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Amino Acids: The Forum for Amino Acid, Peptide and Protein Research (v.31, #3)

Neuropeptides in anxiety and depression by R. Landgraf (pp. 211-213).

CCK and NPY as anti-anxiety treatment targets: promises, pitfalls, and strategies by J. Harro (pp. 215-230).

Short CCK peptides elicit panic attacks in humans and anxiogenic-like effects in some animal models, but CCK receptor antagonists have not been found clinically effective. Yet CCK overactivity appears to be involved in submissive behaviour, and CCK_B receptor expression and binding are increased in suicide victims and animal models of anxiety. Preliminary data suggest that involvement of CCK and its receptor subtypes in anxiety can be better described when focusing on distinct endophenotypes, and considering environmental contingencies and confounds originating from interactions with dopamin-, opioid- and glutamatergic neurotransmission. In contrast, NPY is an anti-anxiety peptide with robust effects in various animal models when administrated into several brain regions. Studies with non-peptide antagonists selective for receptor subtypes have revealed the role of endogenous NPY in active coping. At least Y₁, Y₂ and Y₅ receptors in various brain regions are involved, with the strongest evidence for contribution of Y₁.

Keywords: Keywords: Cholecystokinin – Neuropeptide Y – Anxiety – Panic – Suicide – Brain circuits

Galanin as a modulator of anxiety and depression and a therapeutic target for affective disease by R.-M. Karlsson; A. Holmes (pp. 231-239).

Galanin is a 29 amino-acid (30 in humans) neuropeptide with a close functional relationship with neurotransmitter systems implicated in the pathophysiology and treatment of depression and anxiety disorders. In rodent models of depression-related behavior, treatment with galanin or compounds with agonist actions at galanin receptors has been shown to affect depression-related behaviors and the behavioral and neurochemical effects of antidepressants. Treatment with clinically efficacious antidepressants alters galanin and galanin receptor gene expression in rodents. Rodent anxiety-like behaviors appear to be modulated by galanin in a complex manner, with studies showing either increases, decreases and no effects of galanin treatments and galanin mutations on anxiety-like behavior in various tasks. One concept to emerge from this literature is that galanin recruitment during extreme behavioral and physiological provocations such as stress and opiate withdrawal may serve to attenuate negative emotional states caused by noradrenergic hyperactivation. The specific galanin receptor subtypes mediating the anxiety- and depression-related effects of galanin remains to be determined, with evidence supporting a possible contribution of GalR1, GalR2 and GalR3. While our understanding of the role of galanin as a modulator of emotion remains at an early stage, recent progress in this rapidly evolving field raise possibility of that galanin may represent a target for the development of novel antidepressant and anxiolytic drug treatments.

Keywords: Keywords: Galanin – Neuropeptides – Stress – Anxiety – Depression – Rodent

Corticotropin-releasing factor, vasopressin and receptor systems in depression and anxiety by M. E. Keck (pp. 241-250).

Affective disorders tend to be chronic and life-threatening diseases: suicide is estimated to be the cause of death in 10–15% of individuals with major depressive disorders. Major depression is one of the most prevalent and costly brain diseases with up to 20% of the worldwide population suffering from moderate to severe forms of the disease. Only 50% of individuals with depression show full remission in response to currently available antidepressant drug therapies which are based on serendipitous discoveries made in the 1950s. Previously underestimated, other severe depression-associated deleterious health-related effects have increasingly been recognized. Epidemiological studies have provided substantial evidence that patients with depression have a 2–4-fold increased risk both of developing cardiovascular disease and of mortality after experiencing a myocardial infarction. The majority of patients suffering from affective disorders have measurable shifts in their stress hormone regulation as reflected by elevated secretion of central and peripheral stress hormones or by altered hormonal responses to neuroendocrine challenge tests. In recent years, these alterations have increasingly been translated into testable hypotheses addressing the pathogenesis of illness. Refined molecular technologies and the creation of genetically engineered mice have allowed to specifically target individual genes involved in regulation of corticotropin releasing factor (CRF) and vasopressin (AVP) system elements. The cumulative evidence makes a strong case implicating dysfunction of these systems in the etiology and pathogenesis of depression and pathological anxiety. Translation of these advances into novel therapeutic strategies has already been started.

Keywords: Keywords: Corticotropin releasing factor (CRF) – Vasopressin (AVP) – Depression – Anxiety – CRF receptor antagonist – R121919 – NBI 30775 – CRF receptor type 1 – CRF receptor type 2 – Transgenic mice – Conditional knockout

The role of substance P in stress and anxiety responses by K. Ebner; N. Singewald (pp. 251-272).

