Indispensable amino acid concentrations decrease in tissues of stomachless fish,common carp in response to free amino acid- or peptide-based diets

Summary. The premise that free amino acid or dipeptide based diets will resolve the nutritional inadequacy of formulated feeds for larval and juvenile fish and improve utilization of nitrogen in comparison to protein-based diets was tested in stomachless fish, common carp (Cyprinus carpio L.) larvae. We examined the postprandial whole body free amino acid (FAA) pool in fish that were offered a FAA mixture based diet for the duration of 2 or 4 weeks. We found that the total amount and all indispensable amino acids concentrations in the whole body decreased after a meal. We then fed juvenile carp with dietary amino acids provided in the FAA, dipeptide (PP), or protein (live feed organisms; brine shrimp Artemia salina nauplii, AS) forms. Histidine concentrations in the whole fish body increased in all dietary groups after feeding whereas all other indispensable amino acids decreased in FAA and PP groups in comparison to the AS group. Taurine appears to be the major osmotic pressure balancing free amino acid in larval freshwater fish which may indicate a conditional requirement. We present the first evidence in larval fish that in response to synthetic FAA and PP diets, the whole body indispensable free AA concentrations decreased after feeding. This study shows that amino acids given entirely as FAA or PP cannot sustain stomachless larval fish growth, and may result in depletion of body indispensable AA and most of dispensable AA. The understanding of these responses will determine necessary changes in diet formulations that prevent accelerated excretion of amino acids without protein synthesis.     

N-methyl-d-aspartate andl-aspartate activate distinct receptors in piriform cortex

The effects of ionophoretically applied N-methyl-DL-aspartate (NMDA) and aspartate on identified pyramidal neurons in rat piriform cortex were examined in isolated, submerged, and perfused brain slices. NMDA was more potent than aspartate in eliciting neuronal discharge. Perfusion of the acidic amino acid antagonists, DL-2-amino-5-phosphonovalerate (APV), 10(-6) or 10(-5) M, DL-2-amino-7-phosphonoheptanoate (APH), 10(-5) M, and gamma-D-glutamylglycine (gamma DGG), 10(-5) M, selectively blocked the response to NMDA without effect on the response to aspartate. At higher concentrations which blocked responses to both NMDA and aspartate, gamma DGG blocked kainate responses and depressed glutamate and quisqualate responses. These results suggest that in piriform neurons NMDA and aspartate act at distinct receptor sites, not a common receptor site, and that both of these sites are distinct from those that mediate responses to glutamate, quisqualate, and kainate.     

Studies on Enzymatic Synthesis of Oligosaccharides

The overproduction of tumor necrosis factor-α (TNF-α) was suppressed by orally administering a perilla leaf extract (PLE). When mice were successively injected with OK-432, severe TNF-α was induced in the serum, but this elevated TNF-α level was reduced after an oral administration of PLE (400 μl/mouse). Oral administration of PLE also inhibited TNF-α production that was induced by muramyl dipeptide (500 μg/mouse) and OK-432 (3 KE/mouse). These characteristics were obtained from all strains of perilla. The inhibitory activity against TNF-α production was heat-stable, and the existence of several active molecules was suggested. When PLE was passed through an ultrafilter, the inhibitory activity against TNF-α production was collected in those fractions with a mass of 0.5 to 1 kDa and more than 10 kDa. When PLE was solvent-extracted, the strongest activity was recognized with aqueous preparation, although significant activity was also detected in preparations extracted with n-hexane and ethyl acetate. These findings suggest that the daily use of certain functional foods may be useful for controlling the host defense system.     

