Aminoterminal extension peptides from type I procollagen normalize excessive collagen synthesis of scleroderma fibroblasts

Fibroblasts derived from a skin biopsy of a patient with scleroderma in the sclerotic stage were shown to have a higher rate of DNA synthesis, and to synthesize more collagen than fibroblasts from a healthy control. The addition of procollagen peptides to the culture medium of scleroderma fibroblasts almost normalized the collagen synthesis. This observation indicates that the mechanism for the regulation of collagen synthesis by feed back inhibition of prollagen peptides is functioning in this disease. It is suggested that the level of biologically active procollagen peptides is lowered.     

Spatial distribution of collagen type I mRNA in Paracentrotus lividus eggs and embryos

We have identified the presence of type I collagen (COLL1alpha) mRNA in Paracentrotus lividus unfertilized egg, indicating a maternal origin of this mRNA. By in situ whole mount hybridization the spatial distribution of COLL1alpha mRNA in egg and embryo at different developmental stages was established. Moreover, the presence of COLL1alpha gene in Paracentrotus lividus genome was analyzed by Southern blot experiments. The localization pattern indicates that the maternal mRNA is placed in the fertilized egg in a fixed position, relative to the embryonic axes. Furthermore, the embryonic expression is spatially restricted during development, suggesting involvement in sea urchin embryo cell specification events. The presence of two bands in Southern blot hybridization may indicate that two genes specific for COLL1alpha are present in the sea urchin genome.     

The molecular basis of glucocorticoid-induced skin atrophy: topical glucocorticoid apparently decreases both collagen synthesis and the corresponding collagen mRNA level in human skin in vivo

To evaluate thrombopoiesis in thrombocytopenic disorders, we simultaneously determined reticulated platelet counts in whole blood by FACScan flow cytometry and serum thrombopoietin (TPO) concentrations by a sensitive sandwich ELISA. The subjects were 40 healthy volunteers and 45 thrombocytopenic patients. In idiopathic thrombocytopenic purpura (ITP), the percentage of reticulated platelets was significantly elevated (5.61 +/- 2.02%: mean +/- SD) relative to normal controls (2.17 +/- 0.90%), but serum TPO concentrations (1.91 +/- 1.27 fmol/l) did not differ significantly from the normal range (1.43 +/- 0.62 fmol/l). The patients with aplastic anemia (AA) had decreased reticulated platelet counts and markedly increased serum TPO concentrations (13.65 +/- 10.64 fmol/l). In thrombocytopenic patients with liver cirrhosis (LC), the absolute number of reticulated platelets (1.65 +/- 1.11 x 10(9)/l) decreased similarly that in AA. However, serum TPO concentrations (1.38 +/- 0.50 fmol/l) did not increase in contrast to AA. Our findings suggested a possible dual mechanism of thrombocytopenia in LC; that is, thrombocytopenia in LC results from the decreased TPO production primarily in the liver adding to an increase in platelet sequestration in the spleen.     

Cleavage of type I procollagen by human mast cell chymase initiates collagen fibril formation and generates a unique carboxyl-terminal propeptide

The ability of human mast cell chymase and tryptase to process procollagen was examined. Purified human intestinal smooth muscle cell procollagen was incubated with human mast cell tryptase or human mast cell chymase. Purified chymase, but not tryptase, exhibited procollagen proteinase activity in the presence of EDTA. Addition of purified porcine heparin over a range of 0.1-100 microg/ml did not affect either the rate or the products of procollagen chymase cleavage. The cleavage site of chymase on the pro-alpha1(I) collagen carboxyl terminus was found to be in the propeptide region at Leu-1248-Ser-1249. Cleavage at this site suggested that the collagen products would form fibrils and confirmed the production of a unique carboxyl-terminal propeptide. Turbidometric fibril formation assay demonstrated de novo formation of chymase-generated collagen fibrils with characteristic lag, growth, and plateau phases. When observed by dark field microscopy, these fibrils were similar to fibrils formed by the action of procollagen proteinases. Thus, mast cell chymase, but not tryptase, exhibits procollagen peptidase-like activity as evidenced by its ability to process procollagen to fibril-forming collagen with concurrent formation of a unique carboxyl-terminal propeptide. These data demonstrate that mast cell chymase has a potential role in the regulation of collagen biosynthesis and in the pathogenesis of fibrosis.     

