Synthesis, characterization and hybridization studies of an alternate nucleo-ε/γ-peptide: complexes formation with natural nucleic acids

In order to develop new oligodeoxyribonucleotide (ODN) analogs to be used in biotechnological applications, we report here the synthesis, characterization and nucleic acid binding studies of novel nucleopeptides, that we called ε-lys/γ-dabPNAs, containing a backbone of alternated l-diaminobutyric acid and l-lysine moieties. Exploring the hybridization properties of the new ODN analog, we found, by circular dichroism and UV spectroscopies, that a homothymine ε-lys/γ-dabPNA hexamer binds both DNA and RNA of complementary sequence. Furthermore, human serum stability assays on the alternate nucleopeptide evidenced a noteworthy degradation resistance. These results encourage us to deepen the knowledge of this analog, in order to evaluate its possible use in antigene/antisense or diagnostic applications.     

Arginine in the salt-induced peptide formation reaction: enantioselectivity facilitated by glycine, l- and d-histidine

The salt-induced peptide formation reaction has been proposed as a conceivable preliminary to the prebiotic evolution of peptides. In the present paper, the behaviour of arginine is reported for this reaction together with a discussion of the catalytic effects of glycine, and l- and d-histidine. Importantly, the behaviour of the two histidine enantiomers is different. Both histidine enantiomers perform better than glycine in enhancing the yields of arginine dipeptide with l-histidine being more effective than d-histidine. Yields in the presence of histidine are up to 70 times greater than for arginine solutions alone. This compares with 4.2 times higher in the presence of glycine. This difference is most pronounced in the most concentrated (containing 80 mM arginine) reaction solution where arginine has the lowest reactivity. A distinct preference for dimerisation of l-arginine also appears in the 80 mM cases for catalyses of other amino acids. This phenomenon is different from the behaviour of aliphatic amino acids, which display obvious inherent enantioselectivity for the l-stereomers in the SIPF reaction on their own rather than when catalysed by glycine or histidine.     

Characterization of cationic amino acid transporters (hCATs) 1 and 2 in human skin

In the present study, we characterized the distribution of human cationic amino acid transporters 1 (hCAT1) and 2 (hCAT2) in healthy skin and compared it to psoriatic skin lesions by means of immunohistochemistry. Moreover, we tested the hypothesis that l-arginine and l-ornithine influence the expression and synthesis of hCAT1 and hCAT2 in cell culture experiments by means of real-time-PCR and Western blot. Immunohistochemical comparison between healthy and psoriatic skin revealed a decreased amount of hCAT1, especially in the stratum granulosum of psoriatic skin; the distribution pattern of hCAT2 was not significantly affected in psoriatic skin. Cell culture experiments showed that supraphysiological concentrations of 15 mM l-arginine (72 h) lead to a significant increase of the hCAT1-mRNA and protein expression, whereas other concentrations had no significant influence. In contrast, l-arginine concentrations of 2 mM led to a significant increase of the hCAT2B mRNA-expression after 24 h. However, 48 and 72 h revealed no significant changes and high concentrations (15 mM l-arginine) led to a significant downregulation of the hCAT2B transporter over all time points analyzed. l-ornithine had no effect on the hCAT1 expression of mRNA and protein level. On the other hand the expression of hCAT2B was significantly up regulated at a 5-mM concentration of l-ornithine at all analyzed time points. Other concentrations had no effect. For the first time, the findings yield data about hCAT1 and hCAT2 on protein-level and suggest that l-arginine is a worthwhile object of studies, which investigated l-arginine as a possible therapeutic agent to reduce psoriatic symptoms.     

