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Numerous natural antimicrobial peptides (AMPs) exhibit a cationic amphipathic helical conformation, wherein cationic amino acids, such as lysine and arginine, play pivotal roles in antimicrobial activity by aiding initial attraction to negatively charged bacterial membranes. Expanding on our previous work, which introduced a de novo design of amphipathic helices within cationic heptapeptides using an 'all-hydrocarbon peptide stapling' approach, we investigated the impact of lysine-homologue substitution on helix formation, antimicrobial activity, hemolytic activity, and proteolytic stability of these novel AMPs. Our results demonstrate that substituting lysine with ornithine enhances both the antimicrobial activity and proteolytic stability of the stapled heptapeptide AMP series, while maintaining low hemolytic activity. This finding underscores lysine-homologue substitution as a valuable strategy for optimizing the therapeutic potential of diverse cationic AMPs., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Elsevier Ltd. All rights reserved.)

An amino group at side chain of lysine residue can be targeted for protein modification because of the convenience for covalent bond formation. We have achieved an efficient protein modification by utilizing amine-clickable 6π-azaelectrocyclization, termed RIKEN click reaction recently, which enabled direct click labeling of protein without any introduction of specific functional groups such as alkynes and azides. On the basis of the RIKEN click reaction, we established the double click labeling method. The double click methods composed of copper-free strain-promoted [3 + 2] cyclization or tetrazine ligation and RIKEN click reaction were developed. The double click method realized highly effective proteins including radiolabeling of bioactive peptides and anti-tumor antibodies. In this personal review, the development of double click probes, practical radiolabeling of biological active molecules such as cyclic RGDyK peptides, proteins, and antibodies with α-emission or β-emission radionuclides, and their applications for PET imaging and α-emission cancer treatment are summarized., (Copyright © 2021 Elsevier Ltd. All rights reserved.)

Radioligand therapy (RLT) using prostate-specific membrane antigen (PSMA) targeting ligands is an attractive option for the treatment of Prostate cancer (PCa) and its metastases. We report herein a series of radioiodinated glutamate-urea-lysine-phenylalanine derivatives as new PSMA ligands in which l-tyrosine and l-glutamic acid moieties were added to increase hydrophilicity concomitant with improvement of in vivo targeting properties. Compounds 8, 15, 19a/19b and 23a/23b were synthesized and radiolabeled with 125 I by iododestannylation. All iodinated compounds displayed high binding affinities toward PSMA (IC 50  = 1-13 nM). In vitro cell uptake studies demonstrated that compounds containing an l-tyrosine linker moiety (8, 15 and 19a/19b) showed higher internalization than MIP-1095 and 23a/23b, both without the l-tyrosine linker moiety. Biodistribution studies in mice bearing PC3-PIP and PC3 xenografts showed that [ 125 I]8 and [ 125 I]15 with higher lipophilicity exhibited higher nonspecific accumulations in the liver and intestinal tract, whereas [ 125 I]19a/19b and [ 125 I]23a/23b containing additional glutamic acid moieties showed higher accumulations in the kidney and implanted PC3-PIP (PSMA+) tumors. [ 125 I]23b displayed a promising biodistribution profile with favorable tumor retention, fast clearance from the kidney, and 2-3-fold lower uptake in the liver and blood than that observed for [ 125 I]MIP-1095. [ 125/131 I]23b may serve as an optimal PSMA ligand for radiotherapy treatment of prostate cancer over-expressing PSMA., (Copyright © 2020 Elsevier Ltd. All rights reserved.)

The control of bleaching reaction is important in hair bleaching and laundry detergents to ensure quality of the final product. A better understanding of the reaction mechanisms is needed to minimize product failures. 31 P NMR-spectroscopy-based spin trap technique was employed to detect and quantify the free radical species that were generated in different bleaching solutions. These solutions contained the key actives in an alkaline hair colorant/bleaching product, an ammonium salt and hydrogen peroxide at pH = 10. Generally, the main radical species detected in hair oxidative coloring or bleaching processes, were hydroperoxyl/superoxide radicals HO 2 ·/O 2 .- , amino radicals ·NH 2 and hydroxyl radicals ·OH. Their amounts showed a variation based on the chemical composition of the bleaching systems and the metal ion content. The generation of free radicals from reactions between transition metal ions, such as copper, and hydrogen peroxide at pH = 10 was evaluated. In the absence of chelating agents, the copper ions generated a significant level of hydroxyl radicals in a Fenton-like reaction with hydrogen peroxide at pH = 10. Besides that, an increase in copper ion content led to an increase of amino radical ·NH 2 , whereas the concentration of superoxide radical O 2 ·- decreased which was not yet well reported in the previous literature. The effect of chelating agents like ethylenediaminetetraacetic acid (EDTA), tetrasodium-iminodisuccinate (IDS), a mixture of basic amino acids and dicarboxylic acid on free radical formation was investigated in the presence of binary Cu 2+ -Ca 2+ bleaching systems. As expected, in the binary Cu 2+ -Ca 2+ ion system EDTA did not suppress hydroxyl radical formation effectively, but the mixture containing sodium succinate, lysine and arginine reduced hydroxyl radical formation, whereas IDS (nearly) completely inhibited hydroxyl radical formation. The results indicated that each bleaching solution has its characteristic performance and damage profile. Whereas the reactivity can be controlled by the usage of chelating agents., (Copyright © 2018 Elsevier Inc. All rights reserved.)

Infections caused by Pseudomonas aeruginosa are associated with high morbidity and mortality, especially in immunocompromised patients. These bacteria frequently grow within a biofilm matrix, rendering therapy with conventional antibiotics inefficient; a fact that emphasizes the need for new treatment strategies. Antimicrobial peptidomimetics constitute potential alternatives to traditional antimicrobial agents. However, their application remains limited due to the lack of efficient delivery to their target site in vivo and the risk of high systemic toxicity. Nanogels composed of cross-linked networks of amphiphilic polymers with a therapeutic drug molecule embedded constitute attractive drug delivery systems, as they have been shown to display unique properties such as biocompatibility and biodegrability, as well as confer improved drug stability and reduced drug-mediated cytotoxicity. Here, we report on the first formulation of biopolymer nanogels incorporating a potent antibacterial peptidomimetic. A lysine-based α-peptide/β-peptoid hybrid with potent activity against P. aeruginosa was designed and formulated into a nanogel together with octenyl succinic anhydride-modified hyaluronic acid in order to improve its cell selectivity. Twelve nanogel formulations were prepared by using a design of experiments setup in order to identify the parameters yielding the highest drug loading and the smallest particle size. Encapsulation of the peptidomimetic into nanogels significantly decreased the cytotoxicity of the peptidomimetic to eukaryotes. The most promising formulation with high encapsulation efficiency (88%) of the peptidomimetic demonstrated a three-fold reduction in cytotoxicity towards hepatocytes along with improved bacterial killing kinetics., (Copyright © 2018 Elsevier B.V. All rights reserved.)

Glu-Urea-Lys (GUL) derivatives have been reported as prostate-specific membrane antigen (PSMA) agent. We developed derivatives of GUL conjugated with NOTA or DOTA via a thiourea linker and tested their feasibility as PSMA imaging agents after labeling with 68 Ga. NOTA-GUL and DOTA-GUL were synthesized and labeled with 68 Ga using generator-eluted 68 GaCl 3 in 0.1 M HCl in the presence of 1 M NaOAc at pH 5.5. The stabilities of 68 Ga-labeled compounds in human serum were tested at 37.5 °C. A competitive binding assay was performed using the PSMA-positive prostate cancer cell line 22Rv1 and [ 125 I]MIP-1072 (PSMA-specific binding agent) as a tracer. Biodistribution and micro-PET studies were performed using 22Rv1-xenograft BALB/c nude mice. The radiolabeling efficiency of NOTA-GUL (>99%) was higher than that of DOTA-GUL (92%). The IC 50 of Ga-NOTA-GUL was 18.3 nM. In the biodistribution study, tumor uptake of 68 Ga-NOTA-GUL (5.40% ID/g) was higher than that of 68 Ga-DOTA-GUL (4.66% ID/g) at 1 h. Tumor/muscle and tumor/blood uptake ratios of 68 Ga-NOTA-GUL (31.8 and 135, respectively) were significantly higher than those of 68 Ga-DOTA-GUL (16.1 and 31.1, respectively). The tumor/kidney uptake ratio of 68 Ga-NOTA-GUL was 3.4-fold higher than that of 68 Ga-DOTA-GUL. 68 Ga-NOTA-GUL showed specific uptake to PSMA positive tumor xenograft and was blocked by co-injection of the cold ligand. In conclusion, we successfully synthesized 68 Ga-NOTA-GUL and 68 Ga-DOTA-GUL for prostate cancer imaging. 68 Ga-NOTA-GUL showed better radiochemical and biodistribution results. 68 Ga-NOTA-GUL may be a promising PSMA targeting radiopharmaceutical., (Copyright © 2018 Elsevier Ltd. All rights reserved.)

