The aim of this study was to investigate the usefulness of biochemical markers of bone turnover for monitoring treatment efficacy of Paget's disease of bone, and also to evaluate the utility of ...biological variation data in choosing the best markers for assessment of biochemical response to therapy. Thirty-eight patients with Paget's disease were included in a prospective study. All received 400 mg/day of oral tiludronate for 3 months. In 31 patients that completed treatment, biochemical markers were measured at baseline and at 1 and 6 months after treatment ended. In serum we determined the levels of total alkaline phosphatase (tAP), bone alkaline phosphatase (bAP), procollagen type I N-terminal propeptide (PINP), and C-terminal telopeptide of type I collagen (sCTx). Urine samples were analyzed for hydroxyproline (Hyp) and for C- and N-terminal telopeptides of type I collagen (CTx and NTx, respectively). Quantitative bone scintigraphy was performed at baseline and at 6 months after discontinuation of therapy. A ratio for monitoring response to treatment was obtained for each marker. This ratio reflected the size of treatment response of the marker in relation to the value of its critical difference. Thus, ratio values of >1 indicated a significant decrease of the marker after therapy. In addition, response to therapy was evaluated according to disease activity. Mean values of all markers of bone turnover decreased significantly after therapy. Serum bAP and PINP and urinary NTx showed the highest percentage reduction (between 58% and 68%). Furthermore, serum bAP and PINP showed the highest ratios for monitoring changes induced by treatment, followed by serum tAP and urinary NTx. sCTx and urinary CTx as well as Hyp showed mean ratios for monitoring changes of <1, indicating a low sensitivity for monitoring treatment. Patients with polyostotic disease showed a continuous decrease in mean values for all markers at 6 months from the end of therapy, whereas, in monostotic patients, there was a trend toward increased levels at this timepoint. In conclusion, serum bAP and PINP were the most sensitive markers for monitoring treatment efficacy in Paget's disease, although serum tAP and urinary NTx were also sensitive markers for monitoring changes. Data on biological variation are useful for assessing actual changes induced by treatment.
Summary
Background Keloid pathogenesis involves an altered balance of extracellular matrix metabolism, mainly accumulation of type I collagen. This could be due to excessive synthesis or decreased ...degradation of matrix, or a combination of both processes. Prolidase, an imidodipeptide‐cleaving cytosolic enzyme, plays an important role in the collagen catabolic process by recycling proline for collagen synthesis. Collagen accumulation in keloids is due to an imbalance in the steady state of collagen turnover.
Objectives To investigate prolidase activity and its role in the steady state of collagen turnover between normal skin and keloid tissue and their derived fibroblasts.
Methods Ten sets of keloid and normal skin tissues and their derived fibroblasts were employed. Measurements were made of tissue prolidase activity, free proline level, and concentrations of the collagen synthesis product aminoterminal propeptide of type I procollagen (PINP) and the collagen degradative product carboxyterminal telopeptide of type I collagen (ICTP). Also, synthesis of collagens type I and III and matrix metalloproteinases 1 and 2 was investigated using Western blot analysis.
Results Keloid tissues had a significant increase in prolidase activity, up to fourfold that in normal skin. The elevated prolidase activity was accompanied by an increase in tissue PINP and ICTP concentrations in keloid; in addition, the collagen turnover index (PINP/ICTP) was higher in keloids.
Conclusions The combination of elevated prolidase activity and associated higher collagen synthesis to degradation ratio in keloids suggests a possible metabolic process for the excessive accumulation of type I collagen in keloids.
