Deficiency of CRTAP in non-lethal recessive osteogenesis imperfecta reduces collagen deposition into matrix.

Deficiency of any component of the ER-resident collagen prolyl 3-hydroxylation complex causes recessive osteogenesis imperfecta (OI). The complex modifies the α1(I)Pro986 residue and contains cartilage-associated protein (CRTAP), prolyl 3-hydroxylase 1 (P3H1) and cyclophilin B (CyPB). Fibroblasts normally secrete about 10% of CRTAP. Most CRTAP mutations cause a null allele and lethal type VII OI. We identified a 7-year-old Egyptian boy with non-lethal type VII OI and investigated the effects of his null CRTAP mutation on collagen biochemistry, the prolyl 3-hydroxylation complex, and collagen in extracellular matrix. The proband is homozygous for an insertion/deletion in CRTAP (c.118_133del16insTACCC). His dermal fibroblasts synthesize fully overmodified type I collagen, and 3-hydroxylate only 5% of α1(I)Pro986. CRTAP transcripts are 10% of control. CRTAP protein is absent from proband cells, with residual P3H1 and normal CyPB levels. Dermal collagen fibril diameters are significantly increased. By immunofluorescence of long-term cultures, we identified a severe deficiency (10-15% of control) of collagen deposited in extracellular matrix, with disorganization of the minimal fibrillar network. Quantitative pulse-chase experiments corroborate deficiency of matrix deposition, rather than increased matrix turnover. We conclude that defects of extracellular matrix, as well as intracellular defects in collagen modification, contribute to the pathology of type VII OI.

Osteoblasts extracellular matrix induces vessel like structures through glycosylated collagen I.

Extracellular matrix (ECM) plays a fundamental role in angiogenesis affecting endothelial cells proliferation, migration and differentiation. Vessels-like network formation in vitro is a reliable test to study the inductive effects of ECM on angiogenesis. Here we utilized matrix deposed by osteoblasts as substrate where the molecular and structural complexity of the endogenous ECM is preserved, to test if it induces vessel-like network formation by endothelial cells in vitro. ECM is more similar to the physiological substrate in vivo than other substrates previously utilized for these studies in vitro. Osteogenic ECM, prepared in vitro from mature osteoblasts at the phase of maximal deposition and glycosylation of collagen I, induces EAhy926, HUVEC, and HDMEC endothelial cells to form vessels-like structures and promotes the activation of metalloproteinase-2 (MMP-2); the functionality of the p-38/MAPK signaling pathway is required. Osteogenic ECM also induces a transient increase of CXCL12 and a decrease of the receptor CXCR4. The induction of vessel-like networks is dependent from proper glycosylation of collagens and does not occur on osteogenic ECMs if deglycosylated by -galactosidase or on less glycosylated ECMs derived from preosteoblasts and normal fibroblasts, while is sustained on ECM from osteogenesis imperfecta fibroblasts only when their mutation is associated with over-glycosylation of collagen type I. These data support that post-translational glycosylation has a role in the induction in endothelial cells in vitro of molecules conductive to self-organization in vessels-like structures.

Desmosine as a biomarker of elastin degradation in COPD: current status and future directions.

Desmosine (DES) and isodesmosine (IDES) are two unusual, tetrafunctional, pyridinium ring-containing amino acids involved in elastin cross-linking. Being amino acids unique to mature, cross-linked elastin, they are useful for discriminating peptides derived from elastin breakdown from precursor elastin peptides. According to these features, DES and IDES have been extensively discussed as potentially attractive indicators of elevated lung elastic fibre turnover and markers of the effectiveness of agents with the potential to reduce elastin breakdown. In the present manuscript, immunology-based and separation methods for the evaluation of DES and IDES are discussed, along with studies reporting increased levels of urine excretion in chronic obstructive pulmonary disease (COPD) patients with and without alpha(1)-antitrypsin deficiency. The results of the application of DES and IDES as surrogate end-points in early clinical trials in COPD are also reported. Finally, recent advances in detection techniques, including liquid chromatography tandem mass spectrometry and high-performance capillary electrophoresis with laser-induced fluorescence, are discussed. These techniques allow detection of DES and IDES at very low concentration in body fluids other than urine, such as plasma or sputum, and will help the understanding of whether DES and IDES are potentially useful in monitoring therapeutic intervention in COPD.

Osteogenesis imperfecta: clinical, biochemical and molecular findings.

