Defective folding and stable association with protein disulfide isomerase/prolyl hydroxylase of type I procollagen with a deletion in the pro alpha 2(I) chain that preserves the Gly-X-Y repeat pattern.

We have studied the folding, processing, and association with two endoplasmic reticulum (ER) resident proteins of the abnormal type I procollagen molecules produced by a strain of fibroblasts harboring a 4.5 kilobase deletion in an allele of COL1A2 (Willing, M. C., Cohn, D.H., Starman, B. Holbrook, K.A., Greenberg, C.R., and Byers, P.H. (1988) J. Biol. Chem. 263, 8398-8404). By sequencing cDNA, we found that the mutant allele encodes pro alpha 2(I) chains that are shortened by 180 amino acids but retain the Gly-X-Y repeat pattern crucial for collagen triple helix formation. The type I procollagen molecules that incorporated the shortened chain were retained intracellularly and were stable. The triple helical domain in these molecules did not attain a normal conformation and remained accessible to posttranslational modifying enzymes amino-terminal to the deletion site for a prolonged period. The abnormal molecules folded into a triple helical conformation more slowly than the normal molecules, and the amino-terminal ends of the pro alpha 1(I) chains failed to become protease-resistant. While the abnormal procollagen molecules were not bound by the ER-resident protein BiP, they stably associated with protein disulfide isomerase, the beta-subunit of prolyl-4-hydroxylase. These results indicate that some mutations in type I collagen genes both transiently delay folding and permanently disrupt the structure of the triple helix and suggest that binding to prolyl-4-hydroxylase helps to retain certain abnormal procollagen molecules within the ER.     

In situ hybridization reveals that type I and III collagens are produced by pericytes in the anterior pituitary gland of rats

Type I and III collagens widely occur in the rat anterior pituitary gland and are the main components of the extracellular matrix (ECM). Although ECM components possibly play an important role in the function of the anterior pituitary gland, little is known about collagen-producing cells. Type I collagen is a heterotrimer of two α1(I) chains (the product of the col1a1 gene) and one α2(I) chain (the product of the col1a2 gene). Type III collagen is a homotrimer of α1(III) chains (the product of the col3a1 gene). We used in situ hybridization with digoxigenin-labeled cRNA probes to examine the expression of col1a1, col1a2, and col3a1 mRNAs in the pituitary gland of adult rats. mRNA expression for these collagen genes was clearly observed, and cells expressing col1a1, col1a2, and col3a1 mRNA were located around capillaries in the gland. We also investigated the possible double-staining of collagen mRNA and pituitary hormones, S-100 protein (a marker of folliculo-stellate cells), or desmin (a marker of pericytes). Col1a1 and col3a1 mRNA were identified in desmin-immunopositive cells. Thus, only pericytes produce type I and III collagens in the rat anterior pituitary gland.     

Processing of types I and III procollagen in Ehlers-Danlos syndrome type VII.

The processing of types I and III procollagen was studied in skin fibroblast cultures from type VII A and B of the Ehlers-Danlos syndrome [EDS] and age-matched controls. Synthesis of collagenous proteins was significantly increased in EDS type VII B, and the activities of prolyl-4-hydroxylase and galactosylhydroxylysyl glucosyltransferase were slightly increased in these cell lines, reflecting increased biosynthesis of collagen. The synthesis of collagenous proteins was close to normal in EDS type VII A cells. The synthesis of type III procollagen per cell was increased, as also was the ratio of immunoreactive type III procollagen to total collagen production. The activity of type I procollagen amino-terminal proteinase was decreased in skin fibroblasts of type VII A and normal in those of type VII B relative to cell protein or DNA. Type III amino-terminal proteinase activity was of a level found in normal cells when expressed relative to the protein or DNA, and the release of type III amino-terminal propeptides was nevertheless not disturbed in these EDS type VII cell cultures. The results show that only the conversion of type I procollagen is defective in EDS type VII, and no general defect in procollagen processing can be found in EDS type VII as has been suggested in the case of dermatosparaxis, a connective tissue disorder in animals caused by disturbed procollagen conversion.     

Accumulation of properly folded human type III procollagen molecules in specific intracellular membranous compartments in the yeast Pichia pastoris.

