A Systematic Risk Assessment and Meta-Analysis on the Use of Oral ß-Alanine Supplementation.

ß-Alanine supplementation is one of the world's most commonly used sports supplements, and its use as a nutritional strategy in other populations is ever-increasing, due to evidence of pleiotropic ergogenic and therapeutic benefits. Despite its widespread use, there is only limited understanding of potential adverse effects. To address this, a systematic risk assessment and meta-analysis was undertaken. Four databases were searched using keywords and Medical Subject Headings. All human and animal studies that investigated an isolated, oral, ß-alanine supplementation strategy were included. Data were extracted according to 5 main outcomes, including 1) side effects reported during longitudinal trials, 2) side effects reported during acute trials, 3) effect of supplementation on circulating health-related biomarkers, 4) effect of supplementation on skeletal muscle taurine and histidine concentration, and 5) outcomes from animal trials. Quality of evidence for outcomes was ascertained using the Grading of Recommendations Assessment Development and Evaluation (GRADE) framework, and all quantitative data were meta-analyzed using multilevel models grounded in Bayesian principles. In total, 101 human and 50 animal studies were included. Paraesthesia was the only reported side effect and had an estimated OR of 8.9 [95% credible interval (CrI): 2.2, 32.6] with supplementation relative to placebo. Participants in active treatment groups experienced similar dropout rates to those receiving the placebo treatment. ß-Alanine supplementation caused a small increase in circulating alanine aminotransferase concentration (effect size, ES: 0.274, CrI: 0.04, 0.527), although mean data remained well within clinical reference ranges. Meta-analysis of human data showed no main effect of ß-alanine supplementation on skeletal muscle taurine (ES: 0.156; 95% CrI: -0.38, 0.72) or histidine (ES: -0.15; 95% CrI: -0.64, 0.33) concentration. A main effect of ß-alanine supplementation on taurine concentration was reported for murine models, but only when the daily dose was ≥3% ß-alanine in drinking water. The results of this review indicate that ß-alanine supplementation within the doses used in the available research designs, does not adversely affect those consuming it.

A biochemical correlate of dimorphism in a zygomycete Benjaminiella poitrasii: characterization of purified NAD-dependent glutamate dehydrogenase, a target for antifungal agents.

The fungal organisms, especially pathogens, change their vegetative (Y, unicellular yeast and H, hypha) morphology reversibly for survival and proliferation in the host environment. NAD-dependent glutamate dehydrogenase (NAD-GDH, EC 1.4.1.2) from a non-pathogenic dimorphic zygomycete Benjaminiella poitrasii was previously reported to be an important biochemical correlate of the transition process. The enzyme was purified to homogeneity and characterized. It is a 371 kDa native molecular weight protein made up of four identical subunits. Kinetic studies showed that unlike other NAD-GDHs, it may act as an anabolic enzyme and has more affinity towards 2-oxoglutarate than L-glutamate. Chemical modifications revealed the involvement of single histidine and lysine residues in the catalytic activity of the enzyme. The phosphorylation and dephosphorylation study showed that the NAD-GDH is present in active phosphorylated form in hyphal cells of B. poitrasii. Two of the 1,2,3 triazole linked ß-lactam-bile acid conjugates synthesized in the laboratory (B18, B20) were found to be potent inhibitors of purified NAD-GDH which also significantly affected Y-H transition in B. poitrasii. Furthermore, the compound B20 inhibited germ tube formation during Y-H transition in Candida albicans strains and Yarrowia lipolytica. The possible use of NAD-GDH as a target for antifungal agents is discussed.

Coordinating to three histidine residues: Cu(II) promotes oligomeric and fibrillar amyloid-beta peptide to precipitate in a non-beta aggregation manner.

Cu(II) has been shown in vitro to profoundly promote the aggregation of amyloid-beta peptide (Abeta), a key pathological event in Alzheimer's disease. We investigated both the effect of Cu(II) on the secondary structure transformation of Abeta and the probable residues involved in the chelation to Cu(II). The effects of Cu(II) on Abeta was analyzed by the circular dichroism spectra, Th-T fluorescence and sedimentation assay, and the results indicated that Cu(II) could disrupt the already formed beta-sheet structure, convert beta-sheeted aggregates into non-beta-sheeted aggregates and promote oligomeric Abeta to precipitate in a non-beta-sheeted aggregation way. Additionally, we confirmed that the function of Cu(II) discussed above was achieved through its interaction with His6, His13, and His14 by investigating an Abeta mutant, (23,6,13,14)Abeta(1-40).