Mutations in COL1A1 and COL1A2 genes, encoding the alpha1 and alpha2 chain of type I collagen, respectively, are responsible for the vast majority of cases of osteogenesis imperfecta (OI) (95% of patients with a definite clinical diagnosis). We have investigated 22 OI patients, representing a heterogeneous phenotypic range, at the biochemical and molecular level. A causal mutation in either type I collagen gene was identified in 20 of them: no recurrent mutation was found in unrelated subjects; 15 out of 20 mutations had not been reported previously. In two patients, we could not find any causative mutation in either type I collagen gene, after extensive genomic DNA sequencing. Failure of COL1A1/COL1A2 mutation screening may be due, in a few cases, to further clinical heterogeneity, i.e. additional non-collagenous disease loci are presumably involved in OI types beyond the traditional Sillence's classification.

High levels of desmosines in urine and plasma of patients with pseudoxanthoma elasticum.

BACKGROUND: Pseudoxanthoma elasticum (PXE), a rare heritable disorder caused by mutations of the ABCC6 gene, is characterized by fragmentation and mineralization of elastic fibres. We determined the extent of degradation of elastin by measuring and comparing the amount of desmosines in plasma and urine of PXE patients, healthy carriers and normal subjects. METHODS: Using capillary electrophoresis with laser-induced fluorescence detection (CE-LIF) we measured the amount of desmosines in the urine of 46 individuals (14 PXE patients, 17 healthy carriers and 15 controls) and in the plasma of 56 subjects (18 PXE patients, 23 healthy carriers and 15 controls). Pseudoxanthoma elasticum patients and carriers were identified by clinical, structural and molecular biology analyses. RESULTS: The urinary excretion of desmosines was two-fold higher in PXE patients than in controls (P < 0.01); the values for healthy carriers were intermediate between those of PXE patients and controls. A very similar trend between patients and their relatives was observed for plasma desmosines. There was a significant correlation between the amount of the desmosines in plasma and urine. Moreover, a positive correlation was observed between urinary desmosine content and age of the patients as well as between urinary desmosine content and severity of clinical manifestations. CONCLUSIONS: Both the urinary and plasma desmosine concentrations indicate that elastin degradation is higher in PXE patients and, to a lesser extent, in healthy carriers than in normal subjects. Data seem to indicate that the amount of elastin breakdown products correlates with the age of patients as well as with the severity of the disease.

Therapeutic apheresis exchange in two patients with prolidase deficiency.

BACKGROUND: Prolidase deficiency is a rare genetic disorder for which a cure has not yet been found. OBJECTIVES: To assess the effectiveness of apheresis exchange as a new therapeutic approach. METHODS: Apheresis exchanges were repeated monthly for four consecutive months, in parallel, on two patients, replacing prolidase-deficient red blood cells with normal filtered cells. Prolidase activity and urinary dipeptides were determined at regular intervals. RESULTS: The constant presence of active prolidase inside cells allowed a continuous, although partial, degradation of imidodipeptides, with a concomitant improvement of skin ulceration. CONCLUSIONS: Apheresis exchange could be a reasonable way of obtaining a clinical improvement in these patients.

Urinary desmosine excretion in acute exacerbations of COPD: a preliminary report.

Desmosine (DES) is an elastin-derived, cross-link amino acid, which is not metabolized; hence, its urinary levels reflect elastin breakdown. We hypothesized that elastin degradation should increase as a result of increased lung inflammation during an acute exacerbation of COPD and should decrease after recovery. To test this hypothesis we measured DES in three urine samples from nine COPD subjects during the first 5 days of an acute exacerbation and at 2 months after recovery. We also measured forced expiratory volume in 1 sec (FEV1) to monitor the effects ofthe exacerbation on ventilatory function. The mean (SD) FEV1 was 45 (15)% predicted during the exacerbation and 57.8 (16)% predicted 2 months later (P=0.00001). The mean (SD) DES excretion was 25.3 (9) microg g(-1) creatinine at day 1;23.5 (9) at day 3 and 24 (9) at day 5 of the exacerbation. The mean (SD) urinary DES excretion 60 days after discharge was 20.9 (7) microg g(-1) creatinine (P=0.049) in comparison with the mean of the three acute-phase values. The size of the increase in desmosine excretion during exacerbation is small, 3.2 microg g(-1) creatinine or 16% of the recovery desmosine value. We conclude that there is a small but statistically significant increase in lung elastin breakdown in the body during an acute exacerbation of COPD.

