Amino Acids (v.26, #1)

Recently agmatine, decarboxylated arginine, has been shown to be an important biological compound in several animal tissues. This paper summarizes the known information regarding the transport of arginine, its decarboxylation and the effects of the agmatine formed mainly on NO and polyamine synthesis.
Keywords: Keywords: Arginine – Agmatine – Arginine decarboxylase – Nitric oxide – Polyamines

The human genome maybe limited to about 30 000 genes whereas the human proteome may be represented by a rough estimate of one million proteins. A legion of proteins have been described and information about these structures are readily available in data banks. There remains, however, a large series of unknown or hypothetical proteins (HPs). Many of them have been predicted from nucleic acid sequences only and are therefore named predicted or HPs. Carrying out “protein hunting” by generating large maps of human cell lines, we aimed to find and identify HPs and provide an analytical tool thereof.Cell lysates from human bronchial epithelial, fibroblast, amnion, lymphocyte, mesothelial and kidney cell lines were prepared and proteins run on two-dimensional gel-electrophoresis (2DE) with in-gel digestion and mass spectrometrical analysis using the MALDI-TOF principle.16 HPs were found in these cell lines and some show cell-specific expressional patterns. HPs belong to several protein classes including structural, signaling, transcriptional/translational, chaperone-related and others. We furthermore provide analytical data i.e. pIs that were often different from predicted values in data banks.A list of HPs has been shown to really exist in several human cell lines thus contributing to knowledge on protein machineries and cascades. Observed and predicted pI values are given representing an analytical tool along with unambiguous identification of protein spots by mass spectrometry independent of antibody availability and specificity thus complementing established methods.
Keywords: Keywords: Hypothetical proteins – Structural proteins – Signaling proteins – Transcriptional/translational proteins – Chaperone related proteins

L-3,4-dihydroxyphenylalanine (L-dopa) transport into human Langerhans cells (LC) occurs by a saturable mediation. This plasma membrane agency is, due to its characteristics, distinguishable from systems transporting other neutral, cationic and anionic amino acids into other cells and serves to catalyze the flow of L-dopa, only, into LC. The uphill operation of this L-dopa transport system is believed to occur by down-gradient countermigration of H+. Due to the uniqueness of the L-dopa transport system, the widely used analogue inhibition approach was not applicable. Instead we studied omeprazole and its analogues in our search for suitable inhibitory candidates. Omeprazole and most of its analogues were indeed inhibitory in the concentration range 1–100 μmol/L. Conspicuously, the compounds with strongest polarity were least inhibitory. The inhibitory pattern displayed by omeprazole and the other analogues on L-dopa uptake in LC corresponded to some extent to what has been observed previously for purified H+,K+-ATPase from tubulovesicles of the stomach. No effects of the inhibitors were registered on energy charge and lactate production of epidermal biopsies, nor were any gross alterations of ultrastructure of LC noticed.
Keywords: Keywords: Epidermis – Omeprazole analogues – Langerhans cells – L-dopa transport – Lactate – Energy charge

Fractionation of liver proteins by preparative electrophoresis by M. Fountoulakis; J.-F. Juranville; G. Tsangaris; L. Suter (27-36).
Proteomics offers unique possibilities to investigate changes in the levels and modifications of proteins involved in the pathomechanisms of diseases and toxic events. However, search for potential drug targets and disease or toxicity markers is limited by the fact that mainly the high-abundance, hydrophilic proteins are visualized in two-dimensional gels. Here we studied the enrichment of rat liver cytosolic proteins by preparative electrophoresis. Preparative electrophoresis was performed with the PrepCell apparatus in the presence of 0.1% lithium dodecyl sulfate. Lithium dodecyl sulfate was exchanged against agents compatible with isoelectric focusing prior to the two-dimensional gel electrophoresis. Proteins were identified from two-dimensional gels by matrix-assisted laser desorption ionization time-of-flight mass specrometry. Low- and middle-size proteins and low-abundance proteins, which had not been found before, were enriched by preparative electrophoresis. The present study represents a contribution of proteomics in the quantification of differences in the levels of low-abundance liver proteins in toxicity studies.
Keywords: Keywords: Proteomics – Preparative electrophoresis – Liver – Low-abundance proteins – Mass spectrometry