Understanding the selectivity of fumagillin for the methionine aminopeptidase type II.

The aim of this study is to explain the selectivity of the antiangiogenic drug fumagillin for the eukaryotic enzyme methionine aminopeptidase type II (MetAP-II, EC 3.4.11.18) over the structurally very similar MetAP-I. A homology model for the human MetAP-I is constructed and molecular dynamics simulations are performed on this model with and without a docked fumagillin molecule. These simulations are compared with analogous simulations that were performed on the experimentally determined structure of the human MetAP-II enzyme. We observe an increased flexibility of the active site histidine that is covalently modified by fumagillin in the MetAP-I enzyme. The MetAP-I active site residues, particularly the fumagillin-binding histidine, have a lower probability to be in a conformation that is prone to react with the drug than their MetAP-II counterparts. This result offers an explanation for the selectivity of fumagillin for the eukaryotic MetAP-II enzyme.

A common inhibitory binding site for zinc and odorants at the voltage-gated K(+) channel of rat olfactory receptor neurons.

This study compared the effects of zinc and odorants on the voltage-gated K(+) channel of rat olfactory neurons. Zinc reduced current magnitude, depolarized the voltage activation curve and slowed activation kinetics without affecting inactivation or deactivation kinetics. Zinc inhibition was potentiated by the NO compound, S-nitroso-cysteine. The pH- and diethylpyrocarbonate-dependence of zinc inhibition suggested that zinc acted by binding to histidine residues. Cysteine residues were eliminated as contributing to the zinc-binding site. The odorants, acetophenone and amyl acetate, also reduced current magnitude, depolarized the voltage activation curve and selectively slowed activation kinetics. Furthermore, the diethylpyrocarbonate- and pH-dependence of odorant inhibition implied that the odorants also bind to histidine residues. Zinc inhibitory potency was dramatically diminished in the presence of odorants, implying competition for a common binding site. These observations indicate that the odorants and zinc share a common inhibitory binding site on the external surface of the voltage-gated K(+) channel.

Chemical pathways of peptide degradation. X: effect of metal-catalyzed oxidation on the solution structure of a histidine-containing peptide fragment of human relaxin.

PURPOSE: To elucidate the major degradation products of the metal-catalyzed oxidation of (cyclo S-S) AcCys-Ala-X-Val-Gly-CysNH2 (X = His, cyclic-His peptide), which is a fragment of the protein relaxin, and the effect of this oxidation on its solution structure. METHODS: The cyclic-His peptide and its potential oxidative degradation products, cyclic-Asp peptide (X = Asp) and cyclic-Asn peptide (X = Asn), were prepared by using solid phase peptide synthesis and purified by preparative HPLC. The degradation of the cyclic-His peptide was investigated at pH 5.3 and 7.4 in an ascorbate/cupric chloride/oxygen [ascorbate/Cu(II)/O2] system in the absence or presence of catalase (CAT), superoxide dismutase (SOD), isopropanol, and thiourea. The oxidation of the cyclic-His peptide was also studied in the presence of hydrogen peroxide (H2O2). All reactions were monitored by reversed-phase HPLC. The main degradation product of the cyclic-His peptide formed at pH 7.4 in the presence of ascorbate/Cu(II)/O2 was isolated by preparative HPLC and identified by 1H NMR and electrospray mass spectrometry. The complexation of Cu(II) with the cyclic-His peptide was determined with 1H NMR. The solution structure of the cyclic-His peptide in the presence and absence of Cu(II) at pH 5.3 and 7.4 and the solution structure of the main degradation product were determined using circular dichroism (CD). RESULTS: CAT and thiourea were effective in stabilizing the cyclic-His peptide to oxidation by ascorbate/Cu(II)/O2, while SOD and isopropanol were ineffective. Cyclic-Asp and cyclic-Asn peptides were not observed as degradation products of the cyclic-His peptide oxidized at pH 5.3 and 7.4 in an ascorbate/Cu(II)/O2 system. The main degradation product formed at pH 7.4 was the cyclic 2-oxo-His peptide (X = 2-oxo-His). At pH 5.3, numerous degradation products were formed in low yields, including the cyclic 2-oxo-His peptide. The cyclic 2-oxo-His peptide appeared to have a different secondary structure than did the cyclic-His peptide as determined by CD. 1H NMR results indicate complexation between the cyclic-His peptide and Cu(II). CD results indicated that the solution structure of the cyclic-His peptide in the presence of Cu(II) at pH 5.3 was different than the solution structure observed at pH 7.4. CONCLUSIONS: H2O2 and superoxide anion radical (O(*-)2) were deduced to be the intermediates involved in the ascorbate/Cu(II)/O2-induced oxidation of cyclic-His peptide. H2O2 degradation by a Fenton-type reaction appears to form secondary reactive-oxygen species (i.e., hydroxyl radical generated within complex forms or metal-bound forms of hydroxyl radical) that react with the peptide before they diffuse into the bulk solution. CD results indicate that different complexes are formed between the cyclic-His peptide and Cu(II) at pH 5.3 and pH 7.4. These different complexes may favor the formation of different degradation products. The apparent structural differences between the cyclic-His peptide and the cyclic 2-oxo-His peptide indicate that conformation of the cyclic-His peptide was impacted by metal-catalyzed oxidation.

