Effects of the New Arginase Inhibitor N-Hydroxy-nor- -Arginine on NO Synthase Activity in Murine Macrophages

In stimulated murine macrophage, arginase and nitric oxide synthase (NOS) compete for their common substrate, -arginine. The objectives of this study were (i) to test the new α-amino acid N^ω-hydroxy-nor- -arginine (nor-NOHA) as a new selective arginase inhibitor and (ii) to elucidate the effects of arginase inhibition on -arginine utilization by an inducible NOS. Nor-NOHA is about 40-fold more potent than N^ω-hydroxy- -arginine (NOHA), an intermediate in the -arginine/NO pathway, to inhibit the hydrolysis of -arginine to -ornithine catalyzed by unstimulated murine macrophages (IC₅₀ values 12 ± 5 and 400 ± 50 μM, respectively). Stimulation of murine macrophages with interferon-γ and lipopolysaccharide (IFN-γ + LPS) results in clear expression of an inducible NOS (iNOS) and to an increase in arginase activity. Nor-NOHA is also a potent inhibitor of arginase in IFN-γ + LPS-stimulated macrophage (IC₅₀ value 10 ± 3 μM). In contrast to NOHA, nor-NOHA is neither a substrate nor an inhibitor for iNOS and it appears as a useful tool to study the interplays between arginase and NOS. Inhibition of arginase by nor-NOHA increases nitrite and -citrulline accumulation for incubation times higher than 12 h, under our conditions. Our results allow the determination of the kinetic parameters of the two competitive pathways and the proposal of a simple model which readily explains the differences observed between experiments. This model readily accounts for the observed effects and should be useful to predict the consequences of arginase inhibition in the presence of an active NOS on -arginine availability.     

Building Fluorescent Sensors by Template Polymerization: The Preparation of a Fluorescent Sensor for -Tryptophan

The development of fluorescent sensors for organic molecules is of great practical importance in chemical, biological, and pharmaceutical sciences. Using -tryptophan as an example, we have studied a new way of making polymeric fluorescent sensors using template polymerization or molecular imprinting techniques. The fluorescent polymers were prepared using functional monomers with a fluorescent probe attached to it. The fluorescence of this polymer could be quenched by 4-nitrobenzaldehyde. Addition of the template molecules, -tryptophan, increased the fluorescence intensity of the imprinted polymer/quencher mixture in a concentration-dependent fashion, presumably through the displacement of the quencher from the binding sites by -tryptophan. This fluorescence intensity change upon mixing with -tryptophan allows the binding event to be detected easily. The sensor also exhibited enantioselectivity for the template molecules. For example, the effect of -tryptophan on the fluorescence intensity of the polymer is about 70% that of its -enantiomer. Furthermore, the effect of -phenylalanine and -alanine on the fluorescence intensity change is much smaller than that of -tryptophan. Because the approach used does not require the de novo design of the complementary binding site and does not rely on any specific structural features of the template molecule or prior knowledge of its three-dimensional structure, the same principle could potentially be useful for the future construction of practical fluorescent sensors for many other compounds.     

Chaperone-Assisted Overexpression of an Active -Carbamoylase from Agrobacterium tumefaciens AM 10

The N-carbamoyl- -amino acid amidohydrolase ( -carbamoylase) gene (dcb) from Agrobacterium tumefaciens AM 10 was cloned by polymerase chain reaction in plasmid pET28a and was overexpressed in Escherichia coli JM109 (DE3). However, almost 80% of the enzyme remained trapped in inclusion bodies. To facilitate the expression of the properly folded active enzyme, the chaperones GroEL/ES were coexpressed in plasmid pKY206. This resulted in a 43-fold increase in active enzyme production compared to the wild-type strain. The histidyl-tagged -carbamoylase was purified by a single step nickel-affinity chromatography to a specific activity of 9.5 U/mg protein.     

