Alterations of amino acid metabolism in osteoarthritis: its implications for nutrition and health.

Osteoarthritis (OA) is a common form of arthritis in humans. It has long been regarded as a non-inflammatory disease, but a degree of inflammation is now recognized as being a vital inducer of subpopulation of OA. Besides inflammation, the establishment and development of OA are associated with alterations in metabolism and profiles of amino acids (AA), including glutamate- and arginine-family AA as well as their related metabolites (e.g., creatinine, hydroxyproline, γ-aminobutyrate, dimethylarginines and homoarginine). Functional AA (e.g., glutamine, arginine, glutamate, glycine, proline, and tryptophan) have various benefits (i.e., anti-inflammation and anti-oxidation) in treatment of inflammation-associated diseases, including OA. Thus, these AA have potential as immunomodulatory nutrients for patients with inflammation-induced OA.

Age influence on hypersensitivity pneumonitis induced in mice by exposure to Pantoea agglomerans.

Hypersensitivity pneumonitis (HP) represents the immunologically mediated lung disease induced by repeated inhalations of a wide variety of certain finely dispersed organic antigens. In susceptible subjects, these inhalations provoke a hypersensitivity reaction characterized by intense inflammation of the terminal bronchioles, the interstitium and the alveolar tree. The inflammation often organizes into granulomas and may progress to pulmonary fibrosis. Our previous work indicated that cell extract of gram-negative bacteria Pantoea agglomerans (SE-PA) causes, in young C57BL/6J mice, pulmonary changes that are very similar to the clinical manifestations of HP in men. The purpose of presented studies was to describe the response of mice immune system while exposed to SE-PA. Particular attention was paid to examine the age influence on SE-PA induced inflammation and fibrosis in lung tissue. We used 3- and 18-month-old C57BL/6J mice. Lung samples were collected from untreated mice and animals exposed to harmful agent for 7 and 28 days. HP development was monitored by histological and biochemical evaluation. Using ELISA tests, we examined concentration of pro- and anti-inflammatory cytokines in lung homogenates. Our study demonstrated again that SE-PA provokes in mice changes typical for the clinical picture of HP, and that successive stages of disease (acute, subacute and chronic) might be obtained by modulation of time exposure. Furthermore, we found that animals' age at the time of sensitization influences the nature of observed changes (cytokine expression pattern) and the final outcome (reaction intensity and scale of fibrosis).

The therapeutic efficacy of glutamine for rats with smoking inhalation injury.

Smoke inhalation injury represents a major cause of mortality in burn patients and is associated with a high incidence of pulmonary complications. Glutamine (GLN) is considered a conditionally essential amino acid during critical illness and injury. However, whether GLN could attenuate lung injury caused by smoke inhalation is still unknown. The purpose of this study is to investigate whether GLN has a beneficial effect on smoke inhalation induced lung injury. In our present work, rats were equally randomized into three groups: Sham group (ambient air inhalation plus GLN treatment), Control group (smoke inhalation plus physiological saline) and GLN treatment group (smoke inhalation injury plus GLN treatment). At sampling, bronchoalveolar lavage fluid was performed to determine total protein concentration and pro-inflammatory cytokine levels. Lung tissues were collected for wet/dry ratio, histopathology, hydroxyproline and Western blotting measurement. Our results exhibited that GLN attenuated the lung histopathological alterations, improved pulmonary oxygenation, and mitigated pulmonary edema. At 28days post-injury, GLN mitigated smoke inhalation-induced excessive collagen deposition as evidence by Masson-Goldner trichrome staining and hydroxyproline content. GLN mitigated smoke inhalation-induced lung inflammatory response, and further prevented the activity of NF-kappa-B. More importantly, results from Western blotting and Immunohistochemistry exhibited that GLN enhanced the expression of HSF-1, HSP-70 and HO-1 in lung tissues. Our data demonstrated that GLN protected rats against smoke inhalation-induced lung injury and its protective mechanism seems to involve in inhibition inflammatory response and enhancing HSP expression.

