Dual behavior of histidine during sensitized photo-oxidation of model compounds and proteins.

Histidine (His) photo-oxidation has been widely investigated with several transient and stable products characterized, especially for aerobic conditions. Due to its role and structure, His-side chain can be a key player in the quenching of excited states such as the triplet state of the photosensitizer 3-carboxybenzophenone (3CB*). The capacity of His and its derivatives to quench 3CB* under anaerobic conditions are characterized in the current study by laser flash photolysis, with the resulting oxidation products examined by mass spectrometry to determine the reaction mechanism. The latter include adducts of the 3-carboxybenzophenone ketyl radical (CBH•) to the imidazole ring (Imid-CH2-CBH), His-His dimers, and other products with lower yields. The data obtained with model compounds are compared to those obtained with more complicated systems, including the peptide Exendin-4, and the protein MtHpt1. The data obtained from transient spectroscopy and product analyses indicate that two CB* quenching mechanisms occur: (i) proton-coupled electron transfer (as reported previously) yielding radicals that can recombine to give His-His dimers and CBH-adducts, and (ii) energy transfer yielding 3His* undergoing further reaction leading to formation of Imidazyl-CH2-CBH adduct. The latter, unexpected process only occurs when His and its derivatives have a free α-amino group. This process yielded a novel adduct between the imidazole ring and the CBH• formed by sensitizer reduction.

A novel marker for assessment of liver matrix remodeling: an enzyme-linked immunosorbent assay (ELISA) detecting a MMP generated type I collagen neo-epitope (C1M).

A competitive enzyme-linked immunosorbent assay (ELISA) for detection of a type I collagen fragment generated by matrix metalloproteinases (MMP) -2, -9 and -13, was developed (CO1-764 or C1M). The biomarker was evaluated in two preclinical rat models of liver fibrosis: bile duct ligation (BDL) and carbon tetra chloride (CCL4)-treated rats. The assay was further evaluated in a clinical study of prostate-, lung- and breast-cancer patients stratified according to skeletal metastases. A technically robust ELISA assay specific for a MMP-2, -9 and -13 neo-epitope was produced and seen to be statistically elevated in BDL rats compared to baseline levels as well as significantly elevated in CCL4 rats stratified according to the amount of total collagen in the livers. CO1-764 levels also correlated significantly with total liver collagen and type I collagen mRNA expression in the livers. Finally, the CO1-764 marker was not correlated with skeletal involvement or number of bone metastases. This ELISA has the potential to assess the degree of liver fibrosis in a non-invasive manner.

Assessment of correlated dose and sensitivity profiles on a multi-slice CT scanner.

In the case of computed tomography (CT) scanners as well as other imaging techniques utilising ionising radiation, it is imperative that radiation is confined to the sensitive part of the image detector. Assuring this for a CT scanner requires detailed information about the scanner dose and sensitivity profiles and their spatial correlation. The profiles should ideally be co-centric and tightly fit to each other. Ensuring this inherent performance of the scanner can be seen as one of the fundamental steps in optimising diagnostic examinations with CT. A measurement device using a dedicated liquid ionisation chamber is employed to investigate the performance of a Toshiba Aquilion 16 scanner in this aspect. Dose profile and sensitivity profile pairs for four collimations are presented where each pair of profiles are spatially correlated to each other. The measurement device can be applied to any scanner for fast and accurate assessment of dose and sensitivity profiles and their spatial correlation.

Levels of circulating MMP-7 degraded elastin are elevated in pulmonary disorders.

OBJECTIVES: Elastin is a signature protein of the lungs. Matrix metalloproteinase-7 (MMP-7) is important in lung defence mechanisms and degrades elastin. However, MMP-7 activity in regard to elastin degradation has never been quantified serologically in patients with lung diseases. An assay for the quantification of MMP-7 generated elastin fragments (ELM7) was therefore developed to investigate MMP-7 derived elastin degradation in pulmonary disorders such as idiopathic pulmonary fibrosis (IPF) and lung cancer. DESIGN AND METHODS: Monoclonal antibodies (mABs) were raised against eight carefully selected MMP-7 cleavage sites on elastin. After characterisation and validation of the mABs, one mAB targeting the ELM7 fragment was chosen. ELM7 fragment levels were assessed in serum samples from patients diagnosed with IPF (n=123, baseline samples, CTgov reg. NCT00786201), and lung cancer (n=40) and compared with age- and sex-matched controls. RESULTS: The ELM7 assay was specific towards in vitro MMP-7 degraded elastin and the ELM7 neoepitope but not towards other MMP-7 derived elastin fragments. Serum ELM7 levels were significantly increased in IPF (113%, p<0.0001) and lung cancer (96%, p<0.0001) compared to matched controls. CONCLUSIONS: MMP-7-generated elastin fragments can be quantified in serum and may reflect pathological lung tissue turnover in several important lung diseases.

Hydrolytic properties of a beta-mannosidase purified from Aspergillus niger.