Indicators of l-arginine metabolism and cardiovascular risk factors A cross-sectional study in healthy middle-aged men

Summary. This study examines the relationship between traditional risk factors of coronary artery disease and indicators involved in the metabolism of l-arginine (plasma and urine l-arginine, plasma l-citrulline, serum creatinine and urine orotic acid). Our study population consisted of 40 healthy male volunteers aged between 35 and 55 years. We found an inverse association between serum creatinine and blood pressure, between plasma l-citrulline and blood pressure, as well as between urine l-arginine and blood pressure. We also found a positive association between plasma LDL-cholesterol and urine l-arginine and a negative correlation between plasma l-arginine and LDL-cholesterol. Orotic acid measured from urine was not associated with any of the indicators of l-arginine metabolism. Our results indicate that l-arginine metabolism is of profound significance for cardiovascular health. However, our study does not answer questions relating to causality. Further studies are needed to clarify the causal relationship between cardiovascular risk factors, especially elevated blood pressure and high LDL-cholesterol, and indicators of l-arginine metabolism. Received January 18, 1999     

Threonine aldolases—screening, properties and applications in the synthesis of non-proteinogenic β-hydroxy-α-amino acids

Threonine aldolases (TAs) constitute a powerful tool for catalyzing carbon–carbon bond formations in synthetic organic chemistry, thus enabling an enantio- and diastereoselective synthesis of β-hydroxy-α-amino acids. Starting from the achiral precursors glycine and an aldehyde, two new stereogenic centres are formed in this catalytic step. The resulting chiral β-hydroxy-α-amino acid products are important precursors for pharmaceuticals such as thiamphenicol, a l-threo-phenylserine derivative or l-threo-3,4-dihydroxyphenylserine. TAs are pyridoxal-5-phosphate-dependent enzymes, which, in nature, catalyze the cleavage of l-threonine or l-allo-threonine to glycine and acetaldehyde in a glycine biosynthetic pathway. TAs from a broad number of species of bacteria and fungi have been isolated and characterised as biocatalysts for the synthesis of β-hydroxy-α-amino acids. In this review, screening methods to obtain novel TAs, their biological function, biochemical characterisation and preparative biotransformations with TAs are described.     

Probing of <Emphasis Type="SmallCaps">l</Emphasis>-Arginine as an Additive for the Temperature-Induced Aggregation of Veterinary Growth Hormones: Fluorescence Study

The influence of l-arginine on the temperature-induced aggregation of porcine and mink growth hormones was studied by fluorescence spectroscopy. It was found that l-arginine suppresses the heat-induced aggregation. Moreover, the analysis of l-arginine interaction with the native proteins by fluorescence spectroscopy and circular dichroism spectroscopy revealed no significant changes in their native structure. On the basis of the results, l-arginine could be considered as a potential additive for the prevention of storage and temperature-related denaturation and aggregation of veterinary growth hormones.     

The influence of short-term <Emphasis Type="SmallCaps">l</Emphasis>-arginine supplementation on rats’ muscular and hepatic cells in ischemia–reperfusion syndrome

Due to the complex mechanisms of l-arginine activity, it is difficult to determine the clinical significance of supplementation with this amino acid. The objective of this study was to determine the influence of short-term supplementation with l-arginine in stress conditions, induced by ischemia–reperfusion syndrome, by assessing the damage to muscular and hepatic cells on the basis of creatine kinase (CK), alanine aminotransferase (ALAT) and aspartic aminotransferase (AspAT) activity in blood and the level of oxygen free radicals in analyzed tissues of rats. We observed that induced ischemia of hind limb caused an increase in CK, ALAT and AspAT activity and an increase in the level of free radicals in liver, but not in skeletal muscle. Supplementation with l-arginine led to a reduction in serum activity of CK and AspAT and reduction of the level of free radicals in analysed tissues. Simultaneous supplementation with l-arginine AND l-NAME resulted in a reversal of changes induced by l-arginine supplementation in the case of AspAT and free radicals in skeletal muscle. The results indicate that under conditions of ischemia–reperfusion, short-term administration of l-arginine has a protective effect on skeletal muscle manifesting itself by reduction of CK in the serum and reduction of free radicals level in THIS tissue.     