Effect of N-acetyl- -cysteine on peroxynitrite and superoxide anion production of lung alveolar macrophages in systemic sclerosis

Lung macrophages may play a relevant role in oxidative processes producing both superoxide anion (O(2)(-)) and NO. In this view, an antioxidant therapy can be useful in the treatment of systemic sclerosis (SSc) patients. N-Acetylcysteine (NAC) is able to expand natural antioxidant defenses by increasing intracellular gluthatione concentration and it has been proposed as an antioxidant therapy in respiratory distress syndromes. The aim of our study was to determine whether lung macrophages obtained from SSc patient bronchoalveolar lavage (BAL) express the inducible form of nitric oxide synthase (iNOS) and whether NAC can reduce the peroxynitrite (ONOO(-)) and O(2)(-) production of these cells. Alveolar macrophages were isolated from BAL of 32 patients and used for the immunocytochemical determination of iNOS, and the production of ONOO(-) and O(2)(-) was measured by fluorimetric or spectrophotometric methods, respectively. Lung macrophages obtained from SSc patients expressed a higher level of iNOS compared to healthy subject cells. NAC preincubation (5 x 10(-5)M, 24h) significantly reduced (-21%) the ONOO(-) production in formyl Met-Leu-Phe (fMLP)-activated cells and slightly reduced it under resting conditions, whereas NAC preincubation was unable to modify the release of O(2)(-) both in basal condition and in fMLP-stimulated cells. We conclude that since SSc lung macrophages express high levels of iNOS and produce a significant quantity of ONOO(-), NAC administration reduces ONOO(-) production and can be an useful treatment to alleviate SSc symptoms.     

Structural and mutational characterization of -carnitine binding to human carnitine acetyltransferase

We report the crystal structure of a binary complex of human peroxisomal carnitine acetyltransferase and the substrate l-carnitine, refined to a resolution of 1.8 Angstrom with an R(factor) value of 18.9% (R(free)=22.3%). L-carnitine binds to a preformed pocket in the active site tunnel of carnitine acetyltransferase aligned with His(322). The quaternary nitrogen of carnitine forms a pi-cation interaction with Phe(545), while Arg(497) forms an electrostatic interaction with the negatively charged carboxylate group. An extensive hydrogen bond network also occurs between the carboxylate group and Tyr(431), Thr(444), and a bound water molecule. Site-directed mutagenesis and kinetic characterization reveals that Tyr(431), Thr(444), Arg(497), and Phe(545) are essential for high affinity binding of L-carnitine.     

Stability in -species coevolutionary systems

Stability criteria have recently been developed for coevolutionary Lotka–Volterra systems where individual fitness functions are assumed to be linear in the population state. We extend these criteria as part of a general theory of coevolution (that combines effects of ecology and evolution) based on arbitrary (i.e. nonlinear) fitness functions and a finite number of individual phenotypes. The central role of the stationary density surface where species’ densities are at equilibrium is emphasized. In particular, for monomorphic resident systems, it is shown coevolutionary stability is equivalent to ecological stability combined with evolutionary stability on the stationary density surface. Also discussed is how our theory relates to recent treatments of phenotypic coevolution via adaptive dynamics when there is a continuum of individual phenotypes.     

Use of Galactosyltransferase to Assess the Biological Function of O-linked N-Acetyl- -Glucosamine: A Potential Role for O-GlcNAc during Cell Division

Many cytosolic and nuclear proteins are modified by monomeric O-linked N-acetyl- -glucosamine (O-GlcNAc). The biological functions of this form of glycosylation are unclear but evidence suggests that it heightens regulation of protein function. To assess the biological function of O-GlcNAc addition, we examined the biological effects of galactosyltransferase (GalT) microinjected into the cytoplasm of Xenopus ovarian oocytes. GalT, which catalyzes β1-4-galactose addition to O-GlcNAc, should inhibit deglycosylation and lectin-like interactions requiring unmodified O-GlcNAc residues. Although GalT injection into diplotene-arrested oocytes has no detectable effects on cell viability, it is toxic to oocytes entering meiosis. Cell-cycle-specific toxicity is recapitulated in vitro as GalT inhibits formation of nuclei and microtubule asters from cell-free extracts of ovulated frog eggs. These observations suggest that regulation of O-GlcNAc is important for cell cycle progression and may be important in diseases in which O-GlcNAc metabolism is abnormal. The methods described here outline a viable experimental scheme for ascribing a biological function to this form of glycosylation.     

A spectrophotometric assay of -glucuronate based on Escherichia coli uronate isomerase and mannonate dehydrogenase

Escherichia coli uronate isomerase and mannonate dehydrogenase were overexpressed in E. coli BL21(DE3)pLysS cells and purified to near-homogeneity. The kinetic properties of the two enzymes were investigated. The isomerase was found to be inhibited by EDTA and to be stimulated by Zn(2+), Co(2+), and Mn(2+), but not by Mg(2+) or Ca(2+). Both enzymes were used to develop a sensitive spectrophotometric assay, in which D-glucuronate is converted to D-mannonate with concomitant oxidation of NADH to NAD(+). The sensitivity of this assay permits the detection of less than 1 nmol D-glucuronate. This assay can also be used to determine the concentration of beta-glucuronides and glucuronate 1-phosphate after enzymatic hydrolysis of these compounds with beta-glucuronidase or alkaline phosphatase.     