A beta-mannosidase was purified to homogeneity from the culture filtrate of Aspergillus niger. A specific activity of 500 nkat mg-1 and a 53-fold purification was achieved using ammonium sulfate precipitation, anion-exchange chromatography, and gel filtration. The isolated enzyme has an isoelectric point of 5.0 and appears to be a dimer composed of two 135-kDa subunits. It is a glycoprotein and contains 17% N-linked carbohydrate by weight. Maximal activity was observed at pH 2.4 5.0 and at 70 degrees C. The beta-mannosidase hydrolyzed beta-1,4-linked manno-oligosaccharides of degree of polymerization (DP) 2-6 and also released mannose from polymeric ivory nut mannan and galactomannan. The Km and Vmax values for p-nitrophenyl-beta-D-mannopyranoside were 0.30 mM and 500 nkat mg-1, respectively. Hydrolysis of D-galactose substituted manno-oligosaccharides showed that the beta-mannosidase was able to cleave up to, but not beyond, a side group. An internal peptide sequence of 15 amino acids was highly similar to that of an Aspergillus aculeatus beta-mannosidase belonging to family 2 of glycosyl hydrolases.

Structural aspects in semicrystalline samples of the mannan II family.

A series of samples having the mannan II character were prepared by either (i) desincrusting stems of Acetabularia crenulata, or (ii) acetylating these stems, followed by dissolution and recrystallization under deacetylation conditions, or (iii) recrystallizing at low temperature the alkali soluble fraction of ivory nut mannan. The samples were characterized by transmission electron microscopy, X-ray and electron diffraction analysis together with (13)C CP/MAS NMR spectroscopy. Whereas the A. crenulata stems consisted of a mixture of mannan I and mannan II, the recrystallized samples were all of the hydrated mannan II family and occurred in a ribbonlike morphology where the mannan chains were organized with their molecular axis perpendicular to the ribbon long axis. The recrystallized ivory nut mannan samples presented X-ray and electron diffraction diagrams, together with (13)C solid-state NMR spectra recorded at 95% RH, different from those of recrystallized A. crenulata recorded under the same RH conditions. They corresponded therefore to a new allomorph of the mannan II family. Despite this difference, when the recrystallized samples were in an aqueous environment, they revealed an additional well-defined perhydrated phase, which showed the same (13)C solid-state NMR spectrum for both samples. As this phase, which gave 6-band NMR spectra with narrow line-width and low T1, had no counterpart in X-ray diffraction, it was attributed to specific amorphous segments of mannan chains, gaining some mobility when swollen in water. When the samples were totally dried, their NMR spectra lost their resolution, thus indicating the role played by water for the structural organization of the crystalline and amorphous components of mannan II.

Cloning and characterization of Aspergillus niger genes encoding an alpha-galactosidase and a beta-mannosidase involved in galactomannan degradation.

Alpha-galactosidase (EC 3.2.1.22) and beta-mannosidase (EC 3.2.1.25) participate in the hydrolysis of complex plant saccharides such as galacto(gluco)mannans. Here we report on the cloning and characterization of genes encoding an alpha-galactosidase (AglC) and a beta-mannosidase (MndA) from Aspergillus niger. The aglC and mndA genes code for 747 and 931 amino acids, respectively, including the eukaryotic signal sequences. The predicted isoelectric points of AglC and MndA are 4.56 and 5.17, and the calculated molecular masses are 79.674 and 102.335 kDa, respectively. Both AglC and MndA contain several putative N-glycosylation sites. AglC was assigned to family 36 of the glycosyl hydrolases and MndA was assigned to family 2. The expression patterns of aglC and mndA and two other genes encoding A. niger alpha-galactosidases (aglA and aglB) during cultivation on galactomannan were studied by Northern analysis. A comparison of gene expression on monosaccharides in the A. niger wild-type and a CreA mutant strain showed that the carbon catabolite repressor protein CreA has a strong influence on aglA, but not on aglB, aglC or mndA. AglC and MndA were purified from constructed overexpression strains of A. niger, and the combined action of these enzymes degraded a galactomanno-oligosaccharide into galactose and mannose. The possible roles of AglC and MndA in galactomannan hydrolysis is discussed.

Degradation of mannan I and II crystals by fungal endo-beta-1,4-mannanases and a beta-1,4-mannosidase studied with transmission electron microscopy.

We have used the endo-beta-1,4-mannanase from Trichoderma reesei (Tr Man5A), the endo-beta-1,4-mannanase from Aspergillus niger (An Man5A) and the exo-beta-1,4-mannosidase from A. niger (An Mnd2A) to follow the enzymatic degradation of mannan I and II crystals. The degradation process was studied by transmission electron microscopy and also followed by analysis of the released soluble reducing sugars. The mannan crystals were degraded by the endo-beta-1,4-mannanases and to a lesser extent by the exo-beta-1,4-mannosidase. The observed hydrolysis pattern on mannan I crystals is fully consistent with the current view of the molecular structure of these crystals. The molecular organization of the mannan chains in mannan II crystals is less clear and the digestion results give some further information about the ultrastructure of mannan II. In addition, insight is provided into the mode of the enzymatic attack on the crystals of mannan I and mannan II.