Alterations in neutrophil (PMN) free intracellular alpha-keto acid profiles and immune functions induced by L-alanyl-L-glutamine, arginine or taurine

Summary. The objective of this study was to determine the dose as well as duration of exposure-dependent effects of L-alanyl-L-glutamine, arginine or taurine on polymorphonuclear neutrophil (PMN) free α-keto acid profiles and, in a parallel study, on PMN immune functions. Exogenous L-alanyl-L-glutamine significantly increased PMN α-ketoglutarate, pyruvate PMN superoxide anion (O₂⁻) generation, hydrogen peroxide (H₂O₂) formation and released myeloperoxidase (MPO) activity. Arginine also led to significant increases in α-ketoglutarate, pyruvate, MPO release and H₂O₂ generation. Formation of O₂⁻ on the other hand was decreased by arginine. Incubation with taurine resulted in lower intracellular pyruvate and α-ketobutyrate levels, decreased O₂⁻ and H₂O₂ formation and a concomitant significantly increased MPO activity. We therefore believe that considerable changes in PMN free-α-keto-acid profiles, induced for example by L-alanyl-L-glutamine, arginine or taurine, may be one of the determinants in cell nutrition that considerably modulates the immunological competence of PMN.     

Effects of ornithine on neutrophil (PMN) free amino acid and α-keto acid profiles and immune functions in vitro

Summary. The objective of this study was to determine the effects of ornithine on polymorphonuclear leucocyte (PMN) free amino- and -keto acid profiles, superoxide anion (O₂^–) generation, hydrogen peroxide (H₂O₂) formation and released myeloperoxidase acitivity (MPO). Exogenous ornithine significantly increased PMN asparagine, glutamine, asparatate, glutamate, arginine, citrulline, alanine, -ketoglutarate and pyruvate as intracellular ornithine increased. Concerning PMN immune function markers ornithine increased H₂O₂-generation and MPO acitivity while O₂^–-formation was decreased. We believe therefore that ornithine is important for affecting PMN susceptible free amino- and -keto acid pool although the mechanisms are not yet clear. This may be one of the determinants in PMN nutrition considerably influencing and modulating PMN host defense capability.     

Effects of arginine,L-alanyl-L-glutamine or taurine on neutrophil (PMN) free amino acid profiles and immune functions <Emphasis Type="Italic">in vitro</Emphasis>

Summary. The objective of this study was to determine the effects of arginine, L-alanyl-L-glutamine (Ala-Gln) or taurine on polymorphonuclear leucocyte (PMN) free amino acid profiles, superoxide anion (O_{2 −}) generation, hydrogen peroxide (H₂O₂) formation and released myeloperoxidase activity (MPO). Arginine led to significant increases in PMN arginine, ornithine, citrulline, aspartate, glutamate and alanine concentrations as well as increased H₂O₂-generation and MPO activity while O_{2 −}-formation was decreased. Ala-Gln caused significant increases in PMN free glutamine, alanine, asparagine, aspartate, glutamate, ornithine, arginine, serine and glycine concentrations and increased PMN immune functions. Taurine significantly increased PMN free taurine profiles, reduced PMN neutral amino acid content and decreased H₂O₂- and O_{2 −}-formation while MPO was increased. Altogether, the pharmacological regimens which enhance the supply of arginine, Ala-Gln or taurine in whole blood significantly affect PMN "susceptible free amino acid pool". This may be one of the determinants in PMN nutrition considerably influencing PMN immune functions. Received October 25, 2000 Accepted March 21, 2001     

Novel hemoglobin components and their amino acid sequences from the crab-eating macaque (Macaca fascicularis)

Summary We found two types of hemoglobin, T and R, from the crab-eating macaque and compared those to A and Q previously reported. The 22 animals studied showed six different phenotypes, A, R, QA, QT, QAT, and QAR. Analysis of the complete amino acid sequences for the chains of hemoglobins Q, A, T, and R revealed that amino acids at four positions, 8, 55, 71, and 78 from the N-terminal, are variable. In the ^A chain, Thr, Val, Gly, and Gln occupy these positions, and in the ^Q chain the analogous amino acids are Thr, Val, Asp, and Gln, respectively. In the newly found ^T chain they are Thr, Val, Gly, and His; and in the ^R chain, they are Ser, Ile, Gly, and His, respectively. Two amino acids (8 Thr and 79 Gln) in ^A of the crab-eating macaque were found to be different from those in the chain of the Japanese macaque.     