Upregulation of endogenous glutathione system by 3H-1,2-dithiole-3-thione in pancreatic RINm5F beta-cells as a novel strategy for protecting against oxidative beta-cell injury

This study was undertaken to investigate the inducibility of glutathione (GSH), glutathione reductase (GR) and glutathione peroxidase (GPx) by 3H-1,2-dithiole-3-thione (D3T) in beta-cells, and the resultant cytoprotection against oxidant injury. Incubation of the insulin-secreting RINm5F cells with D3T led to significant induction of GSH, GR and GPx. D3T-mediated induction of GSH was abolished by buthionine sulfoximine (BSO), suggesting a critical involvement of γ-glutamylcysteine ligase (γGCL). Consistently, incubation of RINm5F cells with D3T resulted in increased expression of γGCL protein and mRNA. Pretreatment of RINm5F cells with D3T provided remarkable protection against oxidant-elicited cytotoxicity. On the other hand, depletion of cellular GSH by BSO sensitized RINm5F cells to oxidant injury. Furthermore, cotreatment of RINm5F cells with BSO to reverse D3T-mediated GSH induction abolished the cytoprotective effects of D3T on oxidant injury. Taken together, this study demonstrates that upregulation of glutathione system by D3T is effective for protecting against oxidative beta-cell injury.     

Structural versatility of peptides from Cα, α-disubstituted glycines: Crystal-state conformational analysis of homopeptides from Cα-methyl,Cα-benzylglycine [(αMe)Phe]n

The molecular and crystal structures of one derivative and three homopeptides (from the di-to the tetrapeptide level) of the chiral, C^{α, α}-disubstituted glycine C^α-methyl, C^α-benzylglycine [(αMe)Phe], have been determined by x-ray diffraction. The derivative is mClAc-D -(αMe)Phe-OH, and the peptides are pBrBz-[D -(αMe)Phe]₂-NHMe, pBrBz-[D -(αMe)Phe]₃-OH hemihydrate, and pBrBz-[D -(αMe)Phe]₄-OtBu sesquihydrate. All (αMe)Phe residues prefer ?,ψ torsion angles in the helical region of the conformational map. The dipeptide methylamide and the tripeptide carboxylic acid adopt a β-turn conformation with a 1 ← 4 C?O…?H? N intramolecular H bond. The structure of the tripeptide carboxylic acid is further stabilized by a 1 ← 4 C?O…?H? O intramolecular H bond, forming an “oxy-analogue” of a β-turn. The tetrapeptide ester is folded in a regular (incipient) 3₁₀-helix. In general, the relationship between (αMe)Phe chirality and helix screw sense is opposite to that exhibited by protein amino acids. A comparison is made with the conclusions extracted from published work on homopeptides from other C^α-methylated α-amino acids. © 1993 John Wiley & Sons, Inc.     

Free energies and equilibria of peptide bond hydrolysis and formation

For every n amino acids linked in a protein there are n − 1 peptide bonds. The free energy of peptide bond hydrolysis and formation in aqueous solution defines the equilibrium position between peptide and amino acid hydrolysis products. Yet few experimental values exist. With a minimum of assumptions, this paper deduces the free energies of hydrolysis of a variety of peptide bonds. Formation of a dipeptide from two amino acids is about eight times more difficult than subsequent condensations of an amino acid to a dipeptide or longer chain. Condensation of an amino acid to a peptide of any size is five times more difficult than joining two smaller peptides of at least dipeptide size. Thus in an abiogenesis scenario there is a kind of nucleation in peptide bond formation with the initial condensation of two amino acids to yield a dipeptide more difficult than subsequent condensations to a growing chain. © 1998 John Wiley & Sons, Inc. Biopoly 45: 351–353, 1998     

Conformation of dipeptides

The amide bond in L ,L - and L ,D -α-chloropropionylalanine methyl ester is shown to be trans by molar polarization and infrared spectroscopy. In these dipeptide diastereoisomer analogues, therefore, differences in physical properties, i.e., melting points, crystalline forms, gas chromatographic mobilities, etc., depend on preferred molecular conformations and not peptide bond configuration. Nuclear magnetic resonance spectra of both compounds were identical, indicating that no major chemical environment differences exist, which might have resulted from dissimilar side group interactions. Based on the data reported here and those of others, most dipeptide conformations can be eliminated because of contradiction with limits set by experimental or theoretical considerations. Of the remaining conformational possibilities, a single pair accounts for observed physical differences in dipeptide diastereoisomers, free or blocked. The preferred form contains α-hydrogens trans to each other and in the plane of the peptide bond. In this conformation, R¹–R² and amino–carboxyl distances are minimal in L ,D diastereomers and maximal in L ,L forms.     