Synthesis of hapten and production of a monoclonal antibody against a derivative of L-hydroxyproline, a special amino acid in hydrolyzed animal protein.

China's government has prohibited the addition of simply hydrolyzed animal protein from solid leather waste into milk. The objective of this study was to produce a monoclonal antibody against L-hydroxyproline, a special amino acid in hydrolyzed animal protein. L-hydroxyproline was derivatized with N-acetylsulfanilyl chloride and 5-chlorovaleric acid to synthesize the haptens HP1 and HP2. Then, two immunogens from the two haptens were prepared to produce the antibodies. Results showed that only HP1 was able to stimulate the animal immune system and generate the specific antibody to L-hydroxyproline (as the formation of HP1). The obtained monoclonal antibody from HP1 and the heterologous coating hapten HP2 were incorporated into a competitive indirect enzyme linked immunosorbent assay (ELISA) to determine the antibody's specificity and sensitivity. The IC(50) and the limit of detection for HP1 were 0.16 µg/mL and 0.05 µg/mL respectively. The antibody showed low crossreactivity to parental L-hydroxyproline and showed negligible crossreactivity to D-hydroxyproline and other amino acids. The monoclonal antibody was therefore suitable for the development of an immunoassay to monitor the simply hydrolyzed animal protein from solid leather waste in foodstuffs with L-hydroxyproline as the target analyte.

AGE formation blockade with aminoguanidine does not ameliorate chronic allograft nephropathy.

AIMS: Advanced glycation end products (AGEs) are produced by glycoxidation and lipid peroxidation. AGEs induce oxidative stress and inflammation, and accumulate in tubular cells after kidney transplantation. We hypothesize that the AGE formation blocker aminoguanidine (AG) reduces AGE formation and improves renal transplant function. MAIN METHODS: Fisher 344 kidneys were orthotopically transplanted into Lewis recipients. Recipients were treated with AG (100 mg/kg/day), candesartan (CAND; 5mg/kg/day), or vehicle (VEH) for 24 weeks. The major non-cross linking AGE N(ε)-carboxymethyllysine (CML) was measured post-transplantation with gas chromatography-tandem mass spectrometry or immunohistochemistry. As a marker of systemic lipid peroxidation 8-isoprostane was measured by ELISA. We determined intra-arterial blood pressure, heart weight/body weight ratio, size of cardiomyocytes and cardiac hypertrophy as assessed by echocardiography. For biochemical evaluation of cardiac and renal fibrosis we measured hydroxyproline content. KEY FINDINGS: AG significantly reduced serum CML and 8-isoprostane, but did not reduce signs of chronic allograft nephropathy (CAN) or blood pressure. AG did not alter tubular AGE accumulation. AG reduced heart weight/body weight ratio (AG: 2.7 ± 0.1g/kg; CAND: 2.2 ± 0.1, VEH: 3.0 ± 0.4 g/kg), size of cardiomyocytes (P < 0.05) and showed a tendency to reduce cardiac hypertrophy (wall volume average radial AG 7.072 ± 0.83 cm(3) vs. CAND 6.841 ± 0.66 cm(3) vs. VEH 7.839 ± 0.74 cm(3)). SIGNIFICANCE: Despite effective reduction of serum CML and 8-isoprostane, AG did not ameliorate CAN or reduce renal AGE accumulation. On the other hand AG reduced cardiac size suggesting a supportive cardio-protective action which is blood pressure independent.

Tissue inhibitor of metalloproteinase-1 modulates allergic lung inflammation in murine asthma.