Synthesis of novel 3-pyridinecarbonitriles with amino acid function and their fluorescence properties

Summary. A variety of N-[(4,6-diaryl-3-pyridinecarbonitrile)-2-yl] amino acid esters 2–4 were synthesized through the reaction of 2-bromo-3-pyridinecarbonitriles 1 with the appropriate -amino acid ester hydrochloride in refluxing dioxane in the presence of triethylamine as dehydrohalogenating agent. Similarly, N-glycylglycine analogues 5 were obtained through the reaction of 1 with the dipeptide ester. On the other hand, attempts were made towards the construction of amino acid derivatives 7 through the reaction of 1 with aqueous solution -amino acids 6 in refluxing pyridine, but were unsuccessful, and instead the unexpected 2-amino-3-pyridinecarbonitriles 8 were isolated. The fluorescence properties of the newly synthesized pyridines 2–5 were evaluated. Some of the prepared compounds show considerable antibacterial activity.     

Unexpected Transformations of Natural Chlorins under Treatment with Dichlorodicyanobenzoquinone

The treatment of natural chlorins with 2,3-dichloro-5,6-dicyanobenzoquinone resulted not only in the intramolecular cyclization of the propionic acid residue in position 17 with the formation of an additional -lactone cycle at the pyrrole ring D, but also in the oxygen-assisted oxidation of 8-ethyl group in ring B to an -methoxyethyl substituent.     

Direct and indirect effects of human interferon α on renal cell carcinoma: a new in vitro assay system for evaluating cytokine-mediated antitumor effects

A highly purified natural -interferon (nIFN) was tested in vitro for direct and indirect antiproliferative activity against renal cell carcinoma (RCC), using a modified human tumor clonogenic assay and clinically achievable concentrations. In preclinical experiments, the indirect (cytokine-mediated) antiproliferative activity of nIFN was investigated using ACHN cells (established human RCC cell line). Continuous exposure to nIFN at concentrations of more than 5 IU/ml in the presence of feeder cells (a mixture of 5×10⁴ monocytes/dish and 5×10⁵ lymphocytes/dish, obtained from healthy donors) significantly inhibited colony formation of ACHN cells in comparison with growth inhibition in the absence of feeder cells (P<0.05). Various cytokines were measured in the supernatants lying over the medium on the feeder-layer agarose containing the same conditioned feeder cells. With IFN at 500 IU/ml, tumor necrosis factor (TNF) and IFN were detected at markedly high levels for 2–24 h. Neutralizing anti-TNF monoclonal antibody significantly reduced the indirect antiproliferative activity. Using our modified human tumor clonogenic assay technique, sufficient numbers of colonies for drug testing were observed in 19 of 31 surgical specimens (61.3%). In these clinical materials, nIFN at a clinically achievable concentration (50 IU/ml) significantly inhibited colony growth in the presence of feeder cells consisting of 5×10⁴ monocytes/dish and 5×10⁵ lymphocytes/dish, obtained from the patient whose tumor was examined (P<0.05). In colony-forming cases, a significant correlation between the percentage colony survival and TNF concentration in the supernatant was observed (r=–0.95,P<0.01). These results suggest that this assay system may be an appropriate technique for evaluating the antiproliferative activities of nIFN involving cytokine-mediated action, and that TNF may play an important role in this cytokine-mediated activity.This work was supported in part by a grant-in-aid for promoting research (no. 02771010) from the Ministry of Education     

Rapid and Efficient Synthesis of Peptides Containing α,α-dialkylamino acids employing KOBt

Several Fmoc-,-dialkylamino acids and their acid chlorides have been prepared, isolated and characterised. The synthesis of peptides containing sterically hindered dialkylamino acids has been accomplished using acid chloride/KOBt in dichloromethane. The yields as well as the purity of the peptides were satisfactory.     