The effect of tissue expansion on the expression of collagen type I and type III mRNA in distinct areas of skin in the dog as an animal model

PURPOSE: To reach an optimal treatment result and to avoid damage to critical structures a homogeneous dose distribution in the tumor volume with a rapid decreasing dose to the surrounding structures is necessary. Fractionated interstitial brachytherapy of tumors in the ENT region employing needles depends on exact localization of the target volume during all fractions. Therefore reproducibility of positioning of the needle(s) plays an important role. MATERIAL AND METHODS: We used the ISG Viewing Wand system in combination with the Vogele-Bale-Hohner (VBH) head holder and a new targeting device. Point of entrance, pathway, and target point of the needle were planned and insertion of the needle simulated in advance. To date we have treated 7 patients with inoperable tumors in the ENT region. The actual position of the needle in the control CT was compared to the planned position. RESULTS: The accuracy of positioning of the needle depended on the location of the tumor. In a patient with a recurrent retroorbital adenocarcinoma the mean accuracy was 1 mm. Due to soft tissue displacement in the neck region and the resulting necessity to readjust the targeting device the needle was placed with a mean deviation of 15 mm between the planned and the actual position. CONCLUSIONS: Computer-assisted frameless stereotactic interstitial brachytherapy allows for precise, reproducible and preplanned insertion of hollow needles into target structures closely adherent to the surrounding tissue, thus avoiding damage of neighbouring structures. This technique is of great advantage in treating deeply seated tumors which are fixed to bony structures, especially at the skull base. Inaccuracy in the neck region caused by soft tissue shift requires improvement of the immobilization in this region.     

Immunohistochemical video-microdensitometry of myocardial collagen type I and type III

Collagen is an essential part of the cardiac interstitium. Collagen subtypes, their location, total amount and the architecture of the fibrillar network are of functional importance. Architecture in terms of density of the fibrillar network is assumed to be reflected by the intensity of immunohistochemical staining of collagen. The aim of this study was to evaluate a video-based microdensitometric method for quantifying density expressed as absorbance of collagen subtypes I and III stained with an indirect immunoperoxidase method in myectomy specimens of patients with hypertrophic obstructive cardiomyopathy. Various factors influencing the immunohistochemical staining product and the technical properties of the image analysis system were investigated. Linearity between collagen concentration and the absorbance of the immunohistochemical staining product was demonstrated for collagen I using a dot-blot technique. Immunohistochemical collagen staining and density measurement were easily reproducible. The cardiac disability of the patients was assessed according to the New York Heart Association (NYHA) criteria. There was a significant increase in collagen type I density with higher NYHA class, whereas no significant association was found for total collagen area fraction. Thus, video-based microdensitometry gives further insight into the structural remodelling of myocardial collagens and reveals their significance in the process of heart failure in hypertrophic cardiomyopathy.     

Increased expression of heat-shock protein 47 is associated with overproduction of type I procollagen in systemic sclerosis skin fibroblasts

Albicidins are important factors in systemic pathogenesis by Xanthomonas albilineans, which causes the devastating leaf scald disease of sugar cane. They are also of substantial interest as antibiotics that selectively block prokaryote DNA replication. Albicidin biosynthesis is highly sensitive to medium composition. An optimized, chemically defined medium (SMG3) yielded 30-fold more albicidin from half the accumulated biomass, relative to sucrose peptone (SP) medium. Phosphate starvation stimulated albicidin production in SMG3 and SP media. Addition of other amino acids, ammonium ions or peptones to the defined medium increased the growth rate of X. albilineans XA3, but differentially inhibited albicidin biosynthesis. Knowledge of these factors indicates new approaches to understanding mechanisms of pathogenesis and resistance to sugar cane leaf scald disease, and to strain improvement for production of albicidin antibiotics.     