N-acetyl- -arginine ethyl ester synthesis catalysed by bovine trypsin in organic media

The bovine trypsin-catalysed synthesis of N-acetyl- -arginine ethyl ester from N-acetyl- -arginine and ethanol was studied in various organic solvents (dimethyl sulfoxide, dioxane, dimethylformamide, acetonitrile, acetone, tetrahydrofuran, chloroform, toluene, carbon tetrachloride, cyclohexane and n-hexane). The highest yield was achieved in acetonitrile after incubation for 6 or 24 h. The optimal conditions for ester synthesis in acetonitrile for 6 h were as follows: 5.0 mM N-acetyl- -arginine, 10.0 M ethanol, 7.2 mg trypsin, 2.87% water, total volume 10.3 ml, pH 7.0 and 30°C. The hydrolytic activity of trypsin was determined after incubation for 6 days, when 87.7% of the original activity remained, suggesting that acetonitrile caused little inactivation of the enzyme. The synthetic reaction resulted in a maximal 79.3% conversion under optimized conditions after incubation for 48 h.     

-alkoxyphenol leukotriene B4 receptor antagonists: effect of a chroman carboxylic acid.

Several -alkoxyphenols containing a chroman carboxylic acid sidechain have been prepared as antagonists of leukotriene B₄ receptors. These antagonists were compared to their parent alkoxyphenols containing the tetrazole acid sidechain. These chroman containing antagonists retained their binding potency for human neutrophil receptors; however, showed enhanced potency against guinea pig receptors in both in vitro and in vivo systems.     

Morphological affinities of the al’a 1 frontal bone

The human frontal bone from al’a, Slovak Republic, has previously entered into discussions of the morphological patterns of Central European Neandertals and the origins of early modern humans in that region. A morphological reassessment of its supraorbital region and a morphometric analysis of its overall proportions indicate that it falls well within expected ranges of variation of Late Pleistocene Neandertals and is separate from European earlier Upper Paleolithic early modern human crania. It is similar to the Qafzeh-Skhul sample in some metrical and supraorbital robusticity measures, but it contrasts with them in mid-sagittal curvature and supraorbital torus morphology. In the context of its probable oxygen isotope stage 5 age based on inferred biostratigraphic associations, it should not be employed directly for arguments relating to the emergence of modern humans in Central Europe.     

Inhibition of -tyrosine-induced micronuclei production by phenylthiourea in human melanoma cells

It was previously found that -tyrosine oxidation product(s) are cytotoxic, genotoxic and increase the sister chromatid exchange (SCE) levels in human melanoma cells. In this work, the micronucleus assay has been performed on human melanotic and amelanotic melanoma cell lines (Carl-1 MEL and AMEL) in the presence of 1.0, 0.5 and 0.1 mM -tyrosine concentrations to investigate if melanin synthesis intermediate(s) increase micronuclei production. -Tyrosine oxidation product(s) increased the frequency of micronuclei in melanoma cells; 0.1 mM phenylthiourea (PTU), an inhibitor of -tyrosine oxidation by tyrosinase, lowered the micronucleus production to the control levels. The culture of melanoma cells with high -tyrosine in the culture medium resulted in a positive response to an ELISA-based apoptotic test. For comparison the effect of -tyrosine on micronuclei production in human amelanotic melanoma cells was also investigated; the micronucleus production in the presence of 1 mM -tyrosine in the culture medium was lower than that found with melanotic melanoma cells of the same cell line. The data suggest that melanin synthesis intermediates arising from -tyrosine oxidation may cause micronuclei production in Carl-1 human melanoma cells; the addition of PTU in the presence of -tyrosine decreased the frequency of micronuclei to about the control values thus the inhibition of melanogenesis may have some clinical implication in melanotic melanoma.     

An expedient synthesis of N-protected- -tetrahydrofuranylglycine and its application in the synthesis of novel substrate based inhibitors of HIV-1 protease

Z- and Fmoc-L-tetrahydrofuranylglycines have been obtained from L-vinylglycine through dipolar cycloaddition reaction, and its Fmoc derivative has been applied in the synthesis of modified S9 and S10 substrates of HIV-1 protease. These compounds mostly acted as strong inhibitors, rather than substrates, of the protease, probably due to the favourable interactions of the tetrahydrofuranylglycine moiety at the S(2) site.     