Synthesis of aminobenzyltriethylenetetraaminohexaacetic acid: conjugation of the chelator to protein by an alkylamine linkage

The conjugation of a chelating agent to an antibody as an anchoring site for a radionuclide is the first step in the successful preparation of a radiolabeled antibody for a diagnostic and therapeutic application. The high affinity of the protein bound chelator towards radionuclide ensures a higher selectivity in the delivery of the radionuclide to the targeted tissue. 4-Aminobenzylderivativetriethlenetetraaminohexaacetic acid (TTHA), a hexadentate chelating agent has been now prepared for conjugation with proteins in view of the higher affinity of TTHA metal ions as compared to DTPA. The latent crosslinking potential of -hydroxy aldehydes has been used to conjugate the new chelating agent to proteins through an alkylamine linkage. On incubation of amino benzyl TTHA with glycoladehyde at neutral pH and room temperature, the reagent is converted to oxoethyl amino benzyl TTHA. On addition of albumin to this reaction mixture, the oxo ethylamino benzyl TTHA generates reversible schiff base adducts with the amino groups of albumin. The reduction of the Schiff base adducts of the chelator with the protein by sodium cyanoborohydride stabilizes the schiff base adducts as stable alkylamine linkages. 4-Thiocyanatobenzyl TTHA has also been prepared and conjugated to albumin through a thiocarbamoyl linkage. Both preparations of TTHA conjugated albumin complexed with ^99mTc and ¹¹¹In, with high affinity and no decomposition of the complex was noticed for at least up to 6 hrs after the preparation. The radiolabels complexed with these TTHA -albumin conjugates could not be chased out by free DTPA. A comparison of the biodistribution of ¹¹¹In, bound to the TTHA conjugated through an alkylamine and a thiocarbamoyl linkage showed that ¹¹¹In complexed with alkylamine linked TTHA was retained in blood to a level nearly 17% higher compared to that seen with thicarbamoyl linked TTHA, one hr after the injection into mice. Thus, the alkylamine linkage appears to be more stable under the in vivo conditions. The glycolaldehyde mediated alkylation procedure offers a mild, simple and rapid method for preparation of drug-protein (antibody) conjugates.     

Identification of a GDP-Fuc:Galβ1–3GalNAc-R (Fuc to Gal)α1–2 fucosyltransferase and a GDP-Fuc:Galβ1–4GlcNAc (Fuc to GlcNAc)α1–3 fucosyltransferase in connective tissue of the snailLymnaea stagnalis

Connective tissue of the freshwater pulmonateLymnaea stagnalis was shown to contain fucosyltransferase activity capable of transferring fucose from GDP-Fuc in 1–2 linkage to terminal Gal of type 3 (Gal1–3GalNAc) acceptors, and in 1–3 linkage to GlcNAc of type 2 (Gal1–4GlcNAc) acceptors. The 1–2 fucosyltransferase was active with Gal1–3GalNAc1-OCH₂CH=CH₂ (K _m=12 mM,V _max=1.3 mU ml^–1) and Gal1–3GalNAc (K _m=20 mM,V _max=2.1 mU ml^–1), whereas the 1–3 fucosyltransferase was active with Gal1–4GlcNAc (K _m=23 mM,V _max=1.1 mU ml^–1). The products formed from Gal1–3GalNAc1-OCH₂CH=CH₂ and Gal1–4GlcNAc were purified by high performance liquid chromatography, and identified by 500 MHz¹H-NMR spectroscopy and methylation analysis to be Fuc1–2Gal1–3GalNAc1-OCH₂CH=CH₂ and Gal1–4(Fuc1–3)GlcNAc, respectively. Competition experiments suggest that the two fucosyltransferase activities are due to two distinct enzymes.Abbreviations 2Fuc-T 1–2 fucosyltransferase - 3Fuc-T 1–3 fucosyltransferase - MeO-3Man 3-O-methyl-D-mannose - MeO-3Gal 3-O-methyl-D-galactose     

Proteolytic digestion of α-lactalbumin: Physiological implications

The kinetics of the partial digestion of bovine -lactalbumin (-LA) by trypsin, -chymotrypsin, and pepsin was monitored by lactose synthase activity, HPLC, and difference spectrophotometry. The relative stabilities of the various metal-bound states of -LA to trypsin and chymotrypsin at 37 and 5°C decrease in the following order: Ca(II)--LA>Zn(II), Ca(II)--LA>apo--LA. The HPLC digestion patterns of Ca(II)--LA and Zn(II), Ca(II)--LA at 5 and 37°C were similar, while the corresponding digestion patterns for apo--LA were quite different, reflecting the existence of the thermally induced denaturation states of apo--LA within this temperature region. Occupation of the first Zn(II)-binding site in Ca(II)-loaded -LA slightly alters the HPLC digestion patterns at both temperatures and accelerates the digestion at 37°C due to Zn(II)-induced shift of the thermal transition of -LA, exposing some portion of thermally denatured protein. The results suggest that the binding of Zn(II) to the first Zn(II)- (or Cu(II))-specific site does not cause any drastic changes in the overall structure of -LA. The acidic form of -LA (atpH 2.2 and 37°C) was digested by pepsin at rates similar to that for the apo- or Cu(II), Ca(II)-loaded forms by trypsin or -chymotrypsin at neutralpH. Complexation of -LA with bis-ANS affords protection against pepsin cleavage. It is suggested that the protective effects of similar small lipophilic compounds to -LA may have physiological significance (e.g., for nutritional transport).On leave from the Institute of Biological Physics, USSR Academy of Sciences, Pushchino, Moscow Region, 142292, USSR.     