Plasma membrane properties involved in the photodynamic efficacy of merocyanine 540 and tetrasulfonated aluminum phthalocyanine.

Merocyanine 540 (MC540)-mediated photodynamic damage to erythrocytes was strongly reduced when illumination was performed at pH 8.5 as compared to pH 7.4. This could be explained by high pH-mediated hyperpolarization of the erythrocyte membrane, resulting in decreased MC540 binding at pH 8.5. In accordance, the MC540-mediated photooxidation of open ghosts was not inhibited at pH 8.5. Photoinactivation of vesicular stomatitis virus (VSV) was not inhibited at pH 8.5. This suggests that illumination at increased pH could be an approach to protect red blood cells selectively against MC540-mediated virucidal phototreatment. With tetrasulfonated aluminum phthalocyanine (AIPcS4) as photosensitizer, damage to erythrocytes, open ghosts and VSV was decreased when illuminated at pH 8.5. A decreased singlet oxygen yield at high pH could be excluded. The AIPcS4-mediated photooxidation of fixed erythrocytes was strongly dependent on the cation concentration in the buffer, indicating that the surface potential may affect the efficacy of this photosensitizer. This study showed that altering the environment of the target could increase both the efficacy and the specificity of a photodynamic treatment.

Identification and mechanism of action of two histidine residues underlying high-affinity Zn2+ inhibition of the NMDA receptor.

Zinc (Zn2+) inhibition of N-methyl-D-aspartate receptor (NMDAR) activity involves both voltage-independent and voltage-dependent components. Recombinant NR1/NR2A and NR1/NR2B receptors exhibit similar voltage-dependent block, but voltage-independent Zn2+ inhibition occurs with much higher affinity for NR1/NR2A than NR1/NR2B receptors (nanomolar versus micromolar IC50, respectively). Here, we show that two neighboring histidine residues on NR2A represent the critical determinant (termed the "short spacer") for high-affinity, voltage-independent Zn2+ inhibition using the Xenopus oocyte expression system and site-directed mutagenesis. Mutation of either one of these two histidine residues (H42 and H44) in the extracellular N-terminal domain of NR2A shifted the IC50 for high-affinity Zn2+ inhibition approximately 200-fold without affecting the EC50 of the coagonists NMDA and glycine. We suggest that the mechanism of high-affinity Zn2+ inhibition on the NMDAR involves enhancement of proton inhibition.

HNE interacts directly with JNK isoforms in human hepatic stellate cells.

