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Amino Acids: The Forum for Amino Acid, Peptide and Protein Research (v.31, #4)

On postnatal CNS plasticity by M. Herrera-Marschitz (pp. 357-358).

Subchronic-intermittent caffeine amplifies the motor effects of amphetamine in rats by N. Simola; E. Tronci; A. Pinna; M. Morelli (pp. 359-363).

Caffeine, the most widely consumed psychostimulant drug, acutely stimulates motor behaviour and enhances dopamine agonists actions whilst chronically it induces tolerance to either caffeine- or dopamine agonist-induced motor activating effects. The present study examined whether subchronic caffeine administration (15 mg/kg, on alternate days for 14 days) induces enduring modifications in caffeine- and amphetamine-mediated motor activity. To this end, motor activation and rotational behaviour stimulated by either caffeine or D-amphetamine (0.5, 2 mg/kg), given 3 days after the last caffeine administration, were evaluated in neurologically intact and unilaterally 6-hydroxydopamine-lesioned rats respectively. Subchronic caffeine resulted in an increase in caffeine-induced motor and turning behaviour. Furthermore, caffeine pretreatment potentiated the motor effects of amphetamine in both intact and 6-hydroxydopamine-lesioned rats. These results suggest that subchronic caffeine treatment results in an enhancement of its motor stimulant effects, rather than in tolerance, and induces neuroadaptive facilitatory changes in dopamine transmission.

Keywords: Keywords: Caffeine – Amphetamine – Motor behaviour

The 5-HT_1A receptor active compounds (R)-8-OH-DPAT and (S)-UH-301 modulate auditory evoked EEG responses in rats by K. E. Stevens; H. C. O’Neill; G. M. Rose; J. Luthman (pp. 365-375).

Schizophrenics commonly demonstrate abnormalities in central filtering capability following repetitive sensory stimuli. Such sensory inhibition deficits can be mirrored in rodents following administration of psycho-stimulatory drugs. In the present study, male Sprague-Dawley rats were implanted with brain surface electrodes to record auditory evoked EEG potentials in a paired-stimulus paradigm, using 87 dB clicks delivered 0.5 s apart. Amphetamine (1.83 mg/kg, i.p.) produced the expected loss of sensory inhibition, as defined by an increase in the ratio between test (T) and conditioning (C) amplitudes at N40, a mid-latency peak of the evoked potentials. Also, the 5-HT_1A agonist (R)-8-OH-DPAT caused a significant increase in the TC ratio at the highest dose studied (0.5 mg/kg s.c.), while the 5-HT_1A antagonist (S)-UH-301 did not significantly affect the TC ratio at any dose studied (0.1–5 mg/kg s.c.). When administered with amphetamine, a lower dose of 8-OH-DPAT (0.1 mg/kg) and the highest dose of UH-301 tested (5 mg/kg, s.c.) were able to reverse the amphetamine-induced increase in TC ratio. The findings suggest that 5-HT_1A signaling is involved in sensory inhibition and support the evaluation of 5-HT_1A receptor active compounds in conditions with central filtering deficits, such as schizophrenia.

Keywords: Keywords: Schizophrenia – Sensory inhibition – Auditory evoked potentials – Serotonin – 5-HT_1A receptor – UH-301 – 8-OH-DPAT

Plasticity of the central nervous system (CNS) following perinatal asphyxia: Does nicotinamide provide neuroprotection? by V. Klawitter; P. Morales; D. Bustamante; M. Goiny; M. Herrera-Marschitz (pp. 377-384).

We have investigated the idea that nicotinamide, a non-selective inhibitor of the sentinel enzyme Poly(ADP-ribose) polymerase-I (PARP-1), provides neuroprotection against the long-term neurological changes induced by perinatal asphyxia. Perinatal asphyxia was induced in vivo by immersing foetuses-containing uterine horns removed from ready-to-deliver rats into a water bath for 20 min. Sibling caesarean-delivered pups were used as controls. The effect of perinatal asphyxia on neurocircuitry development was studied in vitro with organotypic cultures from substantia nigra, neostriatum and neocortex, platted on a coverslip 3 days after birth. After approximately one month in vitro (DIV 25), the cultures were treated for immunocytochemistry to characterise neuronal phenotype with markers against the N-methyl-D-aspartate receptor subunit 1 (NR1), the dopamine pacemaker enzyme tyrosine hydroxylase (TH), and nitric oxide synthase (NOS), the enzyme regulating the bioavailability of NO. Nicotinamide (0.8 mmol/kg, i.p.) or saline was administered to asphyctic and caesarean-delivered pups 24, 48 and 72 h after birth.It was found that nicotinamide treatment prevented the effect of perinatal asphyxia on several neuronal parameters, including TH- and NOS-positive neurite atrophy and NOS-positive neuronal loss; supporting the idea that nicotinamide constitutes a therapeutic alternative for the effects produced by sustained energy-failure conditions, as occurring during perinatal asphyxia.