Evaluation of hydrophobicity versus chaperonelike activity of bovine alphaA- and alphaB-crystallin

Calf lens A-crystallin isolated by reversed-phase HPLC demonstrates a slightly more hydrophobic profile than B-crystallin. Fluorescent probes in addition to bis-ANS, like cis-parinaric acid (PA) and pyrene, show higher quantum yields or Ham ratios when bound to A-crystallin than to B-crystallin at room temperature. Bis-ANS binding to both A- and B-crystallin decreases with increase in temperature. At room temperature, the chaperone-like activity of A-crystallin is lower than that of B-crystallin whereas at higher temperatures, A-crystallin shows significantly higher protection against aggregation of substrate proteins compared to B-crystallin. Therefore, calf lens A-crystallin is more hydrophobic than B-crystallin and chaperone-like activity of -crystallin subunits is not quantitatively related to their hydrophobicity.     

Synthesis of peptides employing Fmoc-/Boc-/Z-amino acid fluorides and activated commercial zinc dust

The coupling of urethane protected amino acidfluorides is accomplished in the presence ofactivated, commercial zinc dust to synthesize severaldi- and tripeptides.The coupling was fast and racemization free. The yieldas well as purity of the peptides was satisfactory.The method was extended for the incorporation ofsterically hindered ,-dialkylaminoacids and N-methylamino acids as well.     

The translation elongation factor 1A in tumorigenesis,signal transduction and apoptosis: Review article

Summary. An increasing number of evidences suggest the involvement of the eukaryotic elongation factor 1A, a core component of the protein synthesis machinery, at the onset of cell transformation. In fact, eEF1A is shown to be up-regulated in cell death; moreover, it seems to be involved in the regulation of ubiquitin-mediated protein degradation. In addition, eEF1A undergoes several post-translational modifications, mainly phosphorylation and methylation, that generally influence the activity of the protein. This article summarizes the present knowledges on the several extra-translational roles of eEF1A also in order to understand as the protein synthesis regulatory mechanisms could offer tools for cancer intervention.     

Isolation of a raw starch-binding fragment from barley alpha-amylase

Barley -amylase was purified by ammonium sulfate fraction, ion-exchange, ultrafiltration, and gel filtration to homogeneity. The purified enzyme was partially digested with trypsin, and the reaction mixture was applied to a cyclohepta-amylose epoxy Sepharose 6B column. Bound fragments were eluted by free cyclohepta-amylose, lyophilized, and separated on Tricine gels. Four fragments were shown to interact with -cyclodextrin. The fragment that could be identified on the gel with the lowest molecular weight (11 kDa) was electroblotted onto PVDF membrane for sequencing. The N-terminal sequence of this fragment was determined with the N-terminal amino acid corresponding to Ala283 in the whole protein. The trypsin cleavage was at Lys282/Ala283 and the C-terminal cleavage occurred at Lys354/Ile355 to give a fragment size of 11 kDa as estimated by SDS-PAGE. The fragment would be located at the C-terminal region, forming a majority of the antiparallel -sheets in domain C and the ₇-and ₈-helices of the (/)₈ domain.     

Subunit Composition of Nicotinic Acetylcholine Receptors in Neurons of the Rat Intracardiac Ganglia

The effect of the antibodies against 3, 4, 5, and 7 subunits of neuronal nicotinic acetylcholine receptors (nAChR) on ACh-induced shifts of the membrane potential (ACh potentials) in neurons of the intracardiac ganglia of the rat left atrium was studied using intracellular electrodes. It was found that (i) antibodies against each of the above subunits caused only a partial block of ACh potential; (ii) the blocking effect of each particular antibody could be observed only in some of the examined neurons; (iii) in all neurons under study the block caused by a single antibody was only partial; (iv) the blocking effects of two or more antibodies on the ACh potential recorded from the same neuron were additive. It was concluded that (1) each of the above nAChR subunits is present only in a part of the intracardiac neurons, and (2) each intracardiac neuron has nAChR composed of more than one subunit.     