Nitric oxide is not involved in the control of vasopressin release during acute forced swimming in rats by Dr. Mario Engelmann; G. Wolf; J Putzke; F. E. Bloom; J. Raber; R. Landgraf; M. G. Spina; T. F. W. Horn (37-43).
Neurons of the hypothalamo-neurohypophyseal system (HNS) are known to contain high amounts of neuronal nitric oxide (NO) synthase (nNOS). NO produced by those neurons is commonly supposed to be involved as modulator in the release of the two nonapeptides vasopressin (AVP) and oxytocin into the blood stream. Previous studies showed that forced swimming fails to increase the release of AVP into the blood stream while its secretion into the hypothalamus is triggered. We investigated here whether hypothalamically acting NO contributes to the control of the AVP release into blood under forced swimming conditions. Intracerebral microdialysis and in situ hybridization were employed to analyze the activity of the nitrergic system within the supraoptic nucleus (SON), the hypothalamic origin of the HNS. A 10-min forced swimming session failed to significantly alter the local NO release as indicated both by nitrite and, the main by-product of NO synthesis, citrulline levels in microdialysis samples collected from the SON. Microdialysis administration of NO directly into the SON increased the concentration of AVP in plasma samples collected during simultaneous forced swimming. In an additional experiment the effect of the defined stressor exposure on the concentration of mRNA coding for nNOS within the SON was investigated by in situ hybridization. Forced swimming increased the expression of nNOS mRNA at two and four hours after onset of the stressor compared to untreated controls. Taken together, our results imply that NO within the SON does not contribute to the regulation of the secretory activity of HNS neurons during acute forced swimming. Increased nNOS mRNA in the SON after forced swimming and the increase in AVP release in the presence of exogenous NO under forced swimming points to a possible role of NO in the regulation of the HNS under repeated stressor exposure.
Keywords: In situ hybridization; Microdialysis; Hypothalamoneurohypophyseal system; Stress

An alteration of low density lipoprotein (LDL) apolipoprotein (apo) B-100 structure by direct oxidative modification is an important mechanism involved in atherogenesis. There is difficulty in quantifying this type of modification because a lack of specific assays. The use of N(O,S)-ethoxycarbonyl trifluoroethyl amino acid esters for a rapid and sensitive determination of 6-hydroxy-2-aminocaproic acid (HACA), a highly specific marker of metal catalyzed protein oxidation, by using standard gas chromatography/electron impact mass spectrometry, is discussed. The derivatives are formed by the unlabored reaction of amino acids with ethylchloroformate plus trifluoroethanol plus pyridine. Femtomole levels of HACA can be reproducible measured in different LDL preparations subjected to oxidative damage in the presence of iron or copper. HACA determination compares well with the measurement of carbonyl groups that are generally accepted as a nonspecific index of protein oxidation. Thus, the method could prove to be a sensitive assay for studying specific apoB-100 modification.
Keywords: Keywords: Amino acids – Hemin – Low density lipoprotein – Protein oxidations – Gas chromatography/mass spectrometry

Gender-related differences in carnosine, anserine and lysine content of murine skeletal muscle by R. Peñafiel; C. Ruzafa; F. Monserrat; A. Cremades (53-58).
The aminoacyl-imidazole dipeptides carnosine (β-alanyl-L-histidine) and anserine (β-alanyl-1-methyl-histidine) are present in relatively high concentrations in excitable tissues, such as muscle and nervous tissue. In the present study we describe the existence of a marked sexual dimorphism of carnosine and anserine in skeletal muscles of CD1 mice. In adult animals the concentrations of anserine were higher than those of carnosine in all skeletal muscles studied, and the content of aminoacyl-imidazole dipeptides was remarkably higher in males than in females. Postnatal ontogenic studies and hormonal manipulations indicated that carnosine synthesis was up-regulated by testosterone whereas anserine synthesis increased with age. Regional variations in the concentrations of the dipeptides were observed in both sexes, skeletal muscles from hind legs having higher amounts of carnosine and anserine than those present in fore legs or in the pectoral region. The concentration of L-lysine in skeletal muscles also showed regional variations and a sexual dimorphic pattern with females having higher levels than males in all muscles studied. The results suggest that these differences may be related with the anabolic action of androgens on skeletal muscle.
Keywords: Keywords: Carnosine – Anserine – Lysine – Skeletal muscle – Mice – Sex dimorphism