Synthesis,conformational study,and spectroscopic characterization of the cyclic Cα,α‐disubstituted glycine 9‐amino‐9‐fluorenecarboxylic acid

A series of terminally blocked peptides (to the pentamer level) from l ‐Ala and the cyclic C^α,α‐disubstituted Gly residue Afc and one Gly/Afc dipeptide have been synthesized by solution method and fully characterized. The molecular structure of the amino acid derivative Boc‐Afc‐OMe and the dipeptide Boc‐Afc‐Gly‐OMe were determined in the crystal state by X‐ray diffraction. In addition, the preferred conformation of all of the model peptides was assessed in deuterochloroform solution by FT‐IR absorption and ¹H‐NMR. The experimental data favour the conclusion that the Afc residue tends to adopt either the fully‐extended (C₅) or a folded/helical structure. In particular, the former conformation is highly populated in solution and is also that found in the crystal state in the two compounds investigated. A comparison with the structural propensities of the strictly related C^α,α‐disubstituted Gly residues Ac₅c and Dϕg is made and the implications for the use of the Afc residue in conformationally constrained analogues of bioactive peptides are briefly examined. A spectroscopic (UV absorption, fluorescence, CD) characterization of this novel aromatic C^α,α‐disubstituted Gly residue is also reported. Copyright © 1999 European Peptide Society and John Wiley & Sons, Ltd.     

γ-tocopherol is less effective than α-tocopherol in preventing oxidant-induced sister chromatid exchanges in Chinese hamster V79 cells

Although α-tocopherol (α-TOC) is the most biologically active form of vitamin E and is found at high levels in plasma, γ-tocopherol (γ-TOC) has also been found to be a powerful antioxidant in vitro and constitutes up to 70% of the dietary intake of TOC. Low plasma levels of γ-TOC and a high α-TOC:γ-TOC ratio may be associated with coronary heart disease, suggesting that there may be a positive protective role for the γ-form of TOC. In this study the ability of different forms of vitamin E to protect against sister chromatid exchanges (SCE) induced by either hydrogen peroxide or menadione was investigated. Chinese hamster V79 cells were pre-treated with 10 μM TOC for 24 h, and then challenged with a genotoxin. After a 24 h pre-treatment, there was a greater incorporation of γ-TOC (319.8 ± 66.2 ng/10⁶ cells) into V79 cells compared to α-TOC (66.9 ± 6.4 ng/10⁶ cells). γ-TOC did not protect the cells against SCE induced by either hydrogen peroxide or menadione, α-TOC acetate was partially protective against both genotoxins, whereas α-TOC completely abolished the oxidant induced SCE. These results demonstrate that, despite a greater incorporation of γ-TOC into V79 cells, α-TOC but not γ-TOC was more effective at inhibiting oxidatively-induced SCE in V79 cells.     

Studies on the conformation of amino acids. X. Conformations of norvalyl, leucyl and aromatic side groups in a dipeptide unit

Backbone-side group conformations of amino acid residues including one or two δ-carbons in the side group have been investigated. Conformational energies of norvalyl, leucyl, phenylalanyl, tyrosyl, tryptophenyl, and histidinyl side groups in a dipeptide unit have been calculated by using classical energy expressions. The side group conformations about the C^α—C^β and C^β—C^γ bonds are restricted to specific values of the respective rotational angles. Thus, most favourable positions of γ- and δ-atoms of a linear side-chain (norvalyl) are restricted to (γ_I, δ_II) (γ_II, δ_I), (γ_II, δ_II), (γ_III, δ_II), and (γ_III, δ_III), whereas those of the side-chain branching at a sp³ γ-atom (leueyl) are further restricted. It is also shown that there is a definite correlation between the orientations of the two peptide planes and that of the planar group of the aromatic side chain of phenylalanyl type residues. The studies bring out an important fact that while the γ-atoms have definite and characteristic effects on the backbone rotational angles ? and ψ, the δ atoms and beyond have no effects on the preferred ? and ψ values. Thus, the preferred backbone conformations are independent of the preferred side group conformations beyond the γ-atom and vice versa. The observed ?, ψ, χ¹, and χ² values of amino acids, simple peptides, and of the three protein molecules lysozyme, myoglobin, and chymotrypsin have been compared with the theoretical predictions, and the agreement is found to be excellent.     