Inhibition of human neutrophil elastase by erythromycin and flurythromycin, two macrolide antibiotics.

Fourteen-member-ring macrolides are antibiotics with a variety of anti-inflammatory activities, and have repeatedly been reported to reduce mucus hypersecretion in conditions such as cystic fibrosis and bronchiectasis. Their structure is characterized by a macrocyclic lactone ring. Because human neutrophil elastase (HNE) plays a crucial role in the vicious circle leading to mucus hypersecretion, and lactones are known to be elastase inhibitors, we hypothesized that macrolides might directly inhibit elastase. To investigate this hypothesis we designed a series of spectrophotometric experiments using a chromogenic substrate with two macrolides, erythromycin (Er) and flurythromycin (FE). We determined the 1st order rate constant (k(obs)) by inhibition and competitive substrate assays, the latter allowing us to calculate the substrate binding constant or inhibition constant and the acylation rate constant (k(a)). A proflavine displacement assay was used to determine the deacylation rate constant (k(d)). Both Er and FE are good HNE inhibitors, showing a high k(a) and a low k(d). Because the number of turnovers per inactivation of Er was congruent with 20-fold higher than that of FE, we supposed that the lower reactivation of HNE-FE was due to the formation of a more stable inactivated enzyme. This hypothesis was confirmed by the hydrazine reactivation of the acyl enzyme. For Er we identified a k(d) only, whereas for FE, in addition to the k(d), an alkylation constant (k(2)) was calculated, correlated to a fully inactivated enzyme. From our kinetics data, we therefore conclude that Er acts as an alternate substrate HNE inhibitor, whereas FE acts as an inactivator.

Diagnosis of late-infantile neuronal ceroid lipofuscinosis: a new sensitive method to assay lysosomal pepstatin-insensitive proteinase activity in human and animal specimens by capillary electrophoresis.

Batten disease, or human late-infantile neuronal ceroid lipofuscinosis (LINCL) is a familiar progressive degenerative disease affecting children, caused by a deficiency of a lysosomal proteinase (tripeptidyl peptidase I, TPP-I) and characterized by the accumulation of autofluorescent storage bodies in the brain and other tissues of the body. Current methodology used to diagnose this disease needs to be improved in order to have less invasive techniques with higher resolution and shorter assay time. In this report, we discuss the potential merits of micellar electrokinetic chromatography as an excellent tool that requires minute samples but offers high resolution and a short running time for monitoring TPP-I activity in human and animal specimens.

Alpha1-antitrypsin in serum determined by capillary isoelectric focusing.

A capillary isoelectric focusing (CIEF) method using bare fused-silica capillaries filled with polyethylene oxide (PEO) and carrier ampholyte solutions in the pH 3.5-5.0 range has been developed for the identification of alpha1-antitrypsin (alpha1AT) phenotypes in human serum. This novel procedure was routinely applied to the study of serum samples of five controls whose alpha1AT phenotype was previously identified and of twelve subjects whose alpha1AT phenotype was unknown. The results obtained allowed us to confirm or identify the alpha1AT phenotype in all sera tested. This procedure seems particularly suitable for identification of alpha1AT variants associated with diseases of clinical relevance.

MEKC of desmosine and isodesmosine in urine of chronic destructive lung disease patients.

Degradation of extracellular matrix components is central to many pathological features of chronic destructive lung disorders. Desmosine and isodesmosine are elastin-derived cross-linked amino acids whose urine levels are considered representative of elastin breakdown. The aim of this study was to apply a novel methodology, based on high-performance capillary electrophoresis, to the quantification of desmosine and isodesmosine in 11 patients with stable chronic obstructive pulmonary disease (COPD), 10 with an exacerbation of COPD, nine with alpha1-antitrypsin deficiency, 13 with bronchiectasis, and 11 adults with cystic fibrosis, in comparison to 24 controls. It was found that, in patients with stable COPD, urinary desmosine levels were higher than in controls (p=0.03), but lower than in COPD subjects with an exacerbation (p< or =0.05). The highest desmosine levels were found in subjects with alpha1-antitrypsin deficiency, bronchiectasis and cystic fibrosis (p<0.001 versus stable COPD). In a short-term longitudinal study, five stable COPD patients showed a constant rate of desmosine excretion (mean coefficient of variation <8% over three consecutive days). In conclusion, the present method is simple and suitable for the determination of elastin-derived cross-linked amino acid excretion in urine, giving results similar to those obtained using other separation methods. In addition, evidence is presented that urinary desmosine excretion is increased in conditions characterized by airway inflammation, such as exacerbations of chronic obstructive pulmonary disease, bronchiectasis and cystic fibrosis. Results obtained in subjects with alphal-antitrypsin deficiency suggest that this method might be used to evaluate the putative efficacy of replacement therapy.