We present the case of a 79-year-old man with an elevated postprostatectomy prostate-specific antigen level who was sequentially imaged with positron emission tomography/computed tomography (PET/CT) using 18 F-fluciclovine followed by PSMA-targeted 18 F-DCFPyL. Although both imaging tests successfully identified the same pelvic recurrence, each radiotracer had its relative merits. This case highlights the differences between these two PET radiotracers, which are increasingly being used to image men with recurrent prostate cancer., (Copyright © 2017 Elsevier Inc. All rights reserved.)

A series of bis-3-chloropiperidines containing lysine linkers was synthesised as DNA alkylating model compounds by using a bidirectional synthetic strategy. These novel piperidine mustard based agents have been evaluated for their alkylating properties towards nucleic acids and were shown to alkylate and cleave DNA with strong preference for guanine residues. Our studies reveal that the introduction of aromatic groups in the side chain of the lysine linker has an impact on DNA alkylating activity. Analysis by ESI mass spectrometry enabled the verification of the reactive aziridinium ion formation. Overall, the results confirm our recently proposed reaction mechanism of bis-3-chloropiperidines., (Copyright © 2015 Elsevier Ltd. All rights reserved.)

N(ε)-carboxymethyllysine (CML) is an important driver of diabetic vascular complications and endothelial cell dysfunction. However, how CML dictates specific cellular responses and the roles of protein tyrosine phosphatases and ERK phosphorylation remain unclear. We examined whether endoplasmic reticulum (ER) localization of MAPK phosphatase-3 (MKP-3) is critical in regulating ERK inactivation and promoting NADPH oxidase-4 (Nox4) activation in CML-induced endothelial cell injury. We demonstrated that serum CML levels were significantly increased in type 2 diabetes patients and diabetic animals. CML induced ER stress and apoptosis, reduced ERK activation, and increased MKP-3 protein activity in HUVECs and SVECs. MKP-3 siRNA transfection, but not that of MKP-1 or MKP-2, abolished the effects of CML on HUVECs. Nox4-mediated activation of MKP-3 regulated the switch to ERK dephosphorylation. CML also increased the integration of MKP-3 with ERK, which was blocked by silencing MKP-3. Exposure of antioxidants abolished CML-increased MKP-3 activity and protein expression. Furthermore, immunohistochemical staining of both MKP-3 and CML was increased, but phospho-ERK staining was decreased in the aortic endothelium of streptozotocin-induced and high-fat diet-induced diabetic mice. Our results indicate that an MKP-3 pathway might regulate ERK dephosphorylation through Nox4 during CML-triggered endothelial cell dysfunction/injury, suggesting that therapeutic strategies targeting the Nox4/MKP-3 interaction or MKP-3 activation may have clinical implications for diabetic vascular complications., (Copyright © 2014 Elsevier Inc. All rights reserved.)

As the exopeptidase over-expressed in the cell surface of endothelial cells, aminopeptidase N (APN/CD13) is an essential target for tumor angiogenesis and metastasis. Based on the previous work of L-lysine amide derivatives in our laboratory, we designed and synthesized two series of L-lysine ureido derivatives as APN inhibitors. Within these compounds, one compound, 5d (IC₅₀ = 4.51 μM), showed similar inhibitory effect compared with Bestatin (IC₅₀ = 5.87 μM)., (Copyright © 2010 Elsevier Ltd. All rights reserved.)

Kaempferol has been reported to inhibit nitric oxide synthase and cyclooxygenase enzymes in animal models. The present study was designed to investigate whether kaempferol modulates the cyclooxygenase pathway via inhibition of nitric oxide production, which in turn contributes to its anti-inflammatory activity. Investigations were performed using carrageenan induced rat air pouch model. Inflammation was assessed by measurement of nitrites (nitrite, a breakdown product of nitric oxide), prostaglandin-E(2) levels and cellular infiltration in the pouch fluid exudates. To assess the anti-inflammatory effect of the extract, rat air pouch linings were examined histologically. The levels of nitrite and prostaglandin-E(2) in pouch fluid were measured by using Griess assay and ELISA respectively. Cell counts and differential counts were performed using a Coulter counter and Wright-Giemsa stain respectively. Kaempferol when administered orally at 50 and 100mg/kg dose showed significant inhibition of carrageenan induced production of nitrite (40.12 and 59.74%, respectively) and prostaglandin-E(2) generation (64.23 and 78.55%, respectively). Infiltration of the cells into the rat granuloma air pouch was also significantly inhibited by kaempferol. Modulation of cyclooxygenase pathway via inhibition of nitric oxide synthesis significantly contributes to kaempferol's anti-inflammatory activity. The present study characterizes the effects and mechanisms of naturally occurring phenolic flavonoid kaempferol, on inducible nitric oxide synthase expression and nitric oxide production. These results partially explain the pharmacological efficacy of flavonoids in general and kaempferol in particular as anti-inflammatory compounds., (Copyright (c) 2010 Elsevier B.V. All rights reserved.)

Sirtuins catalyze the NAD(+) dependent deacetylation of N(epsilon)-acetyl lysine residues to nicotinamide, O'-acetyl-ADP-ribose (OAADPR) and N(epsilon)-deacetylated lysine. Here, an easy-to-synthesize Ac-Ala-Lys-Ala sequence has been used as a probe for the screening of novel N(epsilon)-modified lysine containing inhibitors against SIRT1 and SIRT2. N(epsilon)-Selenoacetyl and N(epsilon)-isothiovaleryl were the most potent moieties found in this study, comparable to the widely studied N(epsilon)-thioacetyl group. The N(epsilon)-3,3-dimethylacryl and N(epsilon)-isovaleryl moieties gave significant inhibition in comparison to the N(epsilon)-acetyl group present in the substrates. In addition, the studied N(epsilon)-alkanoyl, N(epsilon)-alpha,beta-unsaturated carbonyl and N(epsilon)-aroyl moieties showed that the acetyl binding pocket can accept rather large groups, but is sensitive to even small changes in electronic and steric properties of the N(epsilon)-modification. These results are applicable for further screening of N(epsilon)-acetyl analogues., (Copyright (c) 2010. Published by Elsevier Ltd.)

The contribution of superoxide-mediated injury to oxidative stress is not fully understood. A potential mechanism is the reaction of superoxide with tyrosyl radicals, which either results in repair of the tyrosine or formation of tyrosine hydroperoxide by addition. Whether these reactions occur with protein tyrosyl radicals is of interest because they could alter protein structure or modulate enzyme activity. Here, we have used a xanthine oxidase/acetaldehyde system to generate tyrosyl radicals on sperm whale myoglobin in the presence of superoxide. Using mass spectrometry we found that superoxide prevented myoglobin dimer formation by repairing the protein tyrosyl radical. An addition product of superoxide at Tyr151 was also identified, and exogenous lysine promoted the formation of this product. In our system, reaction of tyrosyl radicals with superoxide was favored over dimer formation with the ratio of repair to addition being approximately 10:1. Our results demonstrate that reaction of superoxide with protein tyrosyl radicals occurs and may play a role in free radical-mediated protein injury., (Copyright 2010 Elsevier Inc. All rights reserved.)