A consensus model for molecular packing of type I collagen

In this review, recent results from X-ray diffraction studies of tendon are used to develop an understanding of the molecular packing of type I collagen in tendon fibrils. These cover the definition of the unit cell as triclinic, the lateral architecture of molecular packing in a fibril and the molecular packing topology of a structure that gives good agreement with X-ray diffraction data. The proposed model is a 1D staggered left handed microfibril; the molecular orientation of the telopeptides indicates that there are interconnections between microfibrils that may explain the difficulty in isolating individual microfibrillar structures. This is the first structure that defines the absolute molecular packing of molecular segments based on X-ray diffraction data. These results are discussed in the light of direct and indirect evidence relating to molecular packing such as mineralization, natural crosslink position, and biomechanical evidence. The ability of the proposed structure to fulfill many of the structural and biochemical criteria point towards the structure providing a basis for a consensus model of collagen packing.     

Removal of amino-terminal and carboxy-terminal extension peptides from procollagen during synthesis of chick embryo tendon collagen

Plasma phytosterol (plant sterol) levels were studied in 26 infants on various commercial formulas, in 36 infants on breast or cow's milk formulas, in 101 normal and 22 hypercholesterolemic children on a free diet, and in 32 hypercholesterolemic children on a low-cholesterol diet. Commercial formulas, poor in animal fats and enriched with vegetable oils, and low-cholesterol, phytosterol-rich diets generally elevated total plasma phytosterol levels in infants and hypercholesterolemic children from normal mean levels of 2 mg/100 ml to about 9 mg/100 ml. The implications of long-term three- to five-fold elevations of the plasma phytosterols (campesterol, stigmasterol, beta-sitosterol) in infancy and childhood are unknown. Watchful prospective analysis of plasma phytosterol levels may be useful, particularly in regards to otherwise unanticipated long-term effects of cholesterol-poor, phytosterol rich diets.     

The regulation of collagen in fetal skin wounds: mRNA localization and analysis

The deposition of collagen in fetal skin wounds has been shown in several animal models. The authors used a radiolabeled RNA antisense probe, complementary to the mRNA for the alpha-1 chain of human procollagen type I, to assess regulation of this collagen species in fetal and adult rabbit wounds. Dorsal skin wounds were placed on fetal and maternal animals at the beginning of the third trimester, and were harvested 3, 5, and 7 days later. In situ RNA/RNA hybridization was performed on suitable specimens, and morphometric analysis was carried out with a computerized LECO image analyzer. Fetal wounds exhibited an inflow of mesenchymal cells that produced collagen type I at levels higher than the surrounding tissue; this activity was highest on days 3 and 5 after wounding. Adult wounds had increased fibroblast presence by day 7, producing collagen type I at levels higher than those of adjacent unwounded tissue. Morphometric analysis of the signal produced by in situ hybridization and of the number of cells producing the signal in a given field showed that fetal wounds appear to produce collagen type I by an increase in the number of cells in the area of the wound--not by induction of the gene for procollagen type I. In contrast, adult wounds had both fibroblast migration and induction of procollagen type I mRNA synthesis. These findings imply multilevel regulation of collagen production in the adult and posttranslational regulation in the fetus.     

Time-dependent changes of serum carboxy-terminal peptide of type I procollagen and carboxy-terminal telopeptide of type I collagen concentrations in patients with acute myocardial infarction after successful reperfusion: correlation with left ventricular volume indices