Separation of closely related peptide substrates of human proteinases by micellar electrokinetic chromatography with anionic and nonionic surfactants.

In order to use micellar electrokinetic chromatography to determine the proteolytic activity of different proteinases simultaneously present in physiological fluids, the technique must be able to separate mixtures of substrates with closely related structures. In an attempt to determine the best electrophoretic conditions for resolving six p-nitroanilide peptides used as synthetic substrates of the elastolytic enzymes (human neutrophil elastase, cathepsin G, Pseudomonas aeruginosa elastase) most commonly involved in pulmonary diseases, we investigated the efficiency of ionic and nonionic surfactants in achieving the separation of this complex mixture. The results presented here show that, of all the electrophoretic systems tested, 30 mM sodium tetraborate, pH 9.3, containing 25 mM Brij 35 as micellar agent offered the best performance; the separation efficiency of peptides is greater than that obtained with other reagents and all peaks are baseline resolved and unambiguously identifiable. Analysis of the micelle-solute interaction with the surfactants investigated allowed better definition of the mechanism involved in the distribution of these peptides to the micelles and identification of some structural features that determined the magnitude of the micelle peptide complex formation.

Peripheral plasma amino acid abnormalities in rehabilitation patients with severe brain injury.

OBJECTIVE: Acute severe brain injury causes an increased mobilization of amino acids from tissue. The plasma amino acid profile of patients undergoing rehabilitation after brain injury is unknown. This study was aimed at delineating the plasma amino acid profile of rehabilitation patients with brain injury. DESIGN: Peripheral plasma aminogram, lactate, pyruvate, glycerol, ketone body, and carnitine concentrations were determined in 11 patients with brain injury (34.6+/-15 years old, 60+/-16.8 days after injury) and in 8 controls. Resting energy expenditure and nitrogen balance were also determined. RESULTS: (1) All essential amino acids and about 50% of nonessential amino acids were significantly lower in brain injury patients than in controls (p < .05). (2) Plasma amino acids were lower irrespective of either energy and protein intake or nitrogen balance. (3) Total carnitine concentration and esterified/free carnitine ratio were higher in brain injury patients than in controls (p < .05). CONCLUSIONS: Rehabilitation patients with brain injury may have an important reduction of their plasma aminogram. Muscle tissue depletion and the persistence of a hypercatabolic state caused by subclinical infections, pressure sores, and immobility may contribute to this reduction.

Micellar electrokinetic chromatography as a complementary method to sodium dodecyl sulfate-polyacrylamide gel electrophoresis for studying limited proteolysis of proteins.

Micellar electrokinetic chromatography (MEKC) has been utilized as an analytical method to perform investigations on limited proteolysis of proteins. To this purpose partial proteolysis experiments with a series of proteinases were performed, utilizing as model protein pyruvate kinase (PK) from Escherichia coli, an enzyme that is regulated allosterically by fructose 1,6-bisphosphate (FBP). Data obtained with sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and MEKC were compared; the profiles generated by submitting digests of PK treated with different proteinases in the presence and absence of FBP to electrophoretic analysis provided a useful adjunct for a better understanding of the effects of the allosteric activator on the conformation of the model enzyme. MEKC was also found to be a convenient technique for determining the kinetics of substrate proteolysis.

Micellar electrokinetic chromatography for analyzing active site specificity of Pseudomonas aeruginosa elastase.

The geometry of the catalytic site of Pseudomonas aeruginosa elastase was reexamined, exploiting the specific feature of micellar electrokinetic chromatography (MEKC), i.e., its ability to detect a decrease of intact substrate and simultaneous formation of reaction products. We carried out a detailed investigation using two tri- and six tetra-peptide 4-nitroanilides (NA) differing from each other by only one or more amino acids as stable substrates. The kinetic cleavage parameters Km and k(cat) determined by MEKC and the catalytic efficiency Km/k(cat) values calculated allowed us to better define the substrate specificity of this proteinase.