The aims of this study were to evaluate the components of biological variation of the new markers of bone turnover in patients with Paget's bone disease and to compare the results with data obtained ...in healthy subjects. Fifteen patients with Paget's disease in a stable period of the disease and 12 healthy premenopausal women were included for a 1 year follow-up study. Within- and between-subject biological variation, indices of individuality, and critical differences were evaluated for the following biochemical markers: in serum, total (tAP), and bone (bAP) alkaline phosphatases, procollagen type I N-terminal propeptide (PINP) and beta-carboxyterminal telopeptide of type I collagen (sCTx); in urine, hydroxyproline (Hyp), and amino (NTx) and beta-carboxyterminal (CTx) telopeptides of collagen type I. Serum markers of bone turnover showed lower biological variability than urinary markers. Within-subject biological variation was higher in pagetic patients than in healthy subjects for all serum markers. In both groups, bAP presented the lowest within-subject biological variation. In pagetic patients, all markers presented indices of individuality of <0.6, indicating their usefulness for patient monitoring. Critical differences were lower for serum markers than for urinary markers. Among pagetic patients, serum bAP and PINP showed the lowest critical differences with values close to 30%, whereas urinary CTx presented the highest critical differences (near 70%). Conversely, in healthy subjects, tAP was the marker with the lowest critical differences, being two-fold higher in pagetic patients. This study confirms the lower sensitivity of urinary markers to detect significant changes and indicates that data obtained on biological variations from healthy populations cannot always be extrapolated to pathological conditions. In addition, serum bAP and PINP seem to be the markers that best reflect a significant change in activity of Paget's disease.
Cowpea bruchids, when challenged by consumption of the soybean cysteine protease inhibitor scN, reconfigure expression of their major CmCP digestive proteases and resume normal feeding and ...development. Previous evidence indicated that insects selectively induced CmCPs from subfamily B, that were more efficient in autoprocessing and possessed not only higher proteolytic, but also scN-degrading activities. In contrast, dietary scN only marginally up-regulated genes from the more predominant CmCP subfamily A that were inferior to subfamily B. To gain further molecular insight into this adaptive adjustment, we performed domain swapping between the two respective subfamily members B1 and A16, the latter unable to autoprocess or degrade scN even after intermolecular processing. Swapping the propeptides did not qualitatively alter autoprocessing in either protease isoform. Incorporation of either the N- (pAmBA) or C-terminal (pAmAB) mature B1 segment into A16, however, was sufficient to prime autoprocessing of A16 to its mature form. Further, the swap at the N-terminal mature A16 protein region (pAmBA) resulted in four amino acid changes. Replacement of these amino acid residues by the corresponding B1 residues, singly and pair-wise, revealed that autoprocessing activation in pAmBA resulted from cumulative and/or coordinated individual effects. Bacterially expressed isolated propeptides (pA16 and pB1) differed in their ability to inhibit mature B1 enzyme. Lower inhibitory activity in pB1 is likely attributable to its lack of protein stability. This instability in the cleaved propeptide is necessary, although insufficient by itself, for scN-degradation by the mature B1 enzyme. Taken together, cowpea bruchids modulate proteolysis of their digestive enzymes by controlling proCmCP cleavage and propeptide stability, which explains at least in part the plasticity cowpea bruchids demonstrate in response to protease inhibitors.
The single transmembrane receptor SorLA is the mammalian orthologue of the head activator-binding protein, HAB, from hydra. The human neuronal precursor cell line NT2 and the neuroendocrine cell line ...BON produce head activator (HA) and respond to HA by entry into mitosis and cell proliferation. They express SorLA, and bind HA with nanomolar affinity. HA coupled to Sepharose is able to precipitate SorLA specifically proving that SorLA binds HA. Using antisera directed against extra- and intracellular epitopes we find SorLA as membrane receptor and as soluble protein released from cells into the culture medium. Cell lines differ strongly in processing of SorLA, with NT2 cells expressing SorLA mainly as membrane receptor, whereas release predominates in BON cells. Soluble SorLA lacks the intracellular domain and is shed from the transmembrane protein by a metalloprotease. Release from cells and brain slices is stimulated by HA and by phorbol ester, and it is blocked by a metalloprotease inhibitor and by lowering the temperature to 20 degrees C. Blockade of SorLA shedding and treatment of cells with SorLA antisense oligonucleotides lead to a decrease in the rate of cell proliferation. From this we conclude that SorLA is necessary to mediate the mitogenic effect of endogenous HA. HA enhances the translocation of SorLA from internal membranes to the cell surface and its internalization. In addition, HA stimulates SorLA synthesis hinting at an autocatalytic feedback loop in which the ligand activates production, processing, and translocation of its receptor.