肽键的统计分析和蛋白质的二级结构

 本文对蛋白质序列的肽键进行了统计分析,计算了二肽构象参数P_α、P_β、P_c和三肽构象参数Q_α、Q_β、Q_c。在此基础上提出了由氨基酸序列预测二级结构的规则。预测的正确率达90％,优于Chou-Fasman方法。这个结果表明二肽(三肽)关联在形成蛋白质二级结构中具有明显的重要性。     

Use of Sequence-Specific Antisense Oligodeoxynucleotides to Determine the Protozoan Parasite Antigen Recognized by Nonspecific Cytotoxic Cells

The antigen on the protozoan parasite Tetrahymena pyriformis recognized by catfish nonspecific cytotoxic cells (NCC) is a 46- to 48-kDa protein referred to as NKTag. The complete cDNA-derived amino acid sequence of NKTag has been obtained. The antigenic determinant of NKTag corresponding to the NCC binding site has been determined with synthetic peptides in target cell competition experiments. To more directly characterize the mechanism of parasite:effector cell interaction, we applied NKTag sequence-specific antisense oligodeoxynucleotides to Tetrahymena in vitro. NKTag mRNA translation by Tetrahymena was blocked by specific antisense (AS) oligodeoxynucleotides. 5′-3′ sense (S) oligodeoxynucleotide sequences were synthesized corresponding to the first 17 N-terminal amino acids of NKTag (in addition to −2 untranslated codons plus the start codon). Complimentary AS oligodeoxynucleotides were likewise synthesized. To determine the optimum in vitro conditions for AS treatment, we tested parasites at various phases of their growth cycle for the effects of a single AS treatment. At 9 h post-AS treatment (during the linear phase of the growth curve), maximum reduction in membrane expression of NKTag was observed. Eighty-five percent of Tetrahymena were positive for expression of NKTag at 0 time post-AS treatment versus 13% positive at 9 h. Membrane expression of AS-treated parasites returned to normal levels by 24 h post-treatment. In cold target inhibition experiments, the reduced NKTag expression by Tetrahymena at 9 h AS treatment was confirmed by observing a complete inability (compared with S-treated parasites) to compete with IM-9 cells for binding with NCC. These data demonstrated a unique experimental in vitro system to define the antigen determinant on target cells responsible for recognition by cytotoxic effector cells that participate in innate immune responses. Received: 14 June 1999 / Accepted: 4 September 1999     

Preparation and evaluation of a new releasable PEGylated tumor necrosis factor-α (TNF-α) conjugate for therapeutic application

To design a releasable PEGylated TNF-α (rPEG-TNF-α), a cathepsin B-sensitive dipeptide (Val-Cit moiety) was inserted into conventional PEG-modified TNF-α (PEG-TNF-α), facilitating its clinical use for anti-tumor therapy. Comparative pharmacokinetic and pharmacodynamic studies showed that the half-lives of both PEGylated forms of TNF-α were ～60-fold greater than that of unmodified TNF-α. In addition, the in vitro bioactivity of rPEG-TNF-α was greater than that of PEG-TNF-α with the same degree of PEG modification. Release of TNF-α from rPEG-TNF-α in vitro was dependent on the presence of cathepsin B and was inhibited by a cathepsin B inhibitor. Despite the potent cytotoxicity of unmodified TNF-α against normal cells, its PEGylated forms at higher TNF-α concentrations showed low cytotoxic activity against these cells. In contrast, both forms of PEGylated TNF-α showed potent cytotoxic activity against the B16 and L929 cell lines, with rPEG-TNF-α being 5- and 9-fold more potent, respectively, than PEG-TNF-α. Moreover, rPEG-TNF-α was a more potent in vivo antitumor agent than PEG-TNF-α.     