Keywords: Keywords: Perinatal asphyxia – Basal ganglia – Nicotinamide – Poly (ADP-ribose) polymerase-1 (PARP-1) – Organotypic cultures – Neuroprotection – Rat

Long-term effect of moderate and profound hypothermia on morphology, neurological, cognitive and behavioural functions in a rat model of perinatal asphyxia by H. Hoeger; E. Engidawork; D. Stolzlechner; H. Bubna-Littitz; B. Lubec (pp. 385-396).

Background. Perinatal asphyxia is a frequent cause of neurological handicap with no known therapy. However, hypothermic therapy has recently attracted attention owing to its neuroprotective property in brain of immature organisms. Objectives. Hypothermia appears to be promising in reversing the immediate effect of perinatal asphyxia, but data on long-term neuroprotection is still lacking. We therefore intended to test the long-term effect of moderate and profound hypothermia on brain morphology and functions using a well established rat model of perinatal asphyxia. Methods. Rat pups delivered by caesarean section were placed into a water bath, still in patent membranes, at 37 °C and variable hypothermic conditions to induce asphyxia and thereafter given to surrogate mothers. Examinations were performed at the age of three months, consisting of a battery of motor, behavioural, cognition and reflex tests including rota-rod, Morris water maze, multiple T-maze, elevated plus maze and open field studies. Morphological alterations were evaluated by Nissl staining of brain areas known to be hypoxia sensitive. Neurotransmission system markers, including tyrosine hydroxylase, vesicular monoamine transporter, vesicular acetylcholine transporter and excitatory amino acid carrier1 were analyzed by immunohistochemistry. Results. Survival increased with hypothermia. The Nissl stain revealed neuronal loss in hippocampus and hypothalamus of normothermic asphyxiated group (20/37) compared to controls (0/37), but no neuroprotective patterns emerged from hypothermia. An overall inconsistent protection of the neural systems was noted by variable periods of hypothermia. Motor function was significantly impaired in 20/37 as compared to 0/37. In the Morris water maze and multiple T-maze, results were comparable between the groups. In the elevated plus maze, time spent in the closed arm was reduced and in the open field, vertical behaviour was altered in the 20/37 group with horizontal motor behaviour being unaffected. Hypothermia reversed all abnormalities seen in 20/37, with short-term moderate and profound hypothermia being superior to long-term hypothermia. Conclusion. Hypothermia not only significantly increased survival, but also resulted in unimpaired motor as well as improved cognitive functions. Those findings are in contrast to altered brain morphology. As neuronal loss was present in various brain regions, we conclude that deficits may be compensated in the maturing animal. Intrahypoxic hypothermia was able to protect the rat from the devastating effect of perinatal asphyxia not in morphological, but in functional terms.

Keywords: Keywords: Hypothermia – Perinatal asphyxia – Neurotransmitter markers

Preparation and characterization of novel pyrrol-3-ones attached to α/β-amino acids, esters and amides by M. Saçmacı; Ş. H. Üngören; Y. Akçamur (pp. 397-401).

Various α/β amino acid derivatives 5 were attached to compounds 3 to yield 2,3-dihydro-1H-pyrrol-3-ones amino acids derivatives 6. This rare heterocyclic amino acid skeleton including the pyrrolo[1,2-b][1,3]oxazol moiety was also successfully prepared in the esteric form. The structure of the new compounds was characterized by spectroscopic methods.

Keywords: Keywords: Pyrrol-3-ones – Amino acids – Amino acid esters – Amino acid amides – Pyrrolo[2,1-b][1,3]oxazole – Furan-3(2H)-ones

Isotopic effects in the electronic spectra of tryptophan by Yu. A. Zolotarev; Yu. A. Borisov; A. K. Dadayan; N. F. Myasoedov (pp. 403-407).

No influence of isotopic substitution in deuterium-substituted tryptophan on the florescence excitation spectrum has previously been found out. Here, the isotopic effects of electronic excitation of deuterium-substituted tryptophan were experimentally and theoretically analyzed for first time. It was shown a short-wave shift of the UV-absorption maximum at 220 nm corresponding to the 360 cal/mol and short-wave shift for fluorescence spectrum corresponding to the 210 cal/mol. To account for this effect, the quantum chemical calculations of the geometric and electron structure, frequencies of normal vibrations and transition energies have been performed. The isotopic effects originate from the zero-point energies of ground and excited states. It was found that isotopic shifts depend on the position of isotope in the molecule and kind of transition. So, it can be utilized in the analysis of proteins structure and complexation.