4-Hydroxy-2,3-nonenal (HNE) is an aldehydic end product of lipid peroxidation which has been detected in vivo in clinical and experimental conditions of chronic liver damage. HNE has been shown to stimulate procollagen type I gene expression and synthesis in human hepatic stellate cells (hHSC) which are known to play a key role in liver fibrosis. In this study we investigated the molecular mechanisms underlying HNE actions in cultured hHSC. HNE, at doses compatible with those detected in vivo, lead to an early generation of nuclear HNE-protein adducts of 46, 54, and 66 kD, respectively, as revealed by using a monoclonal antibody specific for HNE-histidine adducts. This observation is related to the lack of crucial HNE-metabolizing enzymatic activities in hHSC. Kinetics of appearance of these nuclear adducts suggested translocation of cytosolic proteins. The p46 and p54 isoforms of c-Jun amino-terminal kinase (JNKs) were identified as HNE targets and were activated by this aldehyde. A biphasic increase in AP-1 DNA binding activity, associated with increased mRNA levels of c-jun, was also observed in response to HNE. HNE did not affect the Ras/ERK pathway, c-fos expression, DNA synthesis, or NF-kappaB binding. This study identifies a novel mechanism linking oxidative stress to nuclear signaling in hHSC. This mechanism is not based on redox sensors and is stimulated by concentrations of HNE compatible with those detected in vivo, and thus may be relevant during chronic liver diseases.

Regulation of mammalian Shaker-related K+ channels: evidence for non-conducting closed and non-conducting inactivated states.

1. Using the whole-cell recording mode we have characterized two non-conducting states in mammalian Shaker-related voltage-gated K+ channels induced by the removal of extracellular potassium, K+o. 2. In the absence of K+o, current through Kv1.4 was almost completely abolished due to the presence of a charged lysine residue at position 533 at the entrance to the pore. Removal of K+o had a similar effect on current through Kv1.3 when the histidine at the homologous position (H404) was protonated (pH 6.0). Channels containing uncharged residues at the corresponding position (Kv1.1: Y; Kv1.2: V) did not exhibit this behaviour. 3. To characterize the nature of the interaction between Kv1.3 and K+o concentration ([K+]o), we replaced H404 with amino acids of different character, size and charge. Substitution of hydrophobic residues (A, V and L) either in all four subunits or in only two subunits in the tetramer made the channel insensitive to the removal of K+o, possibly by stabilizing the channel complex. Replacement of H404 with the charged residue arginine, or the polar residue asparagine, enhanced the sensitivity of the channel to 0 mM K+o, possibly by making the channel unstable in the absence of K+o. Mutation at a neighbouring position (400) had a similar effect. 4. The effect of removing K+o on current amplitude does not seem to be correlated with the rate of C-type inactivation since the slowly inactivating G380F mutant channel exhibited a similar [K+]o dependence as the wild-type Kv1.3 channel. 5. CP-339,818, a drug that recognizes only the inactivated conformation of Kv1.3, could not block current in the absence of K+o unless the channels were inactivated through depolarizing pulses. 6. We conclude that removal of K+o induces the Kv1.3 channel to transition to a non-conducting 'closed' state which can switch into a non-conducting 'inactivated' state upon depolarization.

Copper ions promote peroxidation of low density lipoprotein lipid by binding to histidine residues of apolipoprotein B100, but they are reduced at other sites on LDL.

Oxidized LDL is implicated in the pathogenesis of atherosclerosis. A widely studied model for oxidation of the lipid in LDL involves Cu2+. Recent studies suggest that Cu2+ may be reduced to Cu1+ by alpha-tocopherol to initiate LDL lipid peroxidation. LDL demonstrates binding sites for Cu2-, but the nature of these binding sites, as well their role in promoting Cu2+ reduction and lipid peroxidation, has not been established. In the current studies, we used diethylpyrocarbonate (DEPC) to modify the histidine residues of apolipoprotein B100, the major protein in LDL. First, we demonstrated that histidine residues were preferentially modified by DEPC under our experimental conditions. Then we monitored the kinetics of Cu(2+)-promoted oxidation of LDL and DEPC-modified LDL. In both cases, the progress curve of lipid peroxidation exhibited a lag phase and a propagation phase. However, when LDL was modified with DEPC, the length of the lag phase was prolonged whereas the rate of lipid peroxidation during the propagation phase was lower. Studies with LDL oxidized by 2,2'-azobis (2-amidinopropane) hydrochloride and phosphatidylcholine liposomes oxidized with hydroxyl radical established that DEPC was not acting simply as a nonspecific inhibitor of lipid peroxidation. DEPC treatment of LDL almost completely inhibited its ability to bind Cu2+. These observations suggest that peroxidation of the lipids in LDL can proceed with normal kinetics only when Cu2+ binds preferentially to sites on apolipoprotein B100 that contain histidine residues. We also compared the kinetics of Cu2+ reduction in the absence and presence of DEPC. There was no effect of DEPC modification on either the rate or extent of Cu2+ reduction by LDL. Therefore LDL is likely to contain a second class of binding sites for Cu2+ that does not involve histidine residues. Thus, LDL appears to contain at least two classes of Cu(2+)-binding sites: histidine containing sites, which are responsible in part for promoting lipid peroxidation during the propagation phase, and sites at which Cu2+ is reduced without binding to histidine.