EFFECTS OF DIPEPTIDES CONTAINING THE AMINO ACID,PROLINE ON THE CHEMOTAXIS OF TETRAHYMENA PYRIFORMIS. EVOLUTIONARY CONCLUSIONS ON THE FORMATION OF HORMONE RECEPTORS AND HORMONES

Our investigations demonstrate that proline-containing dipeptides can provoke a chemosensory response from the unicellular Tetrahymena pyriformis The chemotactic effects of the dipeptides have a close relationship with the side chain and the lipophilicity of the amino-terminal amino acid. Comparison of ‘mirror’ variants of proline-containing dipeptides points to the fact that dipeptides with small side chain and non-polar character amino acids (Gly-Pro, Ala-Pro) are preferred on the amino-terminal end. In the case of amino acids with very variable side chains, small (Pro-Gly) and the large side chain and non-polar character amino acids (Pro-Leu, Pro-Phe) on the carboxyl-terminal end can induce significant chemotactic responses. With valine on any terminus the proline-containing dipeptide induced a weak repellent effect.     

NEURONAL AND GLIAL SYSTEMS FOR γ-AMINOBUTYRIC ACID METABOLISM

—Bulk prepared neuronal perikarya, nerve endings and glial cells have been used to study amino acid concentrations and GABA metabolism in vitro. All amino acids were more concentrated in synaptosomes and glial cells than in neuronal perikarya. Cell specificity was found with respect to the relative distribution of some amino acids. Glutamate decarboxylase activity was considerably higher in synaptosomes than in glial cells. The inhibitory effect of amino-oxyacetic acid on glutamate decarboxylase activity differed between synaptosomes and glial cells. γ-Aminobutyric acid-α-ketoglutarate transaminase had the highest activity in the glial cell fraction; the inhibition of amino-oxyacetic acid differed between glial and neuronal material. The metabolism of exogenous GABA just accumulated by a cell showed similar time characteristics in neuronal and glial material.     

Mechanisms of action of chloroalanyl antibacterial peptides. Identification of the intracellular enzymes inactivated on treatment of Escherichia coli JSR-O with the dipeptide beta Cl-LAla-beta Cl-LAla

The dipeptide beta Cl-LAla-beta Cl-LAla is an antibacterial agent designed to utilize bacterial peptide transport for intracellular delivery of the alanine racemase inactivator beta Cl-LAla. The minimum inhibitory concentrations (MICs) for the peptide against Gram-negative species grown on enriched agar medium range from 1.56 to 12.5 micrograms/ml; MICs are increased to greater than 100 micrograms/ml when D-alanine is included in the medium, indicating that alanine racemase is, in fact, inhibited in sensitive species. When susceptible Gram-negative cells are grown on a minimal medium, D-alanine supplementation alone does not increase the MICs for beta Cl-LAla-beta Cl-LAla, but complete protection is afforded by supplementation with D-alanine, L-valine, L-leucine, and L-isoleucine. In liquid culture, the peptide is: bactericidal and lytic against Escherichia coli JSR-O growing in enriched medium or in minimal medium supplemented with the branched-chain amino acids; only inhibitory against these cells growing in minimal medium supplemented with D-alanine; and ineffective against these cells in minimal medium containing the branched-chain amino acids plus D-alanine. Cells exposed to beta Cl-LAla-beta Cl-LAla (with the protection of the four amino acids) have specific activities of both alanine racemase and transaminase B that are lower than those of cultures not treated with the peptide. Finally, E. coli JSR-O alanine racemase experiences time-dependent loss of activity when exposed to the dipeptide in the presence of aminopeptidases; the dipeptide alone is not an inactivator of the racemase in vitro. These results suggest the following mechanism of action for beta Cl-LAla-beta Cl-LAla: transport of the dipeptide into the cell; intracellular hydrolysis to give accumulation of beta Cl-LAla; and subsequent inactivation of targeted enzymes. Whether inactivation of the racemase or of the transaminase determines the pathophysiologic effects of the peptide depends on the composition of the growth medium.     