Effector molecules from antitumor macrophages induced with OK-432 and cyclophosphamide

Summary The present study was designed to determine whether antitumor activity of macrophages induced with OK-432 and cyclophosphamide was mainly dependent on their ability to produce a soluble factor, that is,l-arginine-dependent nitric oxide as measured by nitrite concentration. Nitrite production by peritoneal macrophages from NIH Swiss mice pretreated with OK-432 (125 KE/kg) i.p. twice at 1-week intervals and with cyclophosphamide (200 mg/kg) i.p. 2 days before the second OK-432 treatment, increased with time for 24 h, and proportionally depended on macrophage numbers. Nitrite production was inhibited by actinomycin D and puromycin but not by mitomycin C.N ^G-Monomethyl-l-arginine, a specific competitive inhibitor ofl-arginine-dependent nitric oxide synthesis, also inhibited production. There was a close correlation between nitrite production and antitumor activity in macrophages from mice pretreated with either OK-432 and cyclophosphamide, OK-432, or thioglycolate broth. OK-432 increased both nitrite production and antitumor activities when added to the macrophage from mice pretreated with OK-432 but not with thioglycolate broth. Both activities of macrophages from mice pretreated with OK-432 and cyclophosphamide were enhanced with increasing concentrations ofl-arginine (0.125–1 mM) in the culture medium.d-Arginine, however, did not substitute forl-arginine. Neither activity was affected by contact between the macrophage and the EL4 cell. The macrophage showed antitumor activity through a membrane filter though the activity was greatly reduced. This antitumor activity of macrophages through a membrane was also inhibited byN ^G-Monomethyl-l-arginine, and increased by OK-432. However, conditioned media, obtained by culturing macrophages induced with OK-432 and cyclophosphamide, inhibited growth of EL4 cells. This activity was carried out by dialysable and non-dialysable factors. One of the dialysable factors was nitrite, an oxidized product of nitric oxide. The antitumor activity of non-dialysable factors was heat-stable and production of factors was increased byN ^G-Monomethyl-l-arginine and OK-432. Also, non-dialysable factors increased both antitumor and nitrite production activities of OK-432-elicited macrophages, when incubated with factors. Such activity of factors was also heat-stable. The production of factors increased with incubation time of macrophages, and was not inhibited byN ^G-Monomethyl-l-arginine. These results indicate that in vitro antitumor activity of macrophages induced with OK-432 and cyclophosphamide was mainly dependent onl-arginine-dependent nitric oxide, and that macrophageassociated soluble factors other than nitric oxide were also needed to inhibit fully tumor growth in vitro.     

The characteristics of arginine transport by rat cerebellar and cortical synaptosomes

The uptake ofl-[³H]arginine into synaptosomes prepared from rat cerebellum and cortex occurred by a high-affinity carrier-mediated process. The uptake of arginine appeared to be potentiated by removal of extracellular Na⁺, inhibited by high levels of extracellular K⁺, but not by depolarization with veratridine or 4-amino pyridine. The effect of Na⁺ removal or K⁺ elevation did not seem to be due to changes in intracellular Ca²⁺ or pH. In both brain regions, uptake was significantly inhibited byl-arginine,l-lysine,l-ornithine, andl-homoarginine, but not byd-arginine norl-citrulline. Uptake was also inhibited by N^G-monomethyl-l-arginine acetate, but not by N^G-nitro-l-arginine methyl ester nor N^G-nitro-l-arginine except in the cortex at a concentration of 1 mM. The results indicate that the carrier system operating in synaptosomes showed many of the characteristics of the ubiquitous y⁺ system seen in many other tissues, although its apparent sensitivity to variations in extracellular Na⁺ was unusual.     

Construction of an arginine-producing strain of Serratia marcescens

Summary In Serratia marcescens Sr41, l-canavanine was demonstrated to be a weak cell growth inhibitor in minimal medium containing glucose as the sole carbon source. The inhibition of cell growth was enhanced by changing the carbon source from glucose to l-glutamic acid. An arginine regulatory mutant (i.e., argR mutant) in which formation of l-arginine biosynthetic enzymes was genetically derepressed was isolated by selecting for l-canavanine resistance on the glutamate medium. Furthermore, an l-arginine-producing strain was constructed by introducing the mutation leading to feedback-resistant N-acetylglutamate synthase into the argR mutant. The resulting transductant produced about 40 g/l of l-arginine, whereas the wild strain produced no l-arginine and the argR mutant only 3 g/l.     