Effects of taurine supplementation on VDT work induced visual stress by M. Zhang; L. F. Bi; Y. D. Ai; L. P. Yang; H. B. Wang; Z. Y. Liu; M. Sekine; S. Kagamimori (59-63).
In order to evaluate the effects of dietary taurine supplementation on visual fatigue induced by visual display terminals (VDT) work, 25 male college students aged from 20 to 24 years who were not engaged in VDT work were selected to participate in the study. Volunteers were randomly assigned to either the taurine supplementation (n=13) or the placebo supplementation control group (n=12). Before and after 12 days of taurine (3 g/day) or placebo supplementation, two identical 2.5-hr VDT work tests were performed while recording the P100, N75 and N145 latencies and P100 amplitude of pattern visual evoked potential (PVEP) and the frequency of critical flicker fusion (CFF). Following 2.5-hr of VDT work, the P100 and N75 latencies of PVEP increased (P<0.01) while the P100 amplitude decreased significantly (P<0.01). The frequency of CFF also reduced significantly (P<0.01). After 12 days of taurine supplementation, the reduction in P100 amplitude after VDT work alleviated significantly (P<0.05). The results suggest that taurine supplementation alleviates visual fatigue induced by VDT work.
Keywords: Keywords: Taurine – VDT work – Visual fatigue – Pattern visual evoked potential – Critical flicker fusion

Depleting intracellular taurine in heart cells improves their resistance to ischemia and reperfusion injury. The aim of this work was to see whether physiologically low levels of endogenous taurine also reflect a reduced vulnerability of the myocardium to cardiac insults. The myocardial concentration of taurine was measured during different stages of development and compared with vulnerability to ischemia and reperfusion injury in the rat and in pediatric patients undergoing cardiac surgery.Rat hearts with relatively lower levels of taurine were significantly more resistant to an ischemic inult and there was a strong negative correlation between taurine content and recovery. Children’s hearts had significantly lower taurine levels compared to infants’ hearts which was consistent with their known increased resistance to an ischemic cardioplegic insult (Imura et al., 2001). This work shows that the changes in the concentration of myocardial taurine during development correlate with vulnerability to ischemia where low myocardial taurine is associated with improved recovery upon reperfusion.
Keywords: Keywords: Taurine – Heart – Development – Ischemia

Plasma amino acids and sports injuries by M. T. M. van den Baar; D. Fekkes; C. R. van den Hoogenband; H. J. Duivenvoorden; L. Pepplinkhuizen (71-76).
The aim of this study was to explore the relationship between changes in plasma amino acids and the incidence of sports injuries during a soccer season. Fourteen plasma amino acids were assayed at monthly intervals in 12 young soccer players during a whole soccer season. Based on the number and severity of injuries the soccer players were divided into an injury-prone and a non-injury-prone group. The mean plasma level of the amino acid glycine was significantly lower (P=0.009) in the injury-prone group than the other group, while the mean plasma levels of tyrosine, tryptophan and lysine were higher in the injury-prone group during this period (P<0.05). However there were no significant differences in the calculated plasma tryptophan and tyrosine/large neutral amino acids ratios. Significant linear time trends were observed for taurine, ornithine, lysine and the tryptophan/large neutral amino acids ratio.These results indicate that the plasma concentrations of glycine and to a lesser extent those of tyrosine, tryptophan and lysine may be promising peripheral markers for injury-proneness in young soccer players. Whether a role for glycine substitution will be indicative to reduce the occurrence of sports injuries will need to be investigated in future studies.
Keywords: Keywords: Amino acids – Glycine – Tryptophan – Tyrosine – Injury-prone – Plasma

An immunocytochemical investigation of the expression of α1, α6, β2/3, γ2 and δ subunits was performed on rat cerebellum granule cells in culture by the two photon microscopy technique.The first four subunits appear to be expressed abundantly in these cells, whereas the δ one seems to be expressed at a lower level. Another major difference in the distribution of these subunits is that whereas α6, β2/3 and γ2 appear only on plasma membranes α1 and δ are present mainly in the cell bodies cytoplasm. Still another difference was found in that the presence of γ2 on neurites is “polarized”, preferentially labelling neurites with the appearance of dendrites. The subunits α6 and β2/3 appear to label all types of neurites, with β2/3 being by far the most heavily expressed subunit type. A final distinct characteristic is that α6 and, even more, γ2 appear to accumulate in the cytoplasmic domains immediately below the cone of emergence of neurites. This suggests a conspicuous transport of such subunits from the site of synthesis in the cell body to the site of final expression in the neurites (dendrites and axon terminals).
Keywords: Keywords: Fluorescence microscopy – Immunocytochemistry – GABAA receptors – Subunits – Cerebellar granules – In vitro