Sirtuins are NAD(+) dependent deacetylases that modulate various essential cellular functions. Development of peptide based inhibitors of Sir2s would prove useful both as pharmaceutical agents and as effectors by which downstream cellular alterations can be monitored. Click chemistry that utilizes Huisgen's 1,3-dipolar cycloaddition permits attachment of novel modifications onto the side chain of lysine. Herein, we report the synthesis of peptide analogues prepared using click reactions on Nepsilon-propargyloxycarbonyl protected lysine residues and their characterization as inhibitors of Plasmodiumfalciparum Sir2 activity. The peptide based inhibitors exhibited parabolic competitive inhibition with respect to acetylated-peptide substrate and parabolic non-competitive inhibition with NAD(+) supporting the formation of EI(2) and E.NAD(+).I(2) complexes. Cross-competition inhibition analysis with the non-competitive inhibitor nicotinamide (NAM) ruled out the possibility of the NAM-binding site being the second inhibitor binding site, suggesting the presence of a unique alternate pocket accommodating the inhibitor. One of these compounds was also found to be a potent inhibitor of the intraerythrocytic growth of P.falciparum with 50% inhibitory concentration in the micromolar range.

Compound 24, 1-benzyl-N-[3-[spiroisobenzofuran-1(3H),4'-piperidin-1-yl]propyl] pyrrolidine-2-carboxamide was recently identified as a nociceptin/orphanin FQ (N/OFQ) peptide receptor (NOP) ligand. In this study, the in vitro and in vivo pharmacological profiles of Compound 24 were investigated. In vitro studies were performed measuring receptor and [(35)S]GTPgammaS binding and calcium mobilization in cells expressing the recombinant NOP receptor as well as using N/OFQ sensitive tissues. In vivo studies were conducted using the tail withdrawal assay in mice. Compound 24 produced a concentration-dependent displacement of [(3)H]N/OFQ binding to CHO(hNOP) cell membranes showing high affinity (pK(i) 9.62) and selectivity (1000 fold) over classical opioid receptors. Compound 24 antagonized with high potency the following in vitro effects of N/OFQ: stimulation of [(35)S]GTPgammaS binding in CHO(hNOP) cell membranes (pA(2) 9.98), calcium mobilization in CHO(hNOP) cells expressing the Galpha(qi5) chimeric protein (pK(B) 8.73), inhibition of electrically evoked twitches in the mouse (pA(2) 8.44) and rat (pK(B) 8.28) vas deferens, and in the guinea pig ileum (pK(B) 9.12). In electrically stimulated tissues, Compound 24 up to 1 microM did not modify the effects of classical opioid receptor agonists. Finally in vivo, in the mouse tail withdrawal assay, Compound 24 at 10 mg/kg antagonized the pronociceptive and antinociceptive effects of 1 nmol N/OFQ given supraspinally and spinally, respectively. Under the same experimental conditions Compound 24 did not affect the antinociceptive action of 3 nmol endomorphin-1 injected intrathecally. The present study demonstrated that Compound 24 is a pure, competitive, and highly potent non-peptide NOP receptor selective antagonist.

Previous studies from our lab have demonstrated cardiovascular abnormalities such as depressed mean arterial blood pressure and heart rate, endothelial dysfunction and attenuated pressor responses to vasoactive agents in streptozotocin diabetic rats. We investigated whether these abnormalities are due to diabetes-associated chronic activation of inducible nitric oxide synthase (iNOS). Control and streptozotocin (60 mg/kg, iv) diabetic rats were treated with either vehicle or N6-(1-Iminoethyl)-L-lysine dihydrochloride (L-NIL, 3 mg/kg/day, p.o), a specific inhibitor of iNOS for 8 weeks. At the end of treatment, the mean arterial blood pressure and heart rate were measured in freely moving conscious rats. Further, pressor responses to bolus doses of methoxamine were determined. Endothelial nitric oxide synthase (eNOS) and iNOS expression as well as nitrotyrosine (NT) levels were assessed in the heart and superior mesenteric arteries by western blot and immunohistochemistry. Untreated diabetic rats showed depressed mean arterial blood pressure and heart rate and exhibited vascular hyporeactivity that were significantly improved by treatment with L-NIL. Further, decreased eNOS expression and increased iNOS expression and activity were associated with increased NT levels in the heart and superior mesenteric arteries of untreated diabetic rats. L-NIL treatment of diabetic rats normalized the expression of eNOS and NT levels without any effect on iNOS expression in the heart and superior mesenteric arteries. The results of our study suggest that induction of iNOS in cardiovascular tissues contributes significantly to the depressed mean arterial blood pressure, heart rate and pressor responses to vasoactive agents. Chronic inhibition of iNOS in diabetes may prove beneficial in the treatment of cardiovascular abnormalities.

Myxochelin A (1) is an inhibitor of tumor cell invasion produced by the bacterium belonging to the genus Nonomuraea. In order to obtain more potent inhibitors, a series of myxochelin analogues [2 and (S)-3-17] were synthesized through the coupling of lysine or diaminoalkane derivatives and appropriately protected hydroxybenzoate, followed by modification of functional groups and deprotection. These compounds were evaluated for their inhibitory activity against invasion of murine colon 26-L5 carcinoma cells. Among the synthetic analogues tested, compound (S)-6 which possesses carbamoyl group at C-1 was found to be the most potent antiinvasive agent and is considered to be a promising lead molecule for the antimetastasis. Compound (S)-6 was also shown to inhibit gelatinase activities of MMP-2 and MMP-9 and in vivo lung metastasis in mice.

Chemical guiding cues are being exploited to stimulate neuron adhesion and neurite outgrowth. In this study, an amino-functioned PLLA, lysine-capped PLLA [K-(CH(2))(n)-PLLA (n=2, 5, 8)], was synthesized with different length of linking spaces between lysine molecule and PLLA backbone. Drop-cast films were fabricated from K-(CH(2))(n)-PLLA/PLLA blends (10/90, w/w) and amino groups were detected on the surfaces of the resultant films. More amine groups were detected on the surface and the hydrophilicity of the films was obviously improved by annealing the films in water. The representative atomic force microscopy (AFM) images indicated that incorporation of lysine-capped PLLA into PLLA matrix increased the roughness of the films and resulted in a phase separation with distinct two nano-domains which may correspond to the hydrophilic and hydrophobic domains. Furthermore, the laminin-derived peptides, CYIGSR (Cys-Tyr-Ile-Gly-Ser-Arg) and CSIKVAV (Cys-Ser-Ile-Lys-Val-Ala-Val), were jointly tethered to the amine groups of lysine-capped PLLA by a linking reagent sulfo-succinimidyl 4-(N-maleimidomethyl) cyclohexane-1-carboxylate (Sulfo-SMCC). The neonatal mouse cerebellum C17.2 stem cells were seeded on the peptides-grafted K-(CH(2))(n)-PLLA/PLLA (n=2, 5, 8) films and pure PLLA films were used as controls. Improved viability and longer neurites were obtained on the peptide-grafted films than PLLA film over the cultivation period, especially for K-(CH(2))(5)-PLLA/PLLA, which had the highest peptide density of 0.28+/-0.03 microg/cm(2). This study highlights the potential of using the lysine-capped PLLA with laminin-derived peptides for promoting nerve regeneration.

The aim of this study was to clarify the influence of hyperglycemia on the deposition of aggregated protein in the glomeruli of diabetic mice. KK-A(y) mice injected with aggregated bovine serum albumin accumulated more of it in the glomeruli than did ICR mice. There were no histological alterations in the glomeruli of KK-A(y) mice. KK-A(y) mice given voglibose in mouse-chow for 2 weeks had significantly reduced blood glucose, glycated albumin, and hemoglobin A(1C) levels compared with control mice. The voglibose-treated KK-A(y) mice were injected with aggregated bovine serum albumin and accumulated significantly less albumin in the glomeruli than did the control mice. Pioglitazone decreased blood glucose levels compared with the control, and reduced the glomerular deposition of aggregated albumin. Glomerular aggregated bovine serum albumin levels and blood glucose levels were reduced significantly by the injection of insulin. Six times more advanced glycation endproducts were produced from aggregated bovine albumin than from non-aggregated bovine albumin on incubation with glucose and L-lysine in vitro. Glucose-loaded ICR mice generated more advanced glycation endproducts from aggregated albumin, and had more aggregated bovine albumin in the glomeruli. It was suggested that hyperglycemia contributes to an increase in the deposition of aggregated protein in glomeruli even early on in diabetes.