Evidence for NO-dependent vasodilation in the trout (Oncorhynchus mykiss ) coronary system

The effects of l-arginine, and its analogues N ^ω-nitro-l-arginine methyl ester and N ^ω-nitro-l-arginine on vascular resistance were investigated in the intact coronary system of an isolated non-working trout heart preparation. l-Arginine, at 10^–8 mol · l^–1induced a slight vasodilatory effect (max 10%). N ^ω-nitro-l-arginine methyl ester and N ^ω-Nitro-l-arginine in the range 10^–8–10^–4 mol · l^–1 caused dose-dependent increases in coronary resistance. The vasodilatory action of l-arginine was abolished when the preparation was pretreated with 10^–4 mol · l^–1 N ^ω-nitro-l-arginine or N ^ω-nitro-l-arginine methyl ester. Nitroprusside alone at 1 mmol · l^–1 induced a maximum vasodilation (30%) of the coronary system. Methylene blue a known inhibitor of guanylate cyclase, induced a strong vasoconstriction (already significant at 10^–5 mol · l^–1) and was able to overcome the vasodilative effect of nitroprusside. The endothelial nitric oxide agonists acetylcholine and serotonin, established in mammalian vessels, also mediate vasodilation in trout coronary system. In 50% of preparations, acetylcholine induced a biphasic response with vasodilation at low concentration (max 15% at 10^–8 mol · l^–1). Serotonin displayed a dose-response vasodilation in the range 10^–8–10^–4 mol · l^–1 (max 20%). These vasodilative effects were reduced or abolished by 10^–4 mol · l^–1 l-NA. These data support the existence of NO-mediated vasodilation mechanisms in the trout coronary system. Accepted: 1 July 1996     

Evidences for complex formation between L-dabPNA and aegPNA

Continuing our research on the development of nucleopeptides as ODN analogs for biomedical and bioengineering applications, here we report the synthesis and the chemical–physical characterization of a homoadenine hexamer based on a l-diaminobutyric acid (l-DABA) backbone (dabPNA), and its binding studies with a complementary aegPNA. We demonstrated by CD and UV experiments that the l-dabPNA binds the aegPNA forming a complex with good thermal stability, that we identified as a left-handed triplex.     

Reversed-phase liquid chromatographic resolution of diastereomers of protein and non-protein amino acids prepared with newly synthesized chiral derivatizing reagents based on cyanuric chloride

Two new chiral monochloro-s-triazines (MCT) were synthesized [viz N-(4-chloro-6-piperidinyl-[1,3,5]-triazine-2-yl)-l-leucine amide and N-(4-chloro-6-piperidinyl-[1,3,5]-triazine-2-yl)-l-leucine) (CDR 1 and 2, respectively)] by the nucleophilic displacement of chlorine atoms in s-triazine moiety. One of the Cl atoms was replaced with piperidine, and the second Cl atom in the 6-piperidinyl derivative was replaced with amino acid amide (viz l-Leu–NH₂) and amino acid (l-Leu). These reagents were characterized and used as CDRs for chiral separation of protein and non-protein amino acids, and were separated on a reversed-phase C₁₈ column. The reaction conditions were optimized for the synthesis of diastereomers using one MCT reagent. The separation method was validated for limit of detection, linearity, accuracy, precision, and recovery.     

A chemically defined medium for cephalosporin C production by Paecilomyces persicinus

A chemically defined medium was developed for the biosynthesis of cephalosporin C by Paecilomyces persicinus Nicot strain P-10. Glucose served as the major carbon source and nitrogen was supplied by five amino acids, l-arginine, l-aspartic acid, l-glutamic acid, glycine and dl-methionine. Omission of any of the first four diminished or prevented production of cephalosporin C; omission of methionine did not. Methionine is not critical for the production of cephalosporin C in this defined medium. Production of the antibiotic was affected by the concentrations of inorganic salts employed. Biotin was required for growth and cephalosporin C synthesis. The addition of l-lysine precursors to the medium did not influence cephalosporin C levels and l-lysine itself inhibited antibiotic production. Known precursors of -lactam antibiotics as well as oleic acid did not affect biosynthesis of cephalosporin C. Chemical changes occurring in the defined medium revealed that glucose was efficiently utilized after 96 hours incubation whereas total soluble nitrogen levels increased following an initial sharp decrease. Mycelial weight and cephalosporin C production were both maximal after 96 hours incubation. Mycelial nitrogen was highest after 48 hours incubation whereas mycelial lipid levels were greatest after 72 hours.     