Woodward's reagent K inactivation of Escherichia coli L-threonine dehydrogenase: increased absorbance at 340-350 nm is due to modification of cysteine and histidine residues, not aspartate or glutamate carboxyl groups.

L-Threonine dehydrogenase (TDH) from Escherichia coli is rapidly inactivated and develops a new absorbance peak at 347 nm when incubated with N-ethyl-5-phenylisoxazolium-3'-sulfonate (Woodward's reagent K, WRK). The cofactors, NAD+ or NADH (1.5 mM), provide complete protection against inactivation; L-threonine (60 mM) is approximately 50% as effective. Tryptic digestion of WRK-modified TDH followed by HPLC fractionation (pH 6.2) yields four 340-nm-absorbing peptides, two of which are absent from enzyme incubated with WRK and NAD+. Peptide I has the sequence TAICGTDVH (TDH residues 35-43), whereas peptide II is TAICGTDVHIY (residues 35-45). Peptides not protected are TMLDTMNHGGR (III, residues 248-258) and NCRGGRTHLCR (IV, residues 98-108). Absorbance spectra of these WRK-peptides were compared with WRK adducts of imidazole, 2-hydroxyethanethiolate, and acetate. Peptides III and IV have pH-dependent lambda max values (340-350 nm), consistent with histidine modification. Peptide I has pH-independent lambda max (350 nm) indicating that a thiol is modified. WRK, therefore, does not react specifically with carboxyl groups in this enzyme, but rather modifies Cys-38 in the active site of TDH; modification of His-105 and His-255 does not affect enzyme activity. These results are the first definitive proof of WRK modifying cysteine and histidine residues of a protein and show that enzyme inactivation by WRK associated with the appearance of new absorptivity at 340-350 nm does not establish modification of aspartate or glutamate residues, as has been assumed in numerous earlier reports.

Protein kinases and phosphatases that act on histidine, lysine, or arginine residues in eukaryotic proteins: a possible regulator of the mitogen-activated protein kinase cascade.

Phosphohistidine goes undetected in conventional studies of protein phosphorylation, although it may account for 6% of total protein phosphorylation in eukaryotes. Procedures for studying protein N- kinases are described. Genes whose products are putative protein histidine kinases occur in a yeast and a plant. In rat liver plasma membranes, activation of the small G-protein, Ras, causes protein histidine phosphorylation. Cellular phosphatases dephosphorylate phosphohistidine. One eukaryotic protein histidine kinase has been purified, and specific proteins phosphorylated on histidine have been observed. There is a protein arginine kinase in mouse and protein lysine kinases in rat. Protein phosphohistidine may regulate the mitogen-activated protein kinase cascade.

The effects of histidine residue modification on the immune precipitating ability of rabbit IgG.

Treatment of anti-ovalbumin rabbit IgG with diethylpyrocarbonate (DEPC) at concentrations up to 100 microM led to a progressive decrease in the rates of formation of insoluble immune complexes, without affecting the final extent of immune complex formation. DEPC concentrations approximately 10-fold higher were needed to give comparable decreases in the rates of immune complex formation by F(ab')2. Treatment of DEPC-treated IgG with hydroxylamine led to substantial restoration of the rates of formation of insoluble immune complexes. Carbethoxylation of two histidine residues per IgG molecule had little effect on rates of formation of insoluble immune complexes, but these rates were markedly decreased in samples of IgG with four to five histidines per molecule modified. There were parallel decreases in the protein A-binding activity and in the rates of formation of insoluble immune complexes in IgG treated with increasing concentrations of DEPC. The presence of complement protein C1q restored the rates of formation of insoluble immune complexes of DEPC-treated IgG.