Heterochiral Ala/(αMe)Aze sequential oligopeptides: Synthesis and conformational study

α‐Amino acid residues with a ?,ψ constrained conformation are known to significantly bias the peptide backbone 3D structure. An intriguing member of this class of compounds is (αMe)Aze, characterized by an N^α‐alkylated four‐membered ring and C^α‐methylation. We have already reported that (S)‐(αMe)Aze, when followed by (S)‐Ala in the homochiral dipeptide sequential motif ‐(S)‐(αMe)Aze‐(S)‐Ala‐, tends to generate the unprecedented γ‐bend ribbon conformation, as formation of a regular, fully intramolecularly H‐bonded γ‐helix is precluded, due to the occurrence of a tertiary amide bond every two residues. In this work, we have expanded this study to the preparation and 3D structural analysis of the heterochiral (S)‐Ala/(R)‐(αMe)Aze sequential peptides from dimer to hexamer. Our conformational results show that members of this series may fold in type‐II β‐turns or in γ‐turns depending on the experimental conditions.     

Pharmacological antagonists of excitant amino acid action

Pharmacological receptors for excitant amino acids have been classified into three major types found within the vertebrate central nervous system (CNS). The three types of receptor are exemplified by the action of the selective agonists N-methyl-D-aspartate (NMDA), kainate and quisqualate. Several compounds have been discovered which are selective antagonists of NMDA-evoked excitations, the most potent to date being 2-amino-5-phosphonovalerate (APV). Depression of synaptic excitation by NMDA receptor antagonists indicates a physiological role of these receptors in various regions of the CNS.Potent and selective antagonists for kainate or quisqualate receptors have yet to be developed. However, glutamate diethyl ester (GDEE) and γ-D-glutamylglycine (DGG), applied microelectrophoretically, selectively depress quisqualate and kainate-evoked responses, respectively. 2-Amino-4-phosphonobutyrate (APB) and $\text{cis}$-2, 3-piperidine dicarboxylate (PDA) are relatively non-selective antagonists of the three types of excitant receptor. Depression of APV-resistant spinal transmission by PDA and synaptically localized kainate binding in the hippocampus suggest that kainate and/or quisqualate receptors are also involved in excitatory transmission.     

Vaporization of Inorganic Mercury by Cell-free Extracts of Drug Resistant Escherichia coli

The cDNAs encoding venom phospholipase A₂ (PLA₂) inhibitors (PLIs), named Protobothrops elegans (Pe)γPLI-A, PeγPLI-B, PeαPLI-A, and PeαPLI-B, were cloned from the P. elegans liver cDNA library. They were further divided into several constituents due to nucleotide substitutions in their open reading frames. For PeαPLI-A, two constituents, PeαPLI-A_a and PeαPLI-A_b, were identified due to three nonsynonymous substitutions in exon 3. Far-western blot and mass-spectrometry analysis of the P. elegans serum proteins showed the presence of γPLIs, and αPLIs, which can bind venom PLA₂s. In αPLIs from Protobothrops sera, A or B subtype-specific amino acid substitutions are concentrated only in exon 3. A comparison of γPLIs showed that γPLI-As are conserved and γPLI-Bs diversified. Mathematical analysis of the nucleotide sequences of Protobothrops γPLI-B cDNAs revealed that the particular loops in the three-finger motifs diversified by accelerated evolution. Such evolutionary features should have made serum PLIs acquire their respective inhibitory activities to adapt to venom PLA₂ isozymes.