Deregulation of arginine biosynthesis in Synechococcus sp. PCC7942

Summary In order to deregulate arginine biosynthesis in Synechococcus sp. PCC7942, d-arginine-resistant cell lines were selected following ethyl methanesulfonate mutagenesis of wild-type (WT) cells. Three of these arginine-producing mutant (APM) cell lines, APM1, APM31 and APM40, were putative regulatory mutants based upon secretion of l-arginine into their growth medium. HPLC of lyophilized post-harvest supernatants of APM 31 and 40 resolved two predominant amino acids, arginine and citrulline. In-vitro activity of N-acetylglutamate kinase (NAGK), the proposed regulatory enzyme of the arginine pathway, was about 100-fold less sensitive to l-arginine inhibition in extracts from APM 31 and 40 than the enzyme in WT extracts. The enzyme from APM 1 was 20-fold less sensitive to l-arginine inhibition than WT. The most likely site of mutation in each of the APM cell lines is in the gene for NAGK, rendering the enzymes insensitive to l-arginine feedback control. These strains can be utilized for the phototrophic production of arginine. Offprint requests to: S. E. Bingham     

Effects of tensile stress on the α1 nicotinic acetylcholine receptor expression in maxillofacial skeletal myocytes

The present study was to test the hypothesis that 11,12-epoxyeicosatrienoic acid (11,12-EET), a metabolic product of arachidonic acid by cytochrome P450 epoxygenase, regulates nitric oxide (NO) generation of the l-arginine/NO synthase (NOS) pathway in human platelets. Human platelets were incubated in the presence or absence of different concentrations of 11,12-EET for 2 h at 37°C, followed by measurements of activities of the l-arginine/NOS pathway. Incubation with 11,12-EET increased the platelet NOS activity, nitrite production, cGMP content, and the platelet uptake of l-[³H]arginine in a concentration-dependent manner. In addition, 11,12-EET attenuated intracellular free Ca²⁺ accumulation stimulated by collagen, which was at least partly mediated by EET-activated l-arginine/NOS pathway. It is suggested that 11,12-EET regulates platelet function through up-regulating the activity of the l-arginine/NOS/NO pathway.     

Metabolic interactions between restraint stress and <Emphasis Type="SmallCaps">l</Emphasis>-lysine: The effect on urea cycle components

Summary.  We studied the effects of l-lysine on wrap-restraint stress-induced changes in ureagenesis. An exposure to wrap-restraint stress did not affect the plasma concentration of l-lysine, but did decrease plasma urea and arginine. Oral l-lysine (1 g/kg) blocked the effect of stress on ureagenesis, and enhanced the effect of stress on l-arginine. No influence of l-lysine were found in controls. The results imply a stress-specific, ureagenesis-stimulating effect of l-lysine, and suggest an increased requirement for l-arginine during the above conditions. Received December 9, 2002 Accepted January 21, 2003 Published online April 3, 2003 Acknowledgement Authors thank Dr. M. Miura (Ajinomoto Co.) for his help with amino acid analysis and Dr. T. Kimura (Ajinomoto Co.) for discussions on amino acid metabolism. Authors' address: Dr. M. Smriga, Ajinomoto Co. Inc., Institute of Life Sciences, 1-1 Suzuki-cho, 210-8681 Kawasaki, Japan, Fax +81-44-210-5893, E-mail: miroslav_smriga@ajinomoto.com Abbreviations: Arg, l-arginine; Orn, l-ornithine; Lys, l-lysine; p.o., oral; WRS, wrap-restraint stress     