A series of novel l-lysine derivatives were designed, synthesized, and assayed for their inhibitory activities on amino-peptidase N (APN)/CD13 and matrix metalloproteinase-2 (MMP-2). The preliminary biological test showed that most of the compounds displayed a high inhibitory activity against MMP-2 and a low activity against APN except compound B6 which exhibited good potency (IC(50)=13.2microM) similar with APN inhibitor Bestatin (IC(50)=15.5microM), and could be used as lead compound in the future.

Central heme oxigenase-carbon monoxide (HO-CO) pathway has been shown to play a pyretic role in the thermoregulatory response to restraint. However, the specific site in the central nervous system where CO may act modulating this response remains unclear. LC is rich not only in sGC but also in heme oxygenase (HO; the enzyme that catalyses the metabolism of heme to CO, along with biliverdin and free iron). Therefore, the possible role of the HO-CO-cGMP pathway in the restraint-induced-hypothermia by LC neurons was investigated. Body temperature dropped about 0.7 degrees C during restraint. ZnDPBG (a HO inhibitor; 5 nmol, intra-LC) prevented the hypothermic response during restraint. Conversely, induction of the HO pathway in the LC with heme-lysinate (7.6 nmol, intra-LC) intensified the hypothermic response to restraint, and this effect was prevented by pretreatment with ODQ (a sGC inhibitor; given intracerebroventricularly, 1.3 nmol). Taken together, these data suggest that CO in the LC produced by the HO pathway and acting via cGMP is implicated in thermal responses to restraint.

The aim of the present study was to investigate the role of the spinal cord heme oxygenase (HO)-carbon monoxide (CO)-soluble guanylate cyclase (sGC)-cGMP pathway in nociceptive response of rats to the formalin experimental nociceptive model. Animals were handled and adapted to the experimental environment for a few days before the formalin test was applied. For the formalin test 50 microl of a 1% formalin solution was injected subcutaneously in the dorsal surface of the right hind paw. Following injections, animals were observed for 1 h and flinching behavior was measured as the nociceptive response. Thirty min before the test, rats were pretreated with intrathecal injections with the HO inhibitor, zinc deuteroporphyrin 2,4-bis glycol (ZnDPBG) or heme-lysinate, which is known to induce the HO pathway. Control animals were treated with vehicles. We observed a significant increase in nociceptive response of rats treated with ZnDPBG, and a drastic reduction of flinching nociceptive behavioral response in the heme-lysinate treated animals. Furthermore, the HO pathway seems to act via cGMP, since methylene blue (a sGC inhibitor) prevented the reduction of flinching nociceptive behavioral response caused by heme-lysinate. These findings strongly indicate that the HO pathway plays a spinal antinociceptive role during the formalin test, acting via cGMP.

Corneal neovascularization is a significant, sight-threatening complication of many ocular surface disorders. Various growth factors and proteinases are involved in corneal neovascularization. The data supporting a causal role for vascular endothelial growth factor (VEGF) and matrix metalloproteinases (MMPs) are extensive. Inhibition of VEGF and MMPs is a main strategy for treating corneal neovascularization. Several findings have shown that corneal neovascularization can be reduced by using anti-VEGF and anti-MMPs agents. Efficacy of a nutrient mixture (NM) containing lysine, proline, ascorbic acid, and green tea extract has been demonstrated for reducing VEGF and MMPs secretion by various cells. Moreover, NM can inhibit endothelial cell migration and capillary tube formation. We herein note that topical application of NM is potentially useful for inhibiting corneal neovascularization and restoration of corneal clarity. Further investigations in animal models are needed to place NM alongside corneal neovascularization therapeutics.

Trichinella spiralis infection causes hyperexcitability in enteric after-hyperpolarising (AH) sensory neurons that is mimicked by neural, immune or inflammatory mediators known to stimulate adenylyl cyclase (AC)/cyclic 3',5'-adenosine monophosphate (cAMP) signaling. The hypothesis was tested that ongoing modulation and sustained amplification in the AC/cAMP/phosphorylated cAMP related element binding protrein (pCREB) signaling pathway contributes to hyperexcitability and neuronal plasticity in gut sensory neurons after nematode infection. Electrophysiological, immunological, molecular biological or immunochemical studies were done in T. spiralis-infected guinea-pigs (8000 larvae or saline) after acute-inflammation (7 days) or 35 days p.i., after intestinal clearance. Acute-inflammation caused AH-cell hyperexcitability and elevated mucosal and neural tissue levels of myeloperoxidase, mast cell tryptase, prostaglandin E2, leukotrine B4, lipid peroxidation, nitric oxide and gelatinase; lower level inflammation persisted 35 days p.i. Acute exposure to blockers of AC, histamine, cyclooxygenase or leukotriene pathways suppressed AH-cell hyperexcitability in a reversible manner. Basal cAMP responses or those evoked by forskolin (FSK), Ro-20-1724, histamine or substance P in isolated myenteric ganglia were augmented after T. spiralis infection; up-regulation also occurred in AC expression and AC-immunoreactivity in calbindin (AH) neurons. The cAMP-dependent slow excitatory synaptic transmission-like responses to histamine (mast cell mediator) or substance P (neurotransmitter) acting via G-protein coupled receptors (GPCR) in AH neurons were augmented by up to 2.5-fold after T. spiralis infection. FSK, histamine, substance P or T. spiralis acute infection caused a 5- to 30-fold increase in cAMP-dependent nuclear CREB phosphorylation in isolated ganglia or calbindin (AH) neurons. AC and CREB phosphorylation remained elevated 35 days p.i.. Ongoing immune activation, AC up-regulation, enhanced phosphodiesterase IV activity and facilitation of the GPCR-AC/cAMP/pCREB signaling pathway contributes to T. spiralis-induced neuronal plasticity and AH-cell hyperexcitability. This may be relevant in gut nematode infections and inflammatory bowel diseases, and is a potential therapeutic target.

Although in situ hybridization studies have revealed the presence of kainate receptor (KAR) mRNA in neurons of the rat medial entorhinal cortex (mEC), the functional presence and roles of these receptors are only beginning to be examined. To address this deficiency, whole cell voltage clamp recordings of locally evoked excitatory postsynaptic currents (EPSCs) were made from mEC layer II and III neurons in combined entorhinal cortex-hippocampal brain slices. Three types of neurons were identified by their electroresponsive membrane properties, locations, and morphologies: stellate-like "Sag" neurons in layer II (S), pyramidal-like "No Sag" neurons in layer III (NS), and "Intermediate Sag" neurons with varied morphologies and locations (IS). Non-NMDA EPSCs in these neurons were composed of two components, and the slow decay component in NS neurons had larger amplitudes and contributed more to the combined EPSC than did those observed in S and IS neurons. This slow component was mediated by KARs and was characterized by its resistance to either 1-(4-aminophenyl)-4-methyl-7,8-methylenedioxy-5H-2,3-benzodiazepine hydrochloride (GYKI 52466, 100 microM) or 1,2,3,4-tetrahydro-6-nitro-2,3-dioxo-benzo[lsqb]f[rsqb]quinoxaline-7-sulfonamide (NBQX, 1 microM), relatively slow decay kinetics, and sensitivity to 6-cyano-7-nitroquinoxaline-2,3-dione (CNQX, 10-50 microM). KAR-mediated EPSCs in pyramidal-like NS neurons contributed significantly more to the combined non-NMDA EPSC than did those from S and IS neurons. Layer III neurons of the mEC are selectively susceptible to degeneration in human temporal lobe epilepsy (TLE) and animal models of TLE such as kainate-induced status epilepticus. Characterizing differences in the complement of postsynaptic receptors expressed in injury prone versus injury resistant mEC neurons represents an important step toward understanding the vulnerability of layer III neurons seen in TLE.