Matrix metalloproteinases (MMPs) modulate development, inflammation, and repair in lungs. Tissue inhibitors of MMPs (TIMPs) interact with MMPs, controlling the intensity and nature of the response to injury. Absence of MMP-9, -2, and -8 activities is associated with altered lung inflammation during allergic sensitization. To test the hypothesis that the absence of TIMP-1 enhances allergic lung inflammation, airway hyperreactivity (AHR), and lung remodeling in asthma, we studied TIMP-1 null (TIMP-1 KO) mice and their WT controls using an ovalbumin (OVA) asthma model. TIMP-1 KO mice, compared to WT controls, developed an asthma phenotype characterized by AHR, pronounced cellular lung infiltrates, greater reduction in lung compliance, enhanced Th2 cytokine mRNA and protein expression, and altered collagen lung content associated with enhanced MMP-9 activity. Our findings support the hypothesis that TIMP-1 plays a protective role by preventing AHR and modulating inflammation, remodeling, and cytokine expression in an animal model of asthma.

The chaotrope-soluble glycoprotein GP2 is a precursor of the insoluble glycoprotein framework of the Chlamydomonas cell wall.

The cell wall of the unicellular green alga Chlamydomonas reinhardtii consists of an insoluble, hydroxyproline-rich glycoprotein framework and several chaotrope-soluble, hydroxyproline-containing glycoproteins. Up to now, there have been no data concerning the amino acid sequences of the hydroxyproline-containing polypeptides of the insoluble wall fraction. Matrix-assisted laser desorption ionization time-of-flight analyses of peptides released from the insoluble cell wall fraction by trypsin treatment revealed the presence of 14 peptide fragments that could be attributed to non-glycosylated domains of the chaotrope-soluble cell wall glycoprotein GP2. However, these peptides cover only 15% of the GP2 polypeptide backbone. Considerably more information concerning the presence of GP2 in the insoluble cell wall fraction was obtained by an immunochemical approach. For this purpose, 407 overlapping pentadecapeptides covering the whole known amino acid sequence of GP2 were chemically synthesized and probed with a polyclonal antibody raised against the deglycosylated, insoluble cell wall fraction. This particular antibody reacted with 297 of the 407 GP2-derived peptides. The peptides that were recognized by this antibody are distributed over the whole known GP2 sequence. The epitopes recognized by polyclonal antibodies raised against the 64- and 45-kDa constituents purified from the deglycosylation products of the insoluble cell wall fraction are also distributed over the whole GP2 backbone, although the corresponding antigens are considerably smaller than GP2. The significance of the latter results for the structure of the insoluble cell wall fraction is discussed.

Post-translational modifications influence IgE reactivity to the major allergen Phl p 1 of timothy grass pollen.

BACKGROUND: Grass group I consists of very potent allergenic components which are found in the pollen of all temperate grasses. Several post-translational modifications are predicted from the cDNA data. OBJECTIVE: The aim of this study was to identify sequential IgE-binding sites on the allergen Phl p 1 and to determine their influence on IgE reactivity. METHODS: Based on cDNA data and microsequencing results we synthesized overlapping decapeptides covering the complete Phl p 1 molecule and tested them for immunological reactivity by means of the PEPSCAN technique. In a dot test we determined the frequency of IgE reactivities to post-translationally modified structures (hydroxylated proline residues, carbohydrate structure, and disulphide formations). RESULTS: Screening by overlapping peptides demonstrated an IgE binding site on the 10 N-terminal amino acids. Comprehensive studies showed that the two hydroxyproline residues of the native Phl p 1 allergen (at positions 5 and 8) and the N-glycan (at position 9) can result in an increased IgE reactivity; 3.3% of the sera exclusively bound to the hydroxyproline bearing peptide, while only 0.4% bound to the proline containing peptide. With regard to glycosylation, we estimated that 20% of sera recognized protein and carbohydrate epitopes, while one serum exclusively bound to the glycan. The formation of disulphide bonds has no detectable effect on the IgE reactivity to Phl p 1. CONCLUSION: Our results indicate that the post-translational modifications, the carbohydrate structure and the hydroxylation of proline residues, can enhance the IgE reactivity of Phl p 1.