To test the hypothesis that in patients with acute myocardial infarction (AMI), changes in the concentrations of the serum carboxy-terminal peptide of type I procollagen (PICP) and the carboxy-terminal telopeptide of type I collagen (ICTP) reflect extracellular matrix reformation and degradation, respectively, in the infarct healing processes, we measured these serum concentrations by RIA and compared their values with left ventricular (LV) indices obtained by left ventriculography. We studied 13 consecutive patients with their first AMI who underwent successful reperfusion. Blood samples were taken the day of admission and on days 2, 3, 4, 5, 7, and 14. LV volume indices were determined at 1 month after AMI, when LV remodeling was almost completed. The serum concentrations of both PICP and ICTP changed in a time-dependent manner. The average serum PICP concentration was lower than 1 SD below the mean control values on days 2 and 3 and increased thereafter, returning to the lower end of the control range at day 14. The area under the curve (AUC) for PICP was significantly correlated with the LV end systolic (ES) and end diastolic (ED) volume indices and LV ejection fraction for the first 14 days after AMI. The serum PICP on days 5-14 was inversely correlated or tended to be correlated with the LVES and LVED volume indices. The average serum ICTP concentrations on admission were within the control range, began to increase on day 2, and reached maximal concentrations on day 5, remaining at a plateau concentration until day 14. Although the AUC of ICTP for 14 days, the ICTP concentrations on days 1 and 14, and the minimal and maximal concentrations were significantly correlated with creatine kinase (CK) release and the period from AMI onset to the peak CK time, the concentrations were not significantly correlated with any LV indices except for the concentration on day 4, which was weakly correlated with the LVES volume index. The serum concentrations of PICP showed a significant time-dependent change that correlated with LV indices, indicating that PICP may provide additional information for evaluating the healing process because it affects LV remodeling after AMI. Although the serum concentration of ICTP changed in association with CK release, the ICTP concentration was found to be a poor indicator for LV indices.     

Upregulation of IGF-I and collagen I mRNA in human atherosclerotic tissue is not accompanied by changes in type 1 IGF receptor or collagen III mRNA: an in situ hybridization study

BACKGROUND: Immunoreactive insulin-like growth factor-I (IGF-I) has recently been localized to vascular smooth muscle cells in coronary atherectomy plaques, but it remains unclear whether these cells are the source of this growth factor. We therefore investigated the gene expression of this factor, and the expression of the genes for its receptor and two types of collagen known to be regulated by IGF-I, in vascular tissue samples from patients with atherosclerosis or restenosis. METHODS: Gene expression and localization were investigated by in situ hybridization, using 35S-labelled complementary RNA probes specific for IGF-I, its type a receptor, collagen I, and collagen III. The cellular composition of the tissue samples was determined by immunohistochemistry using antibodies specific for smooth muscle cells and macrophages. RESULTS: IGF-I and collagen I messenger RNAs were found in areas containing smooth muscle cells and macrophages, but collagen III and type 1 IGF receptor gene expression could not be detected in any tissue samples. CONCLUSION: IGF-I appears to be involved in the progression of the atherosclerotic plaque, even at an advanced stage, but preliminary data from two restenotic plaques indicate that it may not be involved in the later stages of restenosis.     

Structure and biological activity of chemically modified nisin A species

Nisin, a 34-residue peptide bacteriocin, contains the less common amino acids lanthionine, beta-methyl-lanthionine, dehydroalanine (Dha), and dehydrobutyrine (Dhb). Several chemically modified nisin A species were purified by reverse-phase HPLC and characterized by two-dimensional NMR and electrospray mass spectrometry. Five constituents, [2-hydroxy-Ala5]nisin, [Ile4-amide,pyruvyl-Leu6]des-Dha5-nisin, [Met(O)21]nisin, [Ser33]nisin, and nisin-(1-32)-peptide amide, were found in a commercial nisin sample. A further species, [2-hydroxy-Ala5]nisin-(1-32)-peptide amide, was obtained by freeze drying an acidic nisin solution. These compounds are formed by chemical modification of nisin: the addition of a water molecule to the dehydroalanine residues, which can lead to the cleavage of the polypeptide chain, or the oxidation of methionine residues. The 2-hydroxyalanine-containing products have a limited stability; they are spontaneously converted into the corresponding des-dehydroalanine derivatives. The growth-inhibiting activity of the modified nisins towards different bacteria was determined. The 2-hydroxyalanine-containing species and the des-dehydroalanine derivative show a strong reduction in biological activity as compared to native nisin. [Met(O)21]nisin and [Ser33]nisin show moderate or no reduction in biological activity.     