The aim of the present study was to investigate the role of the peripheral heme oxygenase (HO)-carbon monoxide (CO) pathway on nociceptive response of rats to the formalin experimental model of pain. Animals were handled and adapted to the experimental environment for a few days before the formalin test was applied. For the formalin test, 50 microl of a 1% formalin solution was used and injected subcutaneously in the dorsal surface of the right hind paw. Following injections, animals were observed for 1 h, and flinching behavior was measured as the nociceptive response. Twenty minutes before the test rats were pretreated with podal injections with the HO inhibitor, zinc deuteroporphyrin 2,4-bis glycol (ZnDPBG) or heme-lysinate, which is known to induce the HO pathway. Control animals were treated with vehicles. We observed a significant increase on nociceptive response of rats treated with ZnDPBG, and a drastic reduction of flinching nociceptive behavioral response in the heme-lysinate and CO treated animals. Among the three different HO products, CO seems to account for the heme-lysinate effect because the injection of the gas attenuated the flinching response whereas biliverdine and deferoxanine (an iron chelator) failed to cause any significant change. Furthermore, CO seems to act via cGMP, since methylene blue (a soluble guanylate cyclase inhibitor) prevented the reduction of the flinching nociceptive behavioral response caused by heme-lysinate. These findings strongly indicate that CO is the HO pathway product that plays an antinociceptive role during the formalin test, acting via cGMP.

The ectoenzymes dipeptidyl peptidase IV (DP IV, CD26) and aminopeptidase N (APN, CD13) have been implicated in the regulation of T cell activation and function. Both DP IV and APN serve as targets of efficient enzymatic inhibitors which induce autocrine production of TGF-beta1 and subsequent suppression of T cell proliferation and cytokine release. Here, we tested the hypothesis that the simultaneous inhibition of DP IV and APN enzymatic activity on leukocytes potentiates the anti-inflammatory effect of single DP IV or APN inhibitors. Our data show that the combined application of DP IV and APN inhibitors increased suppression of DNA synthesis in human peripheral blood mononuclear cells and isolated T cells in vitro when compared to the use of a single ectopeptidase inhibitor. Moreover, the combined action of DP IV and APN inhibitors markedly increased TGF-beta1 production associated with the observed immunosuppressive effects. In vivo, targeting DP IV and APN provided a potent therapeutic approach for the treatment of experimental autoimmune encephalomyelitis, an animal model of multiple sclerosis. Taken together, our study suggests that combined DP IV and APN inhibition on pathogenic T cells represents a novel and efficient therapy for autoimmune disease of the central nervous system by a mechanism that involves an active TGF-beta1-mediated anti-inflammatory effect at the site of pathology.

The effect of a lysine-deficient diet on cationic amino acid transporter (CAT1-3) mRNA expression was determined in broiler chickens. Chicks consumed a lysine-adequate (LA; 1.3% lysine) or lysine-deficient (LD; 0.7% lysine) diet. Pair-fed chicks consumed the LA diet in an amount equal to that consumed by LD chicks during the previous day (PLA). CAT 1-3 mRNA expression in the liver, pectoralis and bursa of LD chicks were lower than that of LA and PLA chicks (P<0.05), and levels were not detectable in LD chick thymus. High affinity CAT mRNA expression in isolated bursacytes was 16-fold higher in LD chicks than that of LA chicks (P<0.001). Thymocyte high affinity CAT mRNA expression was 5-fold lower than that of LA chicks (P<0.05). The summed amount of high affinity CAT-1 and CAT-3 mRNA expression in chicks fed a lysine adequate diet was highly correlated (r2=0.51; P<0.001) to a tissue's growth during a lysine deficiency or feed restriction. In the thymus and bursa of LD chicks, CAT mRNA levels differed between resident lymphocytes and their surrounding tissues. By expressing high affinity CAT isoforms, developing lymphocytes may have a greater ability to obtain lysine than their surrounding tissue during a lysine deficiency.

The effect of an acute phase response (APR) on cationic amino acid transporter (CAT1-3) mRNA expression in liver, muscle, bursa and thymus was determined in broiler strain chickens. The APR was initiated by injecting Salmonella typhimurium lipopolysaccharide subcutaneously (LPS; 1 mg/kg bw). In Experiment 1, CAT1-3 mRNA expression was determined at multiple time points following LPS administration. LPS increased bursa and liver total and high affinity CAT mRNA expression (P<0.05) and transiently increased pectoralis total CAT mRNA expression (P<0.05). Total CAT mRNA expression in the thymus decreased 7.7-fold from 0 to 8 h after LPS injection (P<0.05). In Experiment 2, fasted chicks were uninjected or LPS-injected. LPS increased total and high affinity CAT mRNA 2-fold in both the bursa and liver (P<0.05) and did not change thymus total and high affinity CAT mRNA expression (P>0.05). LPS increased liver weight only (P<0.05) and did not alter the plasma lysine and arginine concentration (P>0.05). In Experiments 3 and 4, thymocyte proliferation and total protein content were dependent upon the media lysine concentration (P<0.001). The inability of the thymus to compete for lysine and arginine during the APR may limit the ability of thymocytes to develop during infections.

Carnosine has been characterized as a putative neurotransmitter and implicated as having a possible role in neuron-glia cell interactions. We previously confirmed that central administration of carnosine induced hyperactivity in chicks. In the present study, we investigated the effects of nitric oxide (NO) synthase (NOS) inhibitors on carnosine-induced hyperactivity in chicks. Carnosine-induced (3.2 micromol) hyperactivity was attenuated by intracerebroventricular (i.c.v.) co-administration with a non-selective NOS inhibitor N(G)-nitro-L-arginine methyl ester HCl (200 and 400 nmol) in a dose-dependent manner, while the hyperactivity was not attenuated by the inactive isomer of the NOS inhibitor N(G)-nitro-D-arginine methyl ester HCl (400 nmol). The i.c.v. injection of a selective inhibitor of inducible NOS (iNOS) L-N(6)-(1-iminoethyl) lysine HCl (400 nmol) did not affect carnosine-induced hyperactivity. These results suggest that carnosine-induced hyperactivity may be linked to the constitutive NOS (cNOS), rather than iNOS, in the brain. Central carnosine may regulate brain function and/or behaviors by NO generation via cNOS in chicks.

Accumulation of advanced glycation end products (AGEs) induces alterations in the intracellular redox balance, leading cells to functional injury. Current literature reports that intracellular signaling triggered by the interaction of AGEs with their specific receptors RAGEs depends on the cell type and the state of activation/stress. In this work, NT2 human neurons were exposed for 48 h to glycated fetal serum containing 750-3000 pmol/ml pentosidine; the treatment induced an increase in apoptosis rate linear with AGE concentration up to 1500 pmol/ml, but necrotic death was elicited with the highest AGE amount employed (3000 pmol/ml pentosidine). Pentosidine at 1500 pmol/ml, which was the concentration responsible for the highest apoptotic effect (40% of apoptotic neurons), was able to determine early generation of intracellular reactive oxygen species and increase in RAGE levels. Under these conditions, protein kinase C (PKC) delta activity was increased approximately 2-fold, and DNA binding activity of redox-sensitive transcription factor activator protein-1 (AP-1) was enhanced 2.5-fold. A relationship among oxidative stress, PKCdelta activity, AP-1 activation, and apoptosis was demonstrated by pretreating neurons with 500 muM vitamin E, with 20 mug/ml Ginkgo biloba extract, or with 3 muM Rottlerin, inhibitor of PKCdelta; these pretreatments were able to protect neurons from the glycoxidation-dependent effects.