Keywords: Keywords: Fluorescence – Phosphorescence – Isotopic effects – Electronic transitions – Tryptophan

Laccase-induced derivatization of unprotected amino acid L-tryptophan by coupling with p-hydroquinone 2,5-dihydroxy-N-(2-hydroxyethyl)-benzamide by K. Manda; E. Hammer; A. Mikolasch; D. Gördes; K. Thurow; F. Schauer (pp. 409-419).

We have studied the enzymatic derivatization of amino acids by use of the polyphenol oxidase laccase. Derivatization of L-tryptophan was achieved by enzymatic crosslinking with the laccase substrate 2,5-dihydroxy-N-(2-hydroxyethyl)-benzamide. The main product (yield up to 70%) was identified as the quinoid compound 2-[2-(2-hydroxy-ethylcarbamoyl)-3,6-dioxo-cyclohexa-1,4-dienylamino]-3-(1H-indol-3-yl)- propionic acid and demonstrates that laccase-catalyzed C–N-coupling occurred on the amino group of the aliphatic side chain. These enzyme based reactions provide a simple and fast method for the derivatization of unprotected amino acids.

Keywords: Keywords: Laccase – Cross-coupling – Derivatisation – Tryptophan – Unprotected amino acid – Pycnoporus cinnabarinus

Long-term tryptophan administration enhances cognitive performance and increases 5HT metabolism in the hippocampus of female rats by S. Haider; S. Khaliq; S. P. Ahmed; D. J. Haleem (pp. 421-425).

It has been shown in various studies that increase in serotonergic neurotransmission is associated with increased memory consolidation whereas low brain 5HT impairs memory performance. In the first phase of our study we found that tryptophan (TRP) administration for 6 weeks increased plasma TRP and whole brain TRP, 5HT and 5HIAA levels. Many brain regions are involved in the learning process but particularly the hippocampus is known to have key role in learning and memory.The present study was therefore designed to investigate the effects of TRP loading particularly on hippocampal 5HT metabolism and cognitive performance in rats. TRP-treated rats demonstrated spatial enhancement as evidenced by a significant decrease in time to find the hidden food reward in radial arm maze test (RAM). The important finding of the present study was the greater increase in the 5HT metabolism in hippocampus than in any other brain region of the TRP-treated rats. This increased 5HT metabolism in the hippocampus emphasizes the involvement of this region in memory process.

Keywords: Keywords: Tryptophan – Memory – Hippocampus – Radial arm maze

Domino reactions with fluorinated five-membered heterocycles. α-Trifluoromethyl α-amino acids with unsaturated side-chains by K. Burger; L. Hennig; P. Tsouker; J. Spengler; F. Albericio; B. Koksch (pp. 427-433).

α-Trifluoromethyl α-amino acids with unsaturated side-chains have been prepared from 5-fluoro-4-trifluoromethyloxazole and allyl, propargyl as well as terpene alcohols in a one-pot procedure.

Keywords: Keywords: 5-Fluoro-4-trifluoromethyloxazoles – Allyl alcohols – Propargyl alcohols – Geraniol – Farnesol – Nucleophilic aromatic substitution – Claisen rearrangement – α-Trifluoromethyl α-amino acids – Secondary structure

Mass-spectrometrical analysis of proteins encoded on chromosome 21 in human fetal brain by J.-H. Shin; K. Krapfenbauer; G. Lubec (pp. 435-447).

Overexpression of chromosome 21 genes is directly or indirectly responsible for the Down syndrome phenotype. In order to analyse chromosome 21 gene products (Chr21Ps), we extracted proteins from fetal human brain cortex and applied an ultracentrifugal and chromatographic prefractionation principle followed by two-dimensional gel electrophoresis (2-DE) and mass-spectrometrical analysis using high-throughput automated MALDI-TOF/TOF. Nine Chr21Ps were identified: pyridoxal kinase; superoxide dismutase [Cu/Zn] 1; carbonyl reductase 1; ES1 protein homolog, mitochondrial [Precursor]; cystathionine-beta-synthetase; T-complex protein 1, theta subunit; cystatin B; 6-phosphofructokinase; glycinamide ribonucleotide synthetase. Mass-spectrometric characterisation of Chr21Ps following separation in 2-DE gels is a useful tool for the analysis of these structures in brain, independent of antibody availability and specificity.