Substance P (SP) is one of the most abundant peptides in the central nervous system and has been implicated in a variety of physiological and pathophysiological processes including stress regulation, as well as affective and anxiety-related behaviour. Consistent with these functions, SP and its preferred neurokinin 1 (NK1) receptor has been found within brain areas known to be involved in the regulation of stress and anxiety responses. Aversive and stressful stimuli have been shown repeatedly to change SP brain tissue content, as well as NK1 receptor binding. More recently it has been demonstrated that emotional stressors increase SP efflux in specific limbic structures such as amygdala and septum and that the magnitude of this effect depends on the severity of the stressor. Depending on the brain area, an increase in intracerebral SP concentration (mimicked by SP microinjection) produces mainly anxiogenic-like responses in various behavioural tasks. Based on findings that SP transmission is stimulated under stressful or anxiety-provoking situations it was hypothesised that blockade of NK1 receptors may attenuate stress responses and exert anxiolytic-like effects. Preclinical and clinical studies have found evidence in favour of such an assumption. The status of this research is reviewed here.

Keywords: Keywords: Substance P – Tachykinin – Neurokinin – In vivo release – Anxiety – Depression – Antidepressant – Anxiolytic – Stress – HPA axis – NK1 receptor – NK1 receptor antagonist – Emotional behaviour – Microdialysis – Stress-related disorders

The effect of taurine depletion on the contractile properties and fatigue in fast-twitch skeletal muscle of the mouse by E. J. Hamilton; H. M. Berg; C. J. Easton; A. J. Bakker (pp. 273-278).

Taurine increases force production in skeletal muscle, and taurine levels may fall during exercise. The contractile properties and fatigability of extensor digitorum longus (EDL) muscles depleted of taurine by guanodinoethane sulfonate (GES) treatment were investigated. GES treatment decreased muscle taurine levels to <40% of controls. Peak twitch force levels were 23% of controls in GES treated EDL muscles (p < 0.05), but maximal specific force was unaffected. The force–frequency relationship was examined and significantly less force was produced by the GES treated muscles compared to controls at stimulation frequencies from 50 to 100 Hz (p < 0.05). GES treated EDL muscles exhibited significantly slower rates of fatigue than controls (p < 0.05). In skinned fibres, 20 mM GES had a small but significant effect on force production, indicating that GES may have some minor taurine-like effects. In this study, a fall in taurine levels decreased force output, and increased the endurance of EDL skeletal muscles.

Keywords: Keywords: Muscle performance – Ca²⁺ – Extensor digitorum longus

Selective determination of mimosine and its dihydroxypyridinyl derivative in plant systems by K. Lalitha; S. Rajendra Kulothungan (pp. 279-287).

Our observations on the growth stimulatory nature of mimosine, (β-(3-hydroxy-4-pyridon-1-yl)-L-alanine), the toxic non-protein plant amino acid, in some model experimental systems, warranted sensitive and selective routine estimations. For the determination of both mimosine and DHP, an indirect spectrophotometric method was developed based on their individual reaction with known excess of DZSAM and by estimating the remaining DZSAM with N-(1-naphthyl)ethylene-diamine (NEDA). The resultant decrease in the secondary coupled product was measured at 540 nm. On equimolar basis, DHP had 40% of the reactivity of mimosine while interference from other relevant compounds was 15–35%. The determination of mimosine and DHP in tissue samples under different physiological conditions was effected after paper chromatographic separation of mimosine and DHP with distinctly differing R_f, from other compounds. The indirect method is superior in terms of absolute selectivity, sensitivity and ease of applicability with linear decreases in absorbance, proportional to increasing concentrations of mimosine from 0.1 to 0.75 μM or DHP from 0.2 to 1.5 μM and with recoveries of 99.2 to 100.5%.

Keywords: Keywords: Mimosine – Dihydroxypyridine – Leucaena leucocephala – Reaction with diazotized sulfanilamide – Spectrophotometric determination – Uptake of mimosine – Plant mitochondria

Cytoskeleton changes following differentiation of N1E-115 neuroblastoma cell line by J.-E. Oh; K. Karlmark Raja; J.-H. Shin; A. Pollak; M. Hengstschläger; G. Lubec (pp. 289-298).