Lipopolysaccharides (LPS), otherwise termed 'endotoxins', are outer-membrane constituents of Gram-negative bacteria. Lipopolysaccharides play a key role in the pathogenesis of 'Septic Shock', a major cause of mortality in the critically ill patient. Therapeutic options aimed at limiting downstream systemic inflammatory processes by targeting lipopolysaccharide do not exist at the present time. We have defined the pharmacophore necessary for small molecules to specifically bind and neutralize LPS and, using animal models of sepsis, have shown that the sequestration of circulatory LPS by small molecules is a therapeutically viable strategy. In this paper, the interactions of a focused library of lysine-spermine conjugates with lipopolysaccharide (LPS) have been characterized. Lysine-spermine conjugates with the epsilon-amino terminus of the lysinyl moiety derivatized with long-chain aliphatic hydrophobic substituents in acyl or alkyl linkage bind and neutralize bacterial lipopolysaccharides, and may be of use in the prevention or treatment of endotoxic shock states.

The inducible form of nitric oxide synthase (iNOS) is present in advanced atherosclerotic lesions. The aim of the present paper was to compare the functionality of iNOS in rabbits fed a 0.3% cholesterol-diet for 24 weeks (Baseline), and 36 weeks, with l-arginine (l-Arg) or vehicle supplementation (Saline) for the last 12 weeks. N-iminoethyl-l-lysine (l-NIL; 10 microM), a selective inhibitor of iNOS, potentiated the contractions to phenylephrine in aortas from Baseline, Saline and l-Arg rabbits confirming the presence of a functional iNOS. In l-Arg rabbits, the contractions induced by l-NIL were less pronounced than those noted in Baseline and Saline rabbits; superoxide dismutase (150 U/ml) significantly increased the phenylephrine-induced contractions only in the l-Arg rabbits. In the presence of NADPH, aortas from l-Arg rabbits produced more superoxide anions than aortas from saline rabbits as evidenced by the lucigenin-enhanced chemiluminescence technique. In conclusion, our results show functional and biochemical evidence for an increased superoxide anion production in atherosclerotic aortas from hypercholesterolemic rabbits treated with l-Arg for 12 weeks. These data may thus help to explain the lack of beneficial effects of l-Arg on atherosclerosis progression in long-term experimental hypercholesterolemia as well as in severely atherosclerotic humans.

Oxidative stress has been implicated in the pathophysiology of Alzheimer's disease (AD) and diabetes mellitus (DM). N epsilon-carboxymethyllysine (CML) is an advanced glycation end product (AGE) recently found to be associated with oxidative stress mechanisms. Using immunocytochemical methods we examined the distribution of CML in brain tissue from AD and DM subjects and aging controls. CML reactivity was present in the cytoplasm of neurons, but there were marked differences in the intensity of expression, number of cells, and topographical distribution. CML expression was higher in hippocampus than in frontal and temporal cortex. In the hippocampus, neuronal and, to an extent, glial expression was more marked in CA3 and CA4 than in CA1 and CA2. In AD, CML was found to be coexpressed with tau protein, showing the similar neurofibrillary tangle shape, as well as in neuritic plaques but not in the core of amyloid plaques. We noted an increasing degree of CML expression such that the highest reactivity was evident in those with both AD and DM, followed by AD, DM, and aging controls. There was an inverse relationship between Braak staging and topography of CML expression. Although DM cases did not show Abeta deposition or neurofibrillary tangles, these findings suggest increased CML expression is not limited to AD. Nonetheless, high CML expression in AD with coexistent DM suggests there are additive effects compared with AD alone. It is plausible that the microangiopathy also associated with DM could worsen AD pathogenesis.

L-Lysine HCl is being proposed to be a possible biocompatible adjuvant to enhance immune response by virtue of its probable non-specific bridging action and cellular proliferation properties. This proposal has been tried to be substantiated by carrying out experimentation where L-lysine HCl has been used as an adjuvant (various groups based on mode of application and frequency of booster dose were designed) in tuberculosis vaccination experiments with heat killed Mycobacterium tuberculosis (MTB) and Bacille Calmette Guerin (BCG). Antibody titre has been followed in all the experiments as a measure of immune response. Amongst the various groups designed, group 1A (L-lysine HCl was given at a separate site as that of the antigen; lysine booster was given to this group intermittently, i.e. lysine given on 0th, 7th, 14th, 21st days of immunization) came out as the stand-alone leader. This mode and frequency of application was then compared with a group which received a standard adjuvant, viz. alhydrogel. Results were obtained which showed the following order in terms of decreasing antibody titre: alhydrogel group > lysine group > control group. Considering the biocompatible nature of lysine in comparison with the reportedly hazardous nature of alum adjuvants, we propose L-lysine HCl as a probable adjuvant in vaccination.

The purpose of this study was to investigate the chemical influence of chemo-mechanical caries removal (CMCR) on surface topography of dental ceramic materials. Thirty samples of three different types of ceramics (Vita Omega, Vita Alpha, and Procera AllTitan) were manufactured and used. With an optical profilometer, designed with a confocal setting of its optics, (CLSP). and an atomic force microscope (AFM) the surface topography of the specimens was investigated at two different resolutions. The specimens were exposed to a solution (64 mm NaOCl, 27 mM amino acids, pH = 11) supposed to be more aggressive than Carisolv (34 mm NaOCl, 53mM amino acids, pH = 11), a CMCR system commercially available. After exposure for 5, 10, and 20 min, respectively, areas measured were relocated and the surface topography was reinvestigated. Parameters describing the amplitude, spatial, and hybrid topographical variation were calculated. In general, after 20 min CMCR solution exposure, the AFM topographical parameters were reduced for Vita Omega, and increased for Vita Alpha", whereas the CLSP topographical parameters of Procera AllTitan were slightly reduced. Thus, the results of this study showed minor influence on surface topography after exposure to CMCR solution for 20 min on the dental ceramics Vita Omega, Vita Alpha, and Procera AllTitan.

Objective: Bioprosthetic calcification can be significantly mitigated by both increased concentrations of glutaraldehyde (GA) and the introduction of diamine (DA) bridges. The purpose of the present study was to evaluate whether an optimal effect of DA-enhanced fixation can be achieved by titration of dialdehyde and diamine concentrations., Methods: Porcine aortic roots were fixed at 0.05% GA (under-fixation) or 0.2% GA and 0.7% GA (commercial fixation). An interim step of DA treatment (L-Lysine; 0, 25, 50 or 100 mM; 37 degrees C; 2 days) was followed by completion of the GA fixation (37 degrees C; 5 days). Aortic wall coupons (12 mm) were punched out and implanted subcutaneously into seven-week old Long-Evans rats for 60 days. Calcium content was assessed by atomic absorption spectroscopy and histology., Results: Increasing the L-Lysine concentrations beyond 25 mM was essential to achieve the anti-calcific effect of DA-enhanced fixation. This effect was proportional to the GA concentrations applied. Compared to non-enhanced GA fixation (0 mM DA), calcification increased by 17.4% (p = 0.2114) in 0.05% fixed tissue but decreased by 32.0% (p < 0.0001) and 45.1% (p < 0.0002) in 0.2% and 0.7% GA, respectively, when the DA concentration was 100 mM. Histologically the extent, but not the pattern of calcification, was affected., Conclusion: The calcium mitigating effect of diamine-treatment as an interim step of glutaraldehyde fixation is proportional to the GA concentration applied. Within commercial 0.7% GA fixation 100 mM DA has the potential to practically halve aortic wall calcification.

Nacystelyn (NAL), a recently developed lysine salt of N-acetyl-L-cytokine (NAC) has mucolytic and antioxidant properties. In this study, we investigated the effect of NAL upon oxidant-mediated interleukin (IL)-8 release and the activation of the redox-sensitive transcription factors AP-1, NF-kappaB, and C/EBP in a human alveolar epithelial cell line (A549). NAL (5 mM) enhanced intracellular glutathione (GSH) after 4 h and abolished H(2)O(2)-induced IL-8 release from A549 cells. This was associated with inhibition of NF-kappaB and C/EBP DNA-binding, measured by the Electrophoretic Mobility Shift Assay (EMSA). NAL also abolished the transcriptional activation of IL-8 in an IL-8-chloramphenicol acetyl transferase (CAT) reporter system, transfected into A549 cells. Supernatants obtained from H(2)O(2)-treated A549 cells induced chemotaxis of polymorphonuclear neutrophils, which could be inhibited by co-incubation with NAL. These data indicate that NAL may be used to modulate pro-inflammatory process by inhibiting cytokine release in the lungs and thus has therapeutic potential in inflammatory lung diseases.