The catechol 2,3-dioxygenase gene of Rhodococcus rhodochrous CTM: nucleotide sequence, comparison with isofunctional dioxygenases and evidence for an active-site histidine.

In cell-free extracts of Escherichia coli clones harbouring the 3.5 kb Bg/II fragment of plasmid pTC1 from Rhodococcus rhodochrous CTM a catechol 2,3-dioxygenase (C23O) accepting both 3-methylcatechol and 2,3-dihydroxybiphenyl as substrates could be detected. The plasmid-encoded gene for C23O of R. rhodochrous CTM and its flanking regions were sequenced. In front of the gene a sequence resembling an E. coli promoter was identified, which led to constitutive expression of the cloned gene in E. coli TG1. The derived amino acid sequence of the C23O was compared to that of nine other enzymes, which all catalyse the extradiol cleavage of an aromatic ring. These nine sequences were from different Pseudomonas strains, in contrast to the sequence described here, from a Gram-positive bacterium. The role of four strongly conserved histidines was examined by chemical modification of the histidyl residues of the native enzyme by diethylpyrocarbonate. For that purpose the C23O was purified to homogeneity from E. coli harbouring pSC1701. However, the enzyme lost its activity during the purification. Activity could partially be restored by treatment with Fe2+ and reducing agents.

Clinical and laboratory studies of the photosensitizing potential of norfloxacin, a 4-quinolone broad-spectrum antibiotic.

Cutaneous photosensitivity reactions are a consistent although uncommon feature of the fluoroquinolone group of antibiotics, which are related to nalidixic acid. Objective laboratory and clinical data are now routinely required by regulatory bodies for new drugs suspected of being photosensitizers, but no clear recommendations exist. A series of in vitro tests ranging in complexity revealed a UVA-dependent phototoxic potential for the fluoroquinolone norfloxacin similar to that for ciprofloxacin, and less than that of nalidixic acid. Controlled monochromator phototesting, designed to reveal the clinical characteristics, wavelength dependence and severity of cutaneous reactions in normal subjects showed both norfloxacin and ciprofloxacin to have a weak phototoxic potential which clears within 4 weeks of stopping the drug. UVA wavelengths (335 +/- 30 nm; 365 +/- 30 nm) appear most responsible for producing an asymptomatic erythema which is maximal at 24 h. The clinical study differs from those used previously in being blind, containing positive and negative controls, and phototesting after cessation of drug intake. The methodology has the anticipated limitation of failing to detect idiosyncratic photosensitivity responses.

[Study of the modification of histidine residues, SH- and epsilon-NH2-groups of rat sarcoma-45 myosin by specific reagents]. / Issledovanie modifikatsii spetsificheskimi reagentami ostatkov gistidina, SH- i epsilon-NH2-grupp miozina iz zlokachestvennoi opukholi krys sarkoma-45.

Modification of histidine residues, SH- and epsilon-NH2-groups of myosin from rat sarcoma-45 by specific reagents was studied. It was shown that diethylpyrocarbonate modifies histidine residues essential for the ATPase activity. A kinetic analysis of myosin epsilon-NH2-groups modification by 2,4,6-trinitrobenzene sulfonate revealed that myosin trinitrophenylation and its inactivation by Ca2(+)-ATPase occurs in two steps: a fast and a slow (Km = 2400 and 1.7 s-1 M-1, respectively). Two essential epsilon-NH2-groups of tumour myosin active sites react in the fast reaction. The relatively low concentrations of p-chloromercuribenzoic acid activate rat sarcoma-45 myosin Ca2(+)-ATPase and Mg2(+)-ATPase, whereas higher ones inhibit the enzyme. The data obtained suggest that two SH-groups, SH1 and SH2 are essential for the tumour myosin ATPase function.