No systematic approach to detect expression of differentiation-related elements was published so far. The undifferentiated N1E-115 neuroblastoma cell line was switched into a neuronal phenotype by DMSO treatment and used for proteomic experiments. We used two-dimensional gel electrophoresis followed by unambiguous mass spectrometrical identification of proteins to generate a map of cytoskeleton proteins (CPs), i.e., to search for differentiation-related structures. Alpha-actin, actin-like protein 6A, gamma-tubulin complex component 2, tubulin alpha 3/alpha 7, CLIP associating protein 2, B4 integrin interactor homolog were detectable in the undifferentiated cell line exclusively and neuron-specific CPs drebrin and presynaptic density protein 95, actin-related protein 2/3, alpha and beta-centractin, PDZ-domain actin binding protein, actinin alpha 1, profilin II, ezrin, coactosin-like protein, transgelin 2, myosin light polypeptide 6, tubulin alpha 2, 6 and 7, beta tubulin (94% similar with tubulin beta-2), tubulin beta 3, tubulin tyrosine ligase-like protein 1, lamin B1 and keratin 20 were observed in the differentiated cell line only. We herein identified differentiation-related expressional patterns thus providing new evidence for the role of CPs in the process of neuronal differentiation.

Keywords: Keywords: Cytoskeleton proteins – Actin/actin binding protein – Tubulin – Proteomics – Differentiation – N1E-115 cell line

Effect of melatonin treatment on oxygen consumption by rat liver mitochondria by C. F. Reyes-Toso; I. R. Rebagliati; C. R. Ricci; L. M. Linares; L. E. Albornoz; D. P. Cardinali; A. Zaninovich (pp. 299-302).

The objective of this study was to examine the in vivo effect of melatonin on rat mitochondrial liver respiration. Two experiments were performed: For experiment 1, adult male rats received melatonin in the drinking water (16 or 50 µg/ml) or vehicle during 45 days. For experiment 2, rats received melatonin in the drinking water (50 µg/ml) for 45 days, or the same amount for 30 days followed by a 15 day-withdrawal period. At sacrifice, a liver mitochondrial fraction was prepared and oxygen consumption was measured polarographically in the presence of excess concentration of DL-3-β-hydroxybutyrate or L-succinate. Melatonin treatment decreased Krebs’ cycle substrate-induced respiration significantly at both examined doses. The stimulation of mitochondrial respiration caused by excess concentration of substrate recovered after melatonin withdrawal. Basal state 4 respiration was not modified by melatonin. Melatonin, by curtailing overstimulation of cellular respiration caused by excess Krebs’ cycle substrates, can protect the mitochondria from oxidative damage.

Keywords: Keywords: Melatonin – Oxygen consumption – Free radicals – Liver mitochondria – β-hydroxybutyrate – Succinate

Alterations of taurine in the brain of chronic kainic acid epilepsy model by H. Baran (pp. 303-307).

The aim of the study was to investigate the changes of taurine in the kainic acid (KA, 10 mg/kg, s.c.) chronic model of epilepsy, six months after KA application. The KA-rats used were divided into a group of animals showing weak behavioural response to KA (WDS, rare focal convulsion; rating scale <2 up to 3 h after KA injection) and a group of strong response to KA (WDS, seizures; rating >3 up to 3 h after KA injection). The brain regions investigated were caudate nucleus, substantia nigra, septum, hippocampus, amygdala/piriform cortex, and frontal, parietal, temporal and occipital cortices. KA-rats with rating <2 developed spontaneous WDS which occurred chronically and six months after KA injection increased taurine levels were found in the hippocampus (125.4% of control). KA-rats with rating >3 developed spontaneous recurrent seizures and six months after injection increased taurine levels were found in the caudate nucleus (162.5% of control) and hippocampus (126.6% of control), while reduced taurine levels were seen in the septum (78.2% of control). In summary, increased taurine levels in the hippocampus may involve processes for membrane stabilisation, thus favouring recovery after neuronal hyperactivity. The increased taurine levels in the caudate nucleus could be involved in the modulation of spontaneous recurrent seizure activity.

Keywords: Keywords: Taurine – Epilepsy – Kainic acid

p21^WAF1/CIP1 and 14-3-3 σ gene expression in degenerated aortic valves: A link between cell cycle checkpoints and calcification by O. Golubnitschaja; K. Yeghiazaryan; D. Skowasch; H. Schild; G. Bauriedel (pp. 309-316).