We have developed a reproducible allergic rhinitis model showing biphasic nasal blockage on repetitive inhalation challenge with Japanese cedar pollen in sensitised guinea pigs. The role of nitric oxide (NO) in inducing nasal blockage was evaluated with this model. N(omega)-nitro-L-arginine methyl ester (L-NAME), a non-selective NO synthase (NOS) inhibitor, intravenously administered before the challenge, significantly inhibited both early and late nasal blockage by approximately 80% and 50%, respectively. When L-NAME treatment was performed after the challenge, the late response was inhibited by approximately 70%. This inhibition was completely reversed by co-administration of L-arginine. However, aminoguanidine and L-N(6)-(1-iminoethyl)lysine, selective inhibitors of inducible NOS, negligibly influenced the degree of nasal blockage. Meanwhile, the alpha-adrenergic agonist, naphazoline, strongly suppressed both early and late nasal blockage. These results indicate that NO, likely produced by constitutive rather than inducible NOS, plays a major role in the occurrence of biphasic nasal blockage, primarily by inducing vasodilatation.

Uric acid has been hypothesized as being one of the more important antioxidants in limiting the accumulation of glycosylated endproducts in birds. Study 1 was designed to quantitatively manipulate the plasma concentrations of uric acid using hemin and allopurinol while study 2 determined their effects on skin pentosidine, the shear force value of Pectoralis major muscle, plasma glucose, body weight and chemiluminescence monitored oxidative stress in broiler chickens. Hemin was hypothesized to raise uric acid concentrations thereby lowering oxidative stress whereas allopurinol was hypothesized to lower uric acid concentrations and raise measures of oxidative stress. In study 1 feeding allopurinol (10 mg/kg body weight) to 8-week-old broiler chicks (n=50) for 10 days decreased plasma uric acid by 57%. However, hemin (10 mg/kg body weight) increased uric acid concentrations 20%. In study 2, 12-week-old broiler chicks (n=90) were randomly assigned to either an ad libitum (AL) diet or a diet restricted (DR) group. Each group was further divided into three treatments (control, allopurinol or hemin fed). Unexpectedly, hemin did not significantly effect uric acid concentrations but increased (P<0.05) measures of chemiluminescence dependent oxidative stress in both the DR and AL birds probably due to the ability of iron to generate oxygen radicals. Allopurinol lowered concentrations of uric acid and increased (P<0.05) the oxidative stress in the AL birds at week 22, reduced (P<0.05) body weight in both the AL and DR fed birds at 16 and 22 weeks of age, and markedly increased (P<0.001) shear force values of the pectoralis major muscle. Skin pentosidine levels increased (P<0.05) in AL birds fed allopurinol or hemin fed birds, but not in the diet restricted birds at 22 weeks. The significance of these studies is that concentrations of plasma uric acid can be related to measures of oxidative stress, which can be linked to tissue aging.

7-Nitroindazole, an inhibitor of neuronal nitric oxide synthase, reportedly inhibits hypercapnic dilation, but tetrodotoxin, an inhibitor of neuronal transmission, reportedly does not. Thus, evidence does not uniformly support the hypothesis of a neurogenic link to the hypercapnic response. Others suggest the hypercapnic response is mediated by a K(ATP) ion channel. In the following studies, we observed that topically administered tetrodotoxin inhibited dilations produced by hypercapnia. In addition, topical tetrodotoxin and either topical or intraperitoneal 7-nitroindazole, inhibited dilations produced by the K(ATP) channel openers, cromakalim and pinacidil. Inhibition of hypercapnic dilation and inhibition of dilation by the openers of the K(ATP) channel was immediately reversed by either L-lysine or L-arginine, amino acids previously shown to facilitate opening of the channel. The data strongly supports the previous conclusion that there is a K(ATP) ion channel link in the response of pial arterioles to hypercapnia. The location of the channel is not established by these data, nor is it known whether the action of tetrodotoxin on the channel was direct or indirect.

The aggressiveness in terms of apogenic and necrotic activities of lysine-derived anionic surfactants towards a mammalian cell line (U937) were studied. We used N(alpha)N(epsilon)-dioctanoyl lysine as the model surfactant with lysine, tris, Na(+) or Li(+) as counterions. The aggressiveness of the different surfactants was assessed as the concentrations leading to apoptosis or necrosis after the cells were incubated in the presence of surfactants and then cultivated in their absence. Used in the same conditions, acetates associated with the same cations, had no effects on the cells. Our results show that the aggressiveness of the surfactants depended on the nature of the counterions: it was high when surfactants were associated with small counterions, and low with large counterions.

Amino acid deficiency was modeled by feeding rats a diet deficient in the essential L-amino acid, L-lysine (L-lys). There is a rapid anorectic response to such a diet, and a strong preference for L-lys develops during the deficiency. While the brain appears to trigger this preference, the peripheral pathways that inform the brain about the deficiency are not well understood. One possible information pathway may utilize an "amino acid sensor" in the hepatoportal region. In the present study, we measured in vivo neural activity in normal and L-lys-deficient rat. Compared to the normally fed controls, we found an approximately 100-fold increase in the firing sensitivity of the L-lys sensors in vagal afferent fibers from the hepatoportal region of the L-lys-deficient rats. Injection of 10 mM L-lys into the hepatoportal circulation, but not D-lysine (D-lys), evoked an increase in afferent activity. While L-lys deficiency enhanced the sensitivity of the L-lys sensors, the sensitivity due to other small amino acid sensors remained unchanged. Finally, we observed a time-dependent response of the lysine sensors to lysine deficiency. It required 3-4 days of maintenance on the lysine-deficient diet for the sensitivity of the L-lys sensors to change. Taken together, these results provide additional data to support the existence of putative L-amino acid sensors in the hepatoportal circulation. Additionally, they describe several characteristics of the L-lys sensors and show that these sensors may contribute to the adaptation to dietary L-lys deficiency and to maintenance of L-amino acid homeostasis.

We have qualitatively and quantitatively analysed the anatomical connections within and between rat primary visual cortex (V1) and the rim region surrounding area V1, using both ortho- and retrograde anatomical tracers (biotinylated dextran amine, biocytin, cholera toxin b subunit). From the analysis of the projection patterns, and with the assumption that single points in the rat visual cortex, as in other species, have projection fields made up of multiple patches of terminals, we have concluded that just two V1 recipient areas occupy the entire rim region: an anterolateral area, probably homologous with V2 in other mammals, previously named Oc2L, and a medial area, corresponding to Oc2M. A non-reciprocal projection from the anterolateral area to the medial area was identified. Small injections (300-600microm uptake zone diameter) of the anatomical tracers in area V1, or in the rim region, label orthograde intra-areal connections from each injection site to offset small patches. This is found in all regions of the rim and within at least the relatively expanded central dorsal field representation of V1. From the extent of these projections in V1 and the two rim regions, we have estimated that the neurons at the injection site send diverging laterally spreading projections to other neurons whose receptive fields share any part of the area included in the pooled receptive fields of the neurons at the injection site. Orthogradely labelled inter-areal feedforward projections from V1 to either rim region are estimated to diverge in their projections to neurons that share any part of the area of the pooled receptive fields of the V1 intra-areal connectional field of the same injection. The orthogradely labelled feedback projections to V1, from injection sites in either rim region, reach V1 neurons whose pooled receptive fields match those of the neurons in the rim injection site, i.e. with no divergence. Despite patchy anatomical connectional fields, our estimates indicate that visual space is represented continuously in the receptive fields of neurons postsynaptic to each intra- or inter-areal field of orthograde label. We suggest that, despite the absence of regularly mapped functions in rat V1 (e.g. regularly arranged orientation specificity), which in other species (e.g. primates and cats) relate to the patchy connectional patterns, the rat visual cortex intra- and inter-areal anatomical connections follow similar patterns and scaling factors to those in other species.