Ultrastructural analysis of mineralized matrix from human osteoblastic cells: Effect of tumor necrosis factor-alpha

Recent work by a number of investigators has demonstrated that the process of bone matrix formation and mineralization is under the influence of growth factors and cytokines present in the local environment. Utilizing primary and established osteoblast cell culture systems, these studies have examined the regulation of bone matrix protein synthesis and deposition into the extracellular matrix (ECM) and subsequent mineralization. In previous studies, we have utilized the human osteoblastic cell line, HOS TE85, to study the effects of Tumor Necrosis Factor - alpha (TNF-) on the regulation of matrix proteins and proteolytic function in monolayer cultures as well as during the development and calcification of ECM formed by HOS TE85 cells during extended culture. Our studies demonstrate that TNF- inhibited formation and mineralization of nodules. In the study reported here, we evaluated the ultrastructural morphology of the cell-matrix complex formed by HOS TE85 cells in the presence and absence of TNF- at selected time points during the matrix development process utilizing both transmission electron microscopy and light microscopy. In the presence of TNF-, the cell-matrix complex does not develop normally, with a lack of organization and mineralization, when compared to untreated cells. The lack of mineralization appears to result from the lack of normal collagen fibril deposition and formation of an appropriate ECM essential for the mineralization process. These results support our previous observations that TNF- inhibits HOS TE85 cells from forming a mineralizing ECM by inhibiting incorporation of collagen into the ECM and inducing the synthesis of proteolytic enzymes capable of degrading collagen in the ECM.     

Biological activities of human interferon and 2′–5′ oligoadenylates in plants

Exogenous human interferon 2 (IFN) and 2–5 oligoadenylates (2–5A) have been shown to cause at least a dual physiological effect in tobacco and wheat: (i) increased cytokinin activity and (ii) induced synthesis of numerous proteins, among which members of two groups of stress proteins have been identified, namely pathogenesis-related (PR) and heat shock (HS) proteins. These effects were observed only by low concentrations of these substances: IFN at 0.1–1 u/ml and 2–5A at 1–10 nM.     

Properties of α-galactosidase II2 from Vicia faba seeds

-Galactosidase II² (MW 43 390) from resting Vicia faba L. seeds had been shown to possess d-glucose/d-mannose-specific lectin activity. Inhibition studies with monosaccharides and an examination of the effects of heat and pH on the catalytic and lectin activities of the enzyme indicate that the enzyme substrate and the lectin haptens bind at different sites on the protein. d-Mannosebinding has been investigated by equilibrium dialysis and spectrophotometrically. Both methods yield K_a values of approx. 3·10³ M^-1 for the interaction and there would appear to be two mannosebinding sites per molecule of enzyme protein. The lectin properties of V. faba -galactosidase II² have been discussed in relation to both V. faba lectin (favin) and other legume -galactosidases.Abbreviations con A concanavalin A - CM-cellulose carboxymethyl cellulose - MW molecular weight - PNPG p-nitrophenyl -d-galactoside - SDS sodium dodecyl sulphate - PAGE polyacrylamide-gel electrophoresis     

The association of heterotrimeric GTP-binding protein (Go) with microtubules

The heterotrimeric GTP-binding regulatory proteins (G proteins) play an important role in the regulation of membrane signal transduction. Recently, we identified the association of Go protein with mitotic spindles. Here we have investigated the relationship between Go protein and microtubules. We used temperature-dependent reversible assembly and taxol methods to purify microtubules from bovine brains. Goalpha and Gbeta proteins were identified in the microtubular fraction by both methods. The Goalpha subunit in the microtubular fraction could be ADP ribosylated by pertussis toxin. Co-immunoprecipitation data also revealed that Go protein can interact with microtubules. Exogenous Go protein could be incorporated into the assembled microtubular fraction, and 5 microg/ml (60 nM) of Go protein inhibited 40% of microtubule assembly. Western blot analysis of Goalpha-1 and Goalpha-2 in microtubular fractions showed that only Goalpha-1 is associated with microtubules. We conclude that the Goalpha-1betagamma proteins are associated with microtubules and may play some role in regulating the assembly and disassembly of microtubules.