Localization of carboxy-terminal type II procollagen peptide (pCOL-II-C) and type II collagen in the repair tissue of full-thickness articular cartilage defect

Apoptosis and aging share common mechanisms in oxidative stress and mitochondrial involvement. Treatment of cultured neuroblastoma cells with a radical initiator induced apoptosis; raise in hydrogen peroxide and release of cytochrome c from mitochondria preceded collapse of mitochondrial potential and cell death. In rat hepatocytes treated with adriamycin incubation with exogenous Coenzyme Q10 counteracted the drug-induced increase of hydrogen peroxide and the fall of the mitochondrial potential, thus demonstrating the quinone antioxidant effect. Complex I activity and its rotenone sensitivity decreased in brain cortex non-synaptic mitochondria from old rats; a 5 kb mitochondrial DNA deletion was found only in the old rats. A similar behavior was found in human platelets from old individuals. The postulated energy decline was confirmed by the inhibitor sensitivities of platelet aggregation and lactate production. The lack of the 5 kb deletion in platelets throws doubts on mitochondrial DNA lesions as the only causes of mitochondrial dysfunction in aging.     

Retinoic acid stimulates matrix calcification and initiates type I collagen synthesis in primary cultures of avian weight-bearing growth plate chondrocytes

The effect of retinoic acid (RA) on primary cultures of growth plate chondrocytes obtained from weight-bearing joints was examined, Chondrocytes were isolated from the tibial epiphysis of 6- to 8-week-old broiler-strain chickens and cultured in either serum-containing or serum-free media. RA was administered at low levels either transiently or continuously after the cells had become established in culture. Effects of RA on cellular protein levels, alkaline phosphatase (AP) activity, synthesis of proteoglycan (PG), matrix calcification, cellular morphology, synthesis of tissue-specific types of collagen, and level of matrix metalloproteinase (MMP) activity were explored. RA treatment generally increased AP activity and stimulated mineral deposition, especially if present continuously. RA also caused a shift in cell morphology from spherical/polygonal to spindle-like. This occurred in conjunction with a change in the type of collagen synthesized: type X and II collagens were decreased, while synthesis of type I collagen was increased. There was also a marked increase in the activity of MMP. Contrasting effects of continuous RA treatment on cellular protein levels were seen: they were enhanced in serum-containing media, but decreased in serum-free HL-1 media. Levels of RA as low as 10 nM significantly inhibited PG synthesis and caused depletion in the levels of PG in the medium and cell-matrix layer. Thus, in these appendicular chondrocytes, RA suppressed chondrocytic (PG, cartilage-specific collagens) and enhanced osteoblastic phenotype (cell morphology, type I collagen, alkaline phosphatase, and mineralization).     

Distribution and synthesis of type VII collagen in oral squamous cell carcinoma

Modern reproductive techniques and alternative family structures (with single or homosexual parents and adoption situations) raise questions about the consequences for the growing children involved. Genetic links appear to be less important for the functioning of a family than a strong wish for parenthood; parents who have become parents only through great efforts display a better quality of parenthood than average natural parents. Characteristics of the parent/parents, such as paedagogic qualities, and the quality of the parent-child relationship appear more important than the type of family. Published results of research reveal no reason why lesbian families should be judged differently from heterosexual ones as family types for the raising of children. The main negative factor for the functioning of the child growing up in a single-parent family is the marriage conflicts that have led to the single-parent situation; being raised by a single parent in itself has no adverse effect. Raising adopted children from other countries makes far greater demands on the adoptive parents than parents of biological children have to meet. The raising of a foreign adopted child by a single parent entails additional risks for the child's development. Data on the development of children in alternative family structures frequently concern exceptionally competent parents, which may have biased the findings.