Increased levels of nitric oxide (NO) are found in rejecting renal allografts. Inducible NO synthase (iNOS) in infiltrating monocytes/macrophages could lead to NO bursts. NO may modulate the inflammatory response of early rejection due to its high reactivity with superoxide to yield peroxynitrite. To define the role of iNOS in acute renal allograft, rejection effects of the specific iNOS blockers iminoethyl-lysine and 7-butylhexahydro-1H-azepin-2-imine, monohydrochloride on renal function and morphology were investigated in renal allografts. Lewis rats received Brown Norway grafts with one kidney left in situ. All recipients were treated with low dose cyclosporine-A (2.5 mg/kg BW/day s.c.) to allow moderate rejection. In addition, one group received iminoethyl-lysine (10 mg/kg BW/day gavage) and one group received butylhexahydro-azepin-imine (3.4 mg/kg BW/day i.p.). Sham operated Brown Norway donor rats served as baseline controls. Compared to controls, low dose cyclosporine-A decreased glomerular filtration rate (P<0.05) and numerically increased renal vascular resistance. Adding iminoethyl-lysine to cyclosporine-A improved renal hemodynamics. Adding butylhexahydro-azepin-imine to cyclosporine-A practically restored glomerular filtration rate and renal vascular resistance (P<0.05) to control levels. Grafts treated with cyclosporine-A alone showed vascular, glomerular and tubulointerstitial lesions. Adding iminoethyl-lysine or butylhexahydro-azepin-imine to cyclosporine-A did not significantly reduce vascular and glomerular injury, but diminished tubulointerstitial injury as well as nitrotyrosine staining in tubular epithelium (P<0.05). Thus, adding the iNOS blockers iminoethyl-lysine or butylhexahydro-azepin-imine to cyclosporine-A improved graft function and reduced tubulointerstitial lesions.

To evaluate the influence of atherosclerosis on the global production of NO, we studied the effect of a 0.3% cholesterol-enriched diet on urinary nitrate excretion in rabbits during 69 weeks. To examine whether the inducible nitric oxide synthase (iNOS) present in atherosclerotic lesions could participate in NO excretion, hypercholesterolemic rabbits were treated chronically with the selective iNOS inhibitor, N-iminoethyl-L-lysine (L-NIL; 5 mg/kg/day). Urinary nitrate excretion was higher in hypercholesterolemic than in control rabbits throughout the study period and decreased progressively with time in both groups; L-NIL had no significant effect on urinary nitrate excretion. These data illustrate that systemic NO production is enhanced in hypercholesterolemia and that iNOS, present within the plaque, might not participate in this enhanced NO production.

Lipopolysaccharide (LPS)-induced renal oxidant injury and the role of nitric oxide (NO) were evaluated using the inducible nitric oxide synthase (iNOS) inhibitor L-iminoethyl-lysine (L-NIL). One group of male rats received LPS (Salmonella minnesota; 2 mg/kg, i.v.). A second group received LPS plus L-NIL (3 mg/kg, i.p.). A third group received saline i.v. At 6 hr, iNOS protein was induced in the kidney cortex, and plasma nitrate/nitrite levels were increased from 4 +/- 2 nmol/mL in the Saline group to 431 +/- 23 nmol/mL in the LPS group. The value for the LPS + L-NIL group was reduced significantly to 42 +/- 9 nmol/mL. LPS increased blood urea nitrogen levels from 13 +/- 1 to 47 +/- 3 mg/dL. LPS + L-NIL reduced these levels significantly to 29 +/- 2 mg/dL. Plasma creatinine levels were unchanged in all groups. Tissue lipid peroxidation products in the kidney were increased from 0.16 +/- 0.01 nmol/mg in the Saline group to 0.30 +/- 0.03 nmol/mg in the LPS group. LPS + L-NIL reduced the values significantly to 0.22 +/- 0.02 nmol/mg. Intracellular glutathione levels were decreased in the kidneys from 1.32 +/- 0.1 nmol/mg in the Saline group to 0.66 +/- 0.08 nmol/mg in the LPS group. LPS + L-NIL increased the levels significantly to 0.99 +/- 0.13 nmol/mg. LPS increased the 3-nitrotyrosine-protein adducts in renal tubules as detected by immunohistochemistry, indicating the generation of peroxynitrite. L-NIL decreased adduct formation. These data indicated that LPS-induced NO generation resulted in peroxynitrite formation and oxidant stress in the kidney and that inhibitors of iNOS may offer protection against LPS-induced renal toxicity.

Hypovolemia has been associated with the induction of nitric oxide synthase which is believed to result in an over-production of nitric oxide. In the present study, we have examined the effects of noradrenaline following haemorrhage on cardiac output, blood pressure, mean circulatory filling pressure and vascular resistance in anaesthetized rats after pre-treatment with nitric oxide synthase inhibitor, L-N6-(1-iminoethyl)lysine or dexamethasone. Hypovolemic haemorrhage resulted in induction of nitric oxide synthase, as measured in lungs, and both dexamethasone and L-N6-(1-iminoethyl)lysine inhibited the activity of the inducible form of nitric oxide synthase. An infusion of noradrenaline significantly increased cardiac output, blood pressure and mean circulatory filling pressure in animals pre-treated with L-N6-(1-iminoethyl)lysine and dexamethasone when compared with saline pre-treatment. In addition, the administration of noradrenaline significantly reduced venous resistance in animals pre-treated with L-N6-(1-iminoethyl)lysine when compared with saline pre-treatment. The results of this investigation indicated that the impact of noradrenaline on cardiac output, blood pressure and mean circulatory filling pressure was greater in hypovolemic rats treated with L-N6-(1-iminoethyl)lysine or dexamethasone. In addition, we found that in the hypovolemic state, the greater increase in cardiac output during the infusion of noradrenaline after inhibition of nitric oxide synthase was predominantly due to reduced resistance to venous return.

The kinetics of inactivation of affinity-purified nitric oxide synthase isoforms by N6-iminoethyl-L-lysine (NIL) and N5-iminoethyl-L-ornithine (NIO) has been examined. Each of the agents produced a time and concentration dependent first order inactivation of the nitric oxide synthase isoforms that required exposure of the NO synthase to drug under conditions that supported catalysis, consistent with the proposal that these agents act as alternate substrate, mechanism-based inactivators. As measured at 100 microM arginine, NIL and NIO were equally efficient as inactivators of the cytokine-inducible nitric oxide synthase exhibiting apparent second order inactivation rate constants of 31.5 and 32.0 mM(-1) min(-1) respectively. By contrast, NIL and NIO were less efficient as inactivators of the constitutive neuronal nitric oxide synthase isoform exhibiting apparent second order inactivation rate constants of 0.79 and 8.4 mM(-1) min(-1) respectively. As measured at 100 microM extracellular arginine, NIL and NIO produced a time and concentration dependent inactivation of the NO synthetic capability of cytokine-induced murine macrophage RAW 264.7 cells exhibiting apparent second order inactivation rate constants of 3.1 and 1.8 mM(-1) min(-1). The inactivated RAW cell NO synthetic capability was restored to 30% of its pretreatment value over a 3-h period despite the presence of cycloheximide.

To test our hypothesis that arginine (Arg) and lysine (Lys) enhance immune activities via neuronal control of the thymus and the spleen, a jugular vein was cannulated for amino acid administration in male Wistar rats (approximately 300 g). In one group (n = 5), an efferent nerve filament of the vagal thymus was isolated. In another group (n = 5), splenic nerve efferents were isolated. Efferent firing rates were recorded before and for 60-90 minutes after 10 mM Arg-Lys in 0.5 ml saline intravenously (i.v.). Differences in firing rates were evaluated using analysis of variance (ANOVA) and t-test. I.v. Arg-Lys increased vagal efferent firing rate to the thymus; enhancing thymic lymphocyte release. I.v. Arg-Lys decreased firing rate in splenic efferents; enhancing natural killer (NK) cell activity. Therefore, Arg-Lys are detected by hepatoportal sensors, stimulating hepatic vagal afferents to the hypothalamus, with the efferent neuronal impulses from the hypothalamus modulating immune function in thymus and spleen, thereby demonstrating the mechanism of Arg and Lys immune enhancing activity.