The human surfactant protein B (SP-B) is an small protein produced by the proteolytic processing of a precursor through elimination of two propeptides flanking the mature protein at its NH2- and ...COOH-termini, respectively. The human N-terminal propeptide (SP-BN) sequence has been cloned into the expression vector pMAL-c2x fusioned to maltose-binding protein. Expression of the fusion protein MBP-SP-BN has been achieved in Escherichia coli UT5600 cells after IPTG induction. The fusion protein was purified by affinity chromatography through an amylose resin column and identified by Western blot analysis with anti-MBP and anti-proSP-B antibodies. Proteolytic cleavage with Factor Xa followed by anion exchange chromatography allowed separation of the propeptide SP-BN from MBP. Mass spectrometry gave a molecular mass of 19883Da for the propeptide and its tryptic digestion produced peptides covering partly the propeptide sequence. An amount of 0.18mg of propeptide per L culture was obtained.
The hemopexin-like domain of membrane-type matrix metalloproteinase-1 (MT1-MMP) enables MT1-MMP to form oligomers that facilitate the activation of pro-matrix metalloproteinase-2 (pro-MMP-2) at the ...cell surface. To investigate the role of the MT1-MMP hemopexin domain in the trafficking of MT1-MMP to the cell surface we have examined the activity of two MT1-MT4-MMP chimaeras in which the hemopexin domain of MT1-MMP has been replaced with that of human or mouse MT4-MMP. We show that MT1-MMP bearing the hemopexin domain of MT4-MMP was incapable of activating pro-MMP-2 or degrading gelatin in cell based assays. Furthermore, cell surface biotinylation and indirect immunofluorescence show that transiently expressed MT1-MT4-MMP chimaeras failed to reach the plasma membrane and were retained in the endoplasmic reticulum. Functional activity could be restored by replacing the MT4-MMP hemopexin domain with the wild-type MT1-MMP hemopexin domain. Subsequent analysis with an antibody specifically recognising the propeptide of MT1-MMP revealed that the propeptides of the MT1-MT4-MMP chimaeras failed to undergo proper processing. It has previously been suggested that the hemopexin domain of MT4-MMP could exert a regulatory mechanism that prevents MT4-MMP from activating pro-MMP-2. In this report, we demonstrate unambiguously that MT1-MT4-MMP chimaeras do not undergo normal trafficking and are not correctly processed to their fully active forms and, as a consequence, they are unable to activate pro-MMP-2 at the cell surface.
The use of von Willebrand factor VWF antigen (WF:Ag) measurement as a marker of endothelial cell activation for monitoring hypertensive pregnancies is limited by the poor definition of reference ...values. We reassessed these reference values using different assays, and those of the propeptide (VWF:Ag II) and factor VIII coagulant activity (factor VIII:C), in a large population of normal pregnancies, at 3‐week intervals of gestational age. Plasminogen activator inhibitors (PAI‐1 and PAI‐2) were measured in parallel. Blood was collected at single time points between 12 weeks and delivery in 306 women undergoing normal singleton pregnancy. For clinical purposes, the VWF:Ag reference values were assay independent and the influence of ABO blood group on VWF:Ag or factor VIII:c was found to be limited during the third trimester. VWF:Ag II was not influenced by the ABO blood group. The ratio, VWF:Ag/factor VIII:C was close to 1·0 throughout pregnancy. In contrast, VWF:Ag II increased more slowly than VWF:Ag and the ratio of VWF:Ag II to VWF:Ag in plasma decreased from 1·00 to 0·5 at term. PAI‐1 and PAI‐2 increased with gestational age, but PAI‐2 decreased during the last 2 weeks, indicating physiological placental regression at the very end of pregnancy.
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