L-Histidine is a beneficial adjuvant for antiepileptic drugs against maximal electroshock-induced seizures in mice by R. M. Kamiński; D. Żółkowska; M. Kozicka; Z. Kleinrok; S. J. Czuczwar (85-89).
Endogenous histamine has been reported to be involved in regulation of seizure susceptibility. Enhancement of histamine neurotransmission engendered by L-histidine treatment produces anticonvulsant effects in experimental animals. The present study investigated the influence of L-histidine on the protective effects of carbamazepine and phenytoin against maximal electroshock-induced seizures in mice.L-Histidine, administered at the doses that did not influence the threshold for electroconvulsions (250–500 mg/kg), enhanced by nearly 30% the protective effects of carbamazepine against maximal electroshock-induced seizures. D-Histidine (1000 mg/kg), an inactive isomer of histidine, was without any effect in this regard. L-Histidine (500 mg/kg) also augmented the protective effects of phenytoin. Importantly, the enhancement of the anticonvulsant effects of these antiepileptic drugs produced by L-histidine co-administration was not associated with augmentation of their unwanted effects on memory and motor performance. A pharmacokinetic interaction was also excluded since the free plasma levels of these antiepileptics remained unchanged in the presence of L-histidine. It may be suggested that L-histidine could serve as a beneficial adjuvant for selected antiepileptic drugs.
Keywords: Keywords: L-Histidine – Histamine – Antiepileptic drugs – Mice – Epilepsy

The release of the inhibitory neuromodulator taurine in the hippocampus is markedly enhanced under various neural cell-damaging conditions, including ischemia and exposure to free radicals. The properties and regulation of the release evoked by a medium containing free radicals was investigated in hippocampal slices from adult (3-month-old) and developing (7-day-old) mice, using a superfusion system. The ‘free radical damage’ was induced by applying 0.01% H2O2. The release of [3H]taurine was in both adult and developing hippocampus partly Ca2+-independent, mediated by Na+-dependent transporters and probably resulting from disruption of cell membranes and subsequent ion imbalance. The release in developing mice appeared to be more susceptible to regulation than that in adults, the stimulation by free radicals being in the latter already maximal. The release was reduced by adenosine A1 receptor agonist R(−)N6-(2-phenylisopropyl)adenosine, which effect was, however, abolished by the antagonist 8-cyclopentyl-1,3-dipropylxanthine only in the immature hippocampus, indicating a receptor-mediated process. Moreover, the evoked taurine release in developing mice was potentiated by the ionotropic glutamate receptor agonists N-methyl-D-aspartate, kainate and 2-amino-3-hydroxy-5-methyl-4-isoxazolepropionate in a receptor-mediated manner, since the effects were abolished by their respective antagonists. The metabotropic glutamate receptors are of only minor significance in the release, the agonists of group I and II receptors slightly reducing the release. Furthermore, NO may also be involved in this release, the NO-generating compounds hydroxylamine and S-nitroso-N-acetylpenicillamine being able to enhance the free-radical-evoked release. It seems that the free-radical-stimulated release, potentiated by ionotropic glutamate receptor activation and NO production, could constitute part of the neuroprotective properties of taurine, being important particularly in the developing hippocampus and hence preventing excitotoxicity.
Keywords: Keywords: Hippocampal slices – Developing and adult mice – Taurine release – Free radicals – Glutamate receptors – Adenosine receptors

Evidence for expression of a single distinct form of mammalian cysteine dioxygenase by M. H. Stipanuk; M. Londono; L. L. Hirschberger; C. Hickey; D. J. Thiel; L. Wang (99-106).
Cysteine dioxygenase (CDO) plays a critical role in the regulation of cellular cysteine concentration. Because multiple forms of CDO (∼23 kDa, ∼25 kDa, and ∼68 kDa) have been claimed based upon separation and detection using SDS-PAGE/western blotting (with antibodies demonstrated to immunoprecipitate CDO), we further investigated the possibility of more than one CDO isoform. Using either rabbit antibody raised against purified rat liver CDO or against purified recombinant his6-tagged CDO (r-his6-CDO) and using 15% (wt/vol) polyacrylamide for the SDS-PAGE, we consistently detected the ∼25 kDa band, but never detected a ∼68 kDa band, in rat liver, kidney, lung and brain. Nondenatured gel electrophoresis of r-his6-CDO yielded a molecular mass estimate of 25.7 kDa and no evidence of dimerization. Mass spectrometry of r-his6-CDO yielded two peaks with molecular masses of 24.1 kDa and 24.3 kDa. Anion-exchange FPLC of r-his6-CDO also gave two peaks, with the first containing CDO that was 7.5-times as active as the more anionic form that eluted second. When the two peaks recovered from FPLC were run on SDS/PAGE, the first (more active) CDO fraction yielded two bands (perhaps as an artifact of SDS/PAGE), whereas the second (less active) CDO fraction yielded only the ∼23 kDa band. We conclude that the physiologically active form of CDO is the ∼25 kDa (i.e., 23.5 kDa based on mass spectrometry) monomer and that this active form is probably derived by post-translational modification of the 23 kDa gene product.
Keywords: Keywords: Cysteine dioxygenase – Cysteine – Isozymes – Molecular mass