Simultaneous determination of Pseudomonas aeruginosa elastase, human leukocyte elastase and cathepsin G activities by micellar electrokinetic chromatography.

Micellar electrokinetic chromatography (MEKC) is a new method for analysing proteolytic activities simultaneously present in incubation mixtures. Here we demonstrate that MEKC differentiates between the enzymatic activities of Pseudomonas aeruginosa elastase (PsE) and human leukocyte elastase (HLE) or cathepsin G (Cat G) in assays using the chromogenic peptide substrates Suc-Ala-Ala-Ala-NA or Suc-Ala-Ala-Pro-Phe-NA, respectively (where Suc = succinyl and NA = 4-nitroaniline/u-nitroanilide). When PsE and Cat G were incubated at equimolar ratio with Suc-Ala-Ala-Pro-Phe-NA, the PsE-specific cleavage products PheNA and Suc-Ala-Ala-Pro were detected whereas inhibition of the metalloproteinase PsE with EDTA resulted in detection of NA and Suc-Ala-Ala-Pro-Phe only. Similarly, when PsE and HLE were incubated at equimolar ratio with Suc-Ala-Ala-Ala-NA, the PsE-specific cleavage products Suc-Ala and Ala-Ala-NA were detected whereas at an PsE-HLE ratio 1:50, both the PsE-specific and the HLE-specific cleavage products NA and Suc-Ala-Ala-Ala were separated. MEKC also allowed determination of the kinetic constants for the interactions of PsE, Cat G and HLE with the substrates considered.

Rapid detection of ornithine transcarbamylase activity by micellar electrokinetic chromatography.

A quantitative ultraviolet detection method for determining ornithine transcarbamylase (OTC-ase) activity using micellar electrokinetic chromatography (MEKC) is described. The method is based on the direct determination of citrulline formed upon enzymatic reaction. Using a background electrolyte consisting of 35 mM sodium tetraborate, pH 9.3, containing 65 mM sodium dodecyl sulfate (SDS), the peak of citrulline in the electropherogram was easily identified and integrated. This allowed us to determine the rate of formation of the reaction product and to calculate the kinetic parameter Km of the OTC-ase investigated. The capillary electrophoretic method developed was applied to the determination of OTC-ase activity in plasma samples for citrulline in the nanogram range.

Micellar electrokinetic chromatography: a convenient alternative to colorimetric and high performance liquid chromatographic detection to monitor protease activity.

High performance capillary electrophoresis (HPCE) has been exploited as an analytical method alternative to current procedures for the determination of proteolytic activity of elastases from different sources. Due to some drawbacks with capillary zone electrophoresis (CZE), the mode of operation employed for the assay of elastolytic activity was micellar electrokinetic chromatography (MEKC). Using a background electrolyte consisting of 35 mM sodium tetraborate, pH 9.3, containing 65 mM SDS and 15% v/v methanol, separation of intact peptide substrate from products of proteolytic reaction was easily achieved in a fused-silica capillary of 50 cm effective length x 75 microm ID. This allowed us to determine the rate of hydrolysis of substrates and to calculate the kinetic parameters Km and k(cat) of the proteases investigated. A comparison of these data with those obtained from high performance liquid chromatography (HPLC)-based experiments showed that MEKC is a convenient technique for studying protease kinetics.

Micellar electrokinetic chromatography for the determination of urinary desmosine and isodesmosine in patients affected by chronic obstructive pulmonary disease.

The presence in urine of desmosine (DES) and isodesmosine (IDES), two crosslinked amino acids unique to the elastic fiber network, can be used as a specific indicator of degradation of mature elastin. Compared to methodologies so far available, the capillary electrophoretic technique reported here seems to be suitable and convenient for determining desmosines in urine of patients affected by chronic obstructive pulmonary disease (COPD). By using 35 mM sodium tetraborate pH 9.3 containing 65 mM SDS as the background electrolyte, the peaks of DES and IDES could be detected in hydrolyzed urine samples from controls and patients. Owing to the simultaneous determination of endogenous urinary creatinine used as appropriate internal standard, the amount of these amino acids could be accurately quantified. The results obtained were of the same order of magnitude as the data already reported in the literature for COPD patients. Thus micellar electrokinetic chromatography (MEKC) may be considered as a reliable technique for studying the turnover of the elastic fiber in clinical conditions.