Keywords: Keywords: Human fetal brain – Human chromosome 21 – Ion-exchange chromatography – Two-dimensional gel electrophoresis – MALDI-TOF/TOF

Brain G protein-dependent signaling pathways in Down syndrome and Alzheimer’s disease by M. Lumbreras; C. Baamonde; C. Martínez-Cué; G. Lubec; N. Cairns; J. Sallés; M. Dierssen; J. Flórez (pp. 449-456).

Premature aging and neuropathological features of Alzheimer’s disease (AD) are commonly observed in Down syndrome (DS). Based on previous findings in a DS mouse model, the function of signaling pathways associated with adenylyl cyclase (AC) and phospholipase C (PLC) was assessed in cerebral cortex and cerebellum of age-matched adults with DS, AD, and controls. Basal production of cAMP was reduced in DS but not in AD cortex, and in both, DS and AD cerebellum. Responses to GTPγS, noradrenaline, SKF 38393 and forskolin were more depressed in DS than in AD cortex and cerebellum. Although no differences in PLC activity among control, DS and AD cortex were observed under basal and GTPγS- or Ca-stimulated conditions, the response of DS cortex to serotonergic and cholinergic stimulation was depressed, and that of AD was only impaired at cholinergic stimulation. No differences were documented in cerebellum. Our results demonstrate that PLC and AC were severely disturbed in the aged DS and AD brains, but the alterations in DS were more severe, and differed to some extent from those observed in AD.

Keywords: Keywords: Adenylyl cyclase – Phospholipase C – Cerebral cortex – Cerebellum – Down syndrome – Alzheimer’s disease

Effects of glucocorticoids on polyamine metabolism in liver and spleen of guinea pig during sensitization by G. Bjelakovic; D. Pavlovic; I. Stojanovic; T. Jevtovic; J. Nikolic; G. Kocic (pp. 457-462).

Glucocorticoids are potent anti-inflammatory and immunosuppressive agents. As endogenous inhibitors of cytokine synthesis, glucocorticoids suppress immune activation and uncontrolled overproduction of cytokines, preventing tissue injury. Also, polyamine spermine is endogenous inhibitor of cytokine production (inhibiting IL-1, IL-6 and TNF synthesis). The idea of our work was to examine dexamethasone effects on the metabolism of polyamines, spermine, spermidine and putrescine and polyamine oxidase activity in liver and spleen during sensitization of guinea pigs. Sensitization was done by application of bovine serum albumin with addition of complete Freund’s adjuvant. Our results indicate that polyamine amounts and polyamine oxidase activity increase during immunogenesis in liver and spleen. Dexamethasone application to sensitized and unsensitized guinea pigs causes depletion of polyamines in liver and spleen. Dexamethasone decreases polyamine oxidase activity in liver and spleen of sensitized guinea pigs, increasing at the same time PAO activity in tissues of unsensitized animals.

Keywords: Keywords: Polyamines – Polyamine oxidase – Dexamethasone – Sensitization – Liver – Spleen – Guinea pigs

The relationship between albumin, other plasma proteins and variables, and age in the acute phase response after liver resection in man by I. Giovannini; C. Chiarla; F. Giuliante; M. Vellone; F. Ardito; G. Nuzzo (pp. 463-469).

A large series of plasma albumin (ALB, g/dl) and simultaneous blood and clinical measurements were prospectively performed on 92 liver resection patients, and processed to assess the correlations between ALB, other plasma proteins, additional variables and clinical events. The measurements were performed preoperatively and at postoperative day 1, 3 and 7 in all patients, and subsequently only in those who developed complications or died. In patients who recovered normally ALB was 4.3 ± 0.4 g/dl (mean ± SD) preoperatively, 3.7 ± 0.7 at day 1 and 3, and 3.9 ± 0.4 at day 7. In patients with complications its decrease was more prolonged. In non-survivors it was 3.4 ± 0.4 preoperatively, 3.0 ± 0.4 at day 1, and then decreased further. Regression analysis showed direct correlations between ALB and pseudo-cholinesterase (CHE, U/l, nv 5300-13000), cholesterol (CHOL, mg/dl), iron binding capacity (IBC, mg/dl), prothrombin activity (PA, % of standard reference) and fibrinogen, an inverse correlation with blood urea nitrogen (BUN, mg/dl) for any given creatinine level (CREAT, mg/dl), and weaker direct correlations with hematocrit, other variables and dose of exogenous albumin. An inverse relationship found between ALB and age (AGE, years) became postoperatively (POSTOP) also a function of outcome, showing larger age-related decreases in ALB associated with complications (COMPL: sepsis, liver insufficiency) or death (DEATH). Main overall correlations: CHE = 287.4(2.014)^ALB, r = 0.73; CHOL = 16.5(1.610)^ALB (1.001)^ALKPH, r = 0.71; IBC = 68.6(1.391)^ALB, r = 0.64; PA = 13.8 + 16.0(ALB), r = 0.51; BUN = 21.3 + 20.2(CREAT) – 6.2(ALB), r = 0.91; ALB = 5.0–0.013(AGE) – {0.5 + 0.003(AGE) COMPL + 0.012(AGE) DEATH} POSTOP, r = 0.74 [p < 0.001 for each regression and each coefficient; ALKPH = alkaline phosphatase, U/l, nv 98-279, independent determinant of CHOL; discontinuous variables in italics label the change in regression slope or intercept associated with the corresponding condition]. These results suggest that altered albumin synthesis (or altered synthesis unable to compensate for albumin loss, catabolism or redistribution) is an important determinant of hypoalbuminemia after hepatectomy. The correlations with age and postoperative outcome support the concept that hypoalbuminemia is a marker of pathophysiologic frailty associated with increasing age, and amplified by the challenges of postoperative illness.