Effect of 2-deoxy- -glucose on acetylcholine and histamine levels in gastric juice of pylorus-ligated rats anesthetized with urethane

Acetylcholine (ACh) in gastric juice was detected and measured by pretreatment of acetylcholinesterase inhibitor, 1 mM eserine (1 ml/rat, p.o.), in pylorus-ligated rats, by liquid chromatography with electrochemical detection. In order to elucidate whether or not the ACh level in gastric juice reflects the activity of cholinergic neurons, the effect of 2-deoxy- -glucose (2-DG), a vagus stimulant, on the levels of ACh, histamine and gastric acid in gastric juice was investigated in pylorus-ligated rats anesthetized with urethane (1.25 g/kg, i.p.). Under the non-anesthetic condition, ACh, histamine and gastric acid levels were 100±25 pmol/h, 120±10 ng/h, and 240±32 μequiv./h, respectively. These levels were completely inhibited by urethane anesthesia. Under the anesthetized condition, 2-DG (50–200 mg/kg, i.v.) significantly increased ACh and histamine levels in gastric juice, as well as acid secretion. The 2-DG (200 mg/kg, i.v.)-induced increases in these levels were completely inhibited by vagotomy. These results suggest that ACh level measured in gastric juice reflects the activity of cholinergic transmission. Furthermore, these results also support the conclusion that vagus stimulation facilitates not only cholinergic transmission but also histaminergic transmission related to gastric acid secretion.     

Assay for 5-hydroxylysine and -lysine in human and rat urine and in bone by gas chromatography

An accurate method for the determination of collagen to study its distribution and turn-over in different tissues is described. 5-Hydroxylysine (5Hylys) is an amino acid that is apparently present in no other protein except collagen and, as it is metabolised only to a minor degree compared with 4-hydroxyproline (4Hypro), it has been suggested as a better marker of the collagen metabolism. Interest in this amino acid has increased recently because the levels of 5Hylys in urine and in different tissues may offer a new basis for detecting pathologies of the collagen molecule. This paper describes a method for the quantitative determination of 5Hylys and lysine (Lys) by gas chromatography (GC) in human and rat urine and in rat bone. The limit of detection was 350 pmol ml⁻¹ for 5Hylys and 200 pmol ml⁻¹ for Lys for all the biological samples. This method therefore provides a complete view of the metabolism of this amino acid and of the tissue it comes from.     

-Amino acid aminotransferase: fragmentation at a flexible loop is an efficient method to generate mutant enzymes with new substrate specificities and elevated activities

-Amino acid aminotransferase ( -AAT) (EC 2.6.1.21) catalyzes the interconversion between various -amino acids and -keto acids. A subunit of the homodimeric enzyme from a thermophile, Bacillus sp. YM-1, consists of two distinct structural domains connected by one loop. We previously constructed an active fragmentary enzyme whose backbone was cut at the interdomain loop [J. Biochem. 124 (1998) 905]. In this work, we constructed 13 fragmentary -amino acid aminotransferase genes by inserting a termination codon, an SD sequence, and an initiation codon into the specific positions of the gene corresponding to various loop regions and expressed in Escherichia coli cells. We have obtained six genes producing active fragmentary enzymes, one producing an inactive fragmentary enzyme, four producing only large peptide fragment, and another two that gave no products. The six active fragmentary enzymes purified to near-homogeneity showed various substrate specificities and thermostabilities distinct from each other and also from the wild-type enzyme: two exhibited higher catalytic activity towards -alanine, the most efficient substrate, than the wild-type enzyme. These results suggest that cleavage at a loop region is an efficient method for the alteration of enzyme properties.     

Are prions a relic of an early stage of peptide evolution?

The rather unique properties of prions and their presence in very different kinds of living species suggest that this type of molecule was created at a very early stage of evolution and may even represent a relic from a time where peptide evolution was ongoing and RNA/DNA did not yet exist. A comparison of the most frequently occurring amino acid sequences in known prions with the sequences preferentially formed in the salt-induced peptide formation reaction, the most simple mechanism enabling the formation of peptides under primitive earth conditions, shows a remarkable coincidence that strongly supports this hypothesis.     

Govindjee, Fork, D.C.: Charles Stacy French 1907–1985 National Academy of Sciences, Washington 2006. 28 pp.

Book Reviews

Govindjee, Fork, D.C.: Charles Stacy French 1907–1985 National Academy of Sciences, Washington 2006. 28 pp.