Stimulatory effect of glycine betaine on l-lysine fermentation

Summary The growth rate, sugar consumption rate, and production rate of an l-lysine producing Brevibacterium lactofermentum mutant were stimulated by addition of exogenous glycine betaine. Glycine betaine stimulated the growth rate especially in media of inhibitory osmotic stress, and the stimulation was independent of any specific solute. Therefore growth stimulation by glycine betaine was considered to be an osmoprotective effect. A strong enhancement of the sugar consumption rate and the l-lysine production rate was observed even with resting cells under osmotic stress as well as in a fermentation with growing cells. These data indicated that the osmoprotective effects of glycine betaine on l-lysine production can be independent of protein synthesis. Offprint requests to: Yoshio Kawahara     

Single Taste Stimuli Elicit Either Increases or Decreases in Intracellular Calcium in Isolated Catfish Taste Cells

Taste cells are specialized epithelial cells that respond to stimulation with release of neurotransmitters onto afferent nerves that innervate taste buds. In analogy to neurotransmitter release in other cells, it is expected that neurotransmitter release in taste cells is dependent on an increase in intracellular Ca²⁺ ([Ca²⁺] _i ). We have studied changes in [Ca²⁺] _i elicited by the taste stimuli l- and d-arginine in isolated taste cells from the channel catfish (Ictalurus punctatus). In a sample of 119 cells, we found 15 cells responding to l-arginine, and 12 cells responding to d-arginine with an increase in [Ca²⁺] _i . The response to l-arginine was inhibited by equimolar d-arginine in cells where d-arginine alone did not cause a change in [Ca²⁺] _i , which is consistent with mediation of this response by a previously characterized l-arginine-gated nonspecific cation channel antagonized by d-arginine [31]. However, we also found that these taste stimuli elicited decreases in [Ca²⁺] _i in substantial number of cells (6 for l-Arg, and 2 for d-Arg, n= 119). These observations suggest that stimulation of taste cells with sapid stimuli may result in simultaneous excitation and inhibition of different taste cells within the taste bud, which could be involved in local processing of the taste signal. Received: 25 May 1995/Revised: 29 September 1995     

Uptake of amino acids by actidione-treated yeast cells

Uptake of glyeine,l-cysteine,l-leucine,l-methionine,l-aspartic acid andl-lysine was investigated in resting cells ofSaccharomyces cerevisiae treated with 0.3mm actidione for blocking protein synthesis. The amino acids were taken up against substantial concentration gradients (up to nearly 1,000∶1 for μm l-cysteine and glycine). They were present in the free form inside the cells. Their unidirectional transmembrane fluxes were under a negative feedback control by the intracellular concentration of the amino acid involved. The amino acids tested apparently employed more than one transport agéncies for their membrane passage, the half-saturation constants being 6.2–7.7×10⁻⁴ m for glycine, 2.5×10⁻⁴ m forl-cysteine, 6×10⁻⁵ and 4×10⁻⁴ m forl-lysine, 3×10⁻⁵ and 6×10⁻⁴ m forl-methionine, 7–18×10⁻⁵ and 1.6×10⁻³ m forl-aspartic acid and 6×10⁻⁵ and 2×10⁻³ m forl-leucine. The specificities of the transport systems are overlapping but there emerges a wide-affinity transport system for glycine, alanine, leucine, methionine, serine, cysteine, phenylalanine, aspartic acid, asparagine, glutamic acid and tryptophan (and possibly for other amino acids), and more specific systems for each of the following: glycine, lysine, methionine, histidine, arginine, and aspartic and glutamic acids. Proline had the peculiar effect of stimulating the transport of all the amino acids tested. The amino acids apparently interacted in the uptake not only by competition for the binding site but also by allotopic inhibition (e.g.l-cysteine) and possibly stimulation (l-proline). The initial rate of uptake of amino acids and their steady-state level of distribution were characterized by identical activation energies: 7.5 kcal/mole forl-lysine, 6.9 kcal/mole forl-aspartic acid, and 13.2 kcal/mole for glycine.