic focal localisation in the myotube.     

Prothoracic gland synthesis of 3-dehydroecdysone and its hemolymph 3β-reductase mediated conversion to ecdysone in representative insects

Summary The prothoracic glands of a variety of insects were tested for their ability to synthesize ecdysteroids in vitro. More specifically, they were evaluated for their ability to produce 3-dehydroecdysone and ecdysone using both radioimmunoassay and reverse phase high performance liquid chromatography. Three categories of insect prothoracic glands were noted: a) those producing much more 3-dehydroecdysone than ecdysone; b) glands synthesizing almost equivalent amounts of each of these two ecdysteroids; c) prothoracic glands that yielded more ecdysone than 3-dehydroecdysone. In addition, the 3-oxoecdysteroid 3-reductase activity of the hemolymph of these insects was evaluated for its ability to convert 3-dehydroecdysone to ecdysone. The lepidopteran species tested yielded the most potent enzyme activity, although activity was demonstrated in members of other orders. These data indicate that the dehydroecdysone-ecdysone axis is not restricted to moths and butterflies.     

A new sterol from the spongeHaliclona chilensis (Thiele)

Summary From the spongeHaliclona chilensis (Thiele) a minor keto-sterol was isolated and characterized as 24-keto-cholesta-5,25-dien-3-ol by means of spectroscopic (¹H and¹³C-FT NMR, MS) methods.     

Developmental and ageing changes in aminopeptidase activities in selected tissues of the rat

Aminopeptidase activities, assayed as arylamidase activities, were investigated in selected tissues of 1, 6, 12 and 24-month-old rats. The enzyme activities were found to have a heterogeneous distribution and age-related changes were observed. The highest levels of soluble arginyl-aminopeptidase activity were detected in brain homogenate at all the studied ages, whereas membrane-bound activity presented the highest levels in brain and kidney in the four ages tested. Aspartyl-aminopeptidase activity was detected mainly in the particulate fraction of kidney at all four ages. In 1, 6 and 12-month-old animals, soluble aspartyl-aminopeptidase activity was also higher in the kidney than in the rest of the tissues, whereas in the group of 2-year-old rats, the highest levels were found in both kidney and liver. Age-related changes were observed in all the studied tissues and for all the assayed enzymatic activities. In general, the maximal levels were detected in both the youngest and the oldest animals, and the minimal ones in 6 and 12-month-old rats. However, in the adrenals, the soluble and membrane-bound arginyl-aminopeptidase activity was higher in 6-month and 2-year-old rats than in 1-month and 12-month-old rats. These changes may reflect the functional status of the susceptible endogenous substrates of aminopeptidases.     

Hydrolysis of N-phenylacetyl-α-methyl-α-amino acids by benzylpenicillinacylase

Summary Enzymatic hydrolysis of several racemic N-phenylacetyl--methyl--amino acids containing an additinal aliphatic, aromatic or polar substitutent on the chiral carbon atom, has been studied by using benzylpenicillincylase fromEscherichia coli A.T.C.C. 9637. Both the rate of hydrolysis and the stereoselectivity were found to be considerably lower than in the case of natural -amino acids. Steric and electronic factors in the side chains influencing the stereoselectivity are discussed.