Keywords: Keywords: Plasma albumin – Proteins – Pseudo-cholinesterase – Age – Liver resection – Acute phase response – Iron binding activity

A combination of caffeine and taurine has no effect on short term memory but induces changes in heart rate and mean arterial blood pressure by A. Bichler; A. Swenson; M. A. Harris (pp. 471-476).

Red Bull energy drink has become extraordinarily popular amongst college students for use as a study aid. We investigated the combined effects of Red Bull’s two active ingredients, caffeine and taurine, on short term memory. Studies on the effects of these two neuromodulators on memory have yielded mixed results, and their combined actions have not yet been investigated. In this double-blind study, college student subjects consumed either caffeine and taurine pills or a placebo and then completed a memory assessment. Heart rate and blood pressure were monitored throughout the testing period. The combination of caffeine and taurine had no effect on short term memory, but did cause a significant decline in heart rate and an increase in mean arterial blood pressure. The heart rate decline may have been caused by pressure-induced bradycardia that was triggered by caffeine ingestion and perhaps enhanced by the actions of taurine.

Keywords: Keywords: Caffeine – Taurine – Red Bull – Short term memory – Pressure-induced bradycardia

Polyamines in renal failure by K. Igarashi; S. Ueda; K. Yoshida; K. Kashiwagi (pp. 477-483).

The levels of polyamines (putrescine, spermidine and spermine) and polyamine oxidase in plasma of patients with chronic renal failure were determined. The level of putrescine was increased but the level of spermine was decreased in the plasma of these patients. The patients also had increased plasma polyamine oxidase activity leading to increased degradation of spermine. As acrolein was a major toxic compound produced from spermine by polyamine oxidase, the levels of free and protein-conjugated acrolein in plasma were also measured. Acrolein levels were enhanced in plasma of patients with chronic renal failure. The accumulated acrolein found as protein conjugates was equivalent to 170 µM, which was about 5-fold higher than in plasma of normal subjects. It was found that acrolein is mainly produced by spermine oxidase in plasma. An increase in putrescine, spermine oxidase and acrolein in plasma was observed in all cases such as diabetic nephropathy, chronic glomerulonephritis and nephrosclerosis. After patients with chronic renal failure had undergone hemodialysis, their levels of plasma polyamines, spermine oxidase and acrolein returned towards normal. It is likely that acrolein produced from spermine accumulates in the blood due to decreased excretion into urine and may function as a uremic “toxin”.

Keywords: Keywords: Polyamine – Spermine oxidase – Acrolein – Renal failure – Hemodialysis – Uremic toxin

Application of two-dimensional electrophoresis for monitoring gastrointestinal digestion of milk by M. Carbonaro (pp. 485-488).

Two-dimensional electrophoresis (2-DE) was used for tracing in vivo gastrointestinal digestion of milk proteins in a rapid model system with rats. Contents of stomach and small intestine from digestion trials with rats given a single dose of milk powder were recovered after 1 hour. They were then subjected to 2-DE (IEF and SDS-PAGE). 2-DE showed undigested proteins in a MW range 13.0–66.0 kDa in stomach and 13.0–25.0 kDa in the small intestine, thus indicating that milk proteins are slowly digested. This approach may shed light on pattern of protein digestion and mechanism of amino acid and peptide assimilation.

Keywords: Keywords: 2-DE – In vivo digestion – Milk

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Amino Acids: The Forum for Amino Acid, Peptide and Protein Research (v.31, #4)