The mechanisms underlying aortic valve degeneration are largely unknown. Cardiac tissue responds to a variety of stimuli by hypertrophic growth. Molecular mechanisms resulting in the hypertrophic response indicate similarity and overlap with those involved in both cell growth and death. We hypothesized cell cycle control to be the key event in progression regulation of heart valve degeneration followed by tissue mineralization. Human post-operative tissue samples of native non-rheumatic stenosed aortic valves were categorized according to absence (group 1) or presence of calcification (group 2). The samples were ex vivo examined for cell density and presence of macrophage (CD68), as well as expression of two checkpoint genes, p21^WAF1/CIP1 and 14-3-3 σ, arresting the G₁ and G₂ cell cycle phases, respectively. Expression rates were measured by “Real-Time”-PCR on transcriptional level. Target protein expression was measured and their co-localization in different kinds of valvular cells was tested using immunohistochemical analysis. Whereas macrophages were localized predominantly in sub-endothelial layer of valvular fibrosis, p21^WAF1/CIP1 and 14-3-3 σ expression was observed also in the valvular spongiosa co-localized with alpha-actin positive cells. Significantly higher cell density and inflammation grade were observed in group 2 versus group 1. Accordingly, p21^WAF1/CIP1 and 14-3-3 σ expression was several fold higher in group 1 versus group 2 on both transcription and translation levels. The present findings on degenerated aortic valves show that increased cell density accompanied with consequent calcification might be attributed to the down-regulation of both G₁ and G₂ checkpoint genes.

Keywords: Keywords: Aortic valve degeneration – Calcification – Valvular fibrosis and spongiosa – Cell density and inflammation grade – Molecular monitoring ex vivo – Cell cycle checkpoints

The necessity of functional proteomics: protein species and molecular function elucidation exemplified by in vivo alpha A crystallin N-terminal truncation by W. Hoehenwarter; R. Ackermann; U. Zimny-Arndt; N. M. Kumar; P. R. Jungblut (pp. 317-323).

Ten years after the establishment of the term proteome, the science surrounding it has yet to fulfill its potential. While a host of technologies have generated lists of protein names, there are only a few reported studies that have examined the individual proteins at the covalent chemical level defined as protein species in 1997 and their function. In the current study, we demonstrate that this is possible with two-dimensional gel electrophoresis (2-DE) and mass spectrometry by presenting clear evidence of in vivo N-terminal alpha A crystallin truncation and relating this newly detected protein species to alpha crystallin activity regulation by protease cleavage in the healthy young murine lens. We assess the present state of technology and suggest a shift in resources and paradigm for the routine attainment of the protein species level in proteomics.

Keywords: Keywords: Alpha crystallin – Lp82 – Protein species – Proteomics – Tertiary structure

Taurine trophic modulation of goldfish retinal outgrowth and its interaction with the optic tectum by S. Cubillos; L. Lima (pp. 325-331).

Goldfish retinal explant outgrowth in the presence of fetal calf serum is stimulated by taurine. In the absence of it, but with glucose in the medium, length of neurites is still elevated by the amino acid. Using the medium in the presence of glucose, but in the absence of fetal calf serum, we explored the effect of optic tectum medium from cultures of them coming from goldfish without crush of the optic nerve or 3, 5, 10, 14 and 20 days after crush. Retinal explants, intact or from goldfish with crush of the optic nerve 10 days prior to starting the culture, were employed in order to measure the possible effect of optic tectum media and the inter action with taurine. In other type of experiments the optic nerve was crushed 1, 2, 4, 7 and 10 days before dissection of the optic tectum, and then co-cultured with intact or 10 days post-crush retinal explants. Optic tectum media produced a time-dependent effect on outgrowth in lesioned retinas with a maximum effect around 5 days after the lesion for the corresponding optic tectum. Taurine, 4 mM, did not further affect the outgrowth in the presence of optic tectum media, but did significantly increase length of neurites either in intact or in post-lesion retinas. Co-culture of optic tectum at different days post-lesion and retinas at 10 days post-lesion increased the outgrowth around 4 days post-lesion, in a preparation resulting in mutual effects of both types of tissues. The addition of taurine in these conditions did not further increase outgrowth, rather inhibited it according to the time after lesion of optic nerve corresponding to the co-cultured optic tectum. The effect of taurine was concentration-dependent, since 0.2 mM was more effective than 2 or 4 mM in the presence of optic tectum with lesion of 2 days. These results demonstrate the time-course of the regeneration processes in the visual system of goldfish, indicating the crucial periods after crush in which the tectum could produce stimulation and later decrease or no effect on outgrowth from the retina. In addition, they are evidences of the interaction between taurine and optic tectum production of time-produced specific agents. The mechanisms underlying these effects are closely related to calcium, as it was demonstrated by the addition of extracellular or intracellular chelators to the medium, which inhibited the effects of the optic tectum and the trophic properties of taurine in this system. The inhibitor of taurine transport, guanidoethylsulfonate, also decreased the stimulatory effects of the optic tectum and of taurine, indicating an interaction of substances produced by the tectum with taurine, and an effect of taurine mediated through its entrance to the cells. Overall, retinal explants outgrowth in the absence of fetal calf serum, the interaction of agents of the optic tectum and taurine modulates outgrowth from the retina, and these effects are mediated by calcium levels and by the levels of intracellular taurine.

Keywords: Keywords: Co-culture – Optic nerve crush – Optic tectum – Retinal outgrowth – Taurine

The fragmentation mechanism of β-(N-alkyl/arylamino)-α,β-unsaturated carboxylates under electrospray ionization conditions by W. Yin; Y. Ma; Y. Zhao (pp. 333-336).

The positive ion mass spectrometric behavior of six β-(N-alkyl/arylamino)-α,β-unsaturated carboxylates, α-(1-alkyl/arylaminoethylidene)-γ-lactones, has been studied under electrospray ionization conditions. Their fragmentation pathways are described and supported by tandem mass spectrometry. The protonated compounds are apt to eliminate a water, and a water plus a oxacyclopent-2-yne molecule. Some of the compounds show a tendency to undergo a four-membered ring contraction rearrangement to lose a carbon dioxide. The fragmentation patterns of these compounds exhibit a strong substituent dependency.

Keywords: Keywords: β-(N-alkyl/arylamino)-α,β-unsaturated carboxylate – Mass spectrometry – β-amino-α,β-unsaturated acid – β-amino acid

Preparation of β-nitroalanine using the Easton three-component coupling method by K. M. Soapi; C. A. Hutton (pp. 337-339).

A simple one-step preparation of β-nitroalanine has been developed using the Easton three-component coupling method. To date one limitation of this method has been that use of nitromethane as the nitroalkane component does not yield β-nitroalanine. We report that use of the dipotassium salt of nitroacetic acid in the Easton three-component coupling gives β-nitroalanine in high yield, presumably via facile decarboxylation of a β-nitroaspartate intermediate.

Keywords: Keywords: Amino acid – Nitroalanine – Three-component coupling – Decarboxylation

Non-irradiation-derived reactive oxygen species (ROS) and cancer: therapeutic implications by E. Agostinelli; N. Seiler (pp. 341-355).

Owing to their chemical reactivity, radicals have cytocidal properties. Destruction of cells by irradiation-induced radical formation is one of the most frequent interventions in cancer therapy. An alternative to irradiation-induced radical formation is in principle drug-induced formation of radicals, and the formation of toxic metabolites by enzyme catalysed reactions. Although these developments are currently still in their infancy, they nevertheless deserve consideration. There are now numerous examples known of conventional anti-cancer drugs that may at least in part exert cytotoxicity by induction of radical formation. Some drugs, such as arsenic trioxide and 2-methoxy-estradiol, were shown to induce programmed cell death due to radical formation. Enzyme-catalysed radical formation has the advantage that cytotoxic products are produced continuously over an extended period of time in the vicinity of tumour cells. Up to now the enzymatic formation of toxic metabolites has nearly exclusively been investigated using bovine serum amine oxidase (BSAO), and spermine as substrate. The metabolites of this reaction, hydrogen peroxide and aldehydes are cytotoxic. The combination of BSAO and spermine is not only able to prevent tumour cell growth, but prevents also tumour growth, particularly well if the enzyme has been conjugated with a biocompatible gel. Since the tumour cells release substrates of BSAO, the administration of spermine is not required. Combination with cytotoxic drugs, and elevation of temperature improves the cytocidal effect of spermine metabolites. The fact that multidrug resistant cells are more sensitive to spermine metabolites than their wild type counterparts makes this new approach especially attractive, since the development of multidrug resistance is one of the major problems of conventional cancer therapy.

Keywords: Keywords: Radicals – Reactive oxygen species (ROS) – Polyamines – Bovine serum amine oxidase – Cancer

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Amino Acids: The Forum for Amino Acid, Peptide and Protein Research (v.31, #3)