It was recently reported that co-expression of the proalpha1(III) chain of human type III procollagen with the subunits of human prolyl 4-hydroxylase in Pichia pastoris produces fully hydroxylated and properly folded recombinant type III procollagen molecules (Vuorela, A., Myllyharju, J., Nissi, R., Pihlajaniemi, T., Kivirikko, K.I., 1997. Assembly of human prolyl 4-hydroxylase and type III collagen in the yeast Pichia pastoris: formation of a stable enzyme tetramer requires coexpression with collagen and assembly of a stable collagen requires coexpression with prolyl 4-hydroxylase. EMBO J. 16, 6702-6712). These properly folded molecules accumulated inside the yeast cell, however, only approximately 10% were found in the culture medium. We report here that replacement of the authentic signal sequence of the human proalpha1(III) with the Saccharomyces cerevisiae alpha mating factor prepro sequence led only to a minor increase in the amount secreted. Immunoelectron microscopy studies indicated that the procollagen molecules accumulate in specific membranous vesicular compartments that are closely associated with the nuclear membrane. Prolyl 4-hydroxylase, an endoplasmic reticulum (ER) lumenal enzyme, was found to be located in the same compartments. Non-helical proalpha1(III) chains produced by expression without recombinant prolyl 4-hydroxylase likewise accumulated within these compartments. The data indicate that properly folded recombinant procollagen molecules accumulate within the ER and do not proceed further in the secretory pathway. This may be related to the large size of the procollagen molecule.     

Biosynthesis and degradation of collagen in X-irradiated mouse lung

Fibrosis, characterized by accumulation of collagen, is a delayed result of radiation injury in many tissues, including lung. To investigate its development, synthesis and degradation of collagen were measured in lungs of mice after X irradiation of the whole thorax. The ratio of type I (coarse fibered) to type III (meshwork) collagen was also determined. Synthesis of procollagen, measured as the activities of prolyl-4-hydroxylase and protein disulfide isomerase in lung tissue, was increased at 2 months after X-ray doses of 5, 7.5, and 9 Gy. Maximal increases were observed 6 to 7 months after doses of 9 Gy and persisted up to 15 months after exposure. Increases after 5 and 7.5 Gy were more gradual, but by 1 year after irradiation they had reached levels similar to those after 9 Gy. X irradiation had no effect on the degradation of collagen as assessed by collagenase activity in lung. The ratio of type I to type III collagen, analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis of collagen-derived cyanogen bromide peptides, was the same in irradiated lungs as in age-matched controls. Therefore, increased synthesis of procollagen, rather than decreased degradation of collagen or changes in collagen type, is an important factor in the accumulation of collagen in irradiated lung.     

dpy-18 encodes an alpha-subunit of prolyl-4-hydroxylase in caenorhabditis elegans

Collagen is an extracellular matrix (ECM) component encoded by a large multigene family in multicellular animals. Procollagen is post-translationally modified by prolyl-4-hydroxylase (EC 1.14.11.2) before secretion and participation in ECM formation. Therefore, collagen processing and regulation can be studied by examining this required interaction of prolyl-4-hydroxylase with procollagen. High-resolution polymorphism mapping was used to place the Caenorhabditis elegans dpy-18 gene on the physical map, and we show that it encodes a prolyl-4-hydroxylase alpha catalytic subunit. The Dpy phenotype of dpy-18(e364) amber mutants is more severe when this mutation is in trans to the noncomplementing deficiency tDf7, while the dpy-18(e499) deletion mutant exhibits the same phenotype as dpy-18(e499)/tDf7. Furthermore, dpy-18 RNA interference (RNAi) in wild-type worms results in Dpy progeny, while dpy-18 (RNAi) in dpy-18(e499) mutants does not alter the Dpy phenotype of their progeny. These observations suggest that the dpy-18 null phenotype is Dpy. A dpy-18::gfp promoter fusion construct is expressed throughout the hypodermis within the cells that abundantly produce the cuticle collagens, as well as in certain head and posterior neurons. While prolyl-4-hydroxylase has been studied extensively by biochemical techniques, this is the first report of a mutationally defined prolyl-4-hydroxylase in any animal.     

Epidermal growth factor increases collagen production in granulation tissue by stimulation of fibroblast proliferation and not by activation of procollagen genes.

The effects of epidermal growth factor (EGF) on granulation-tissue formation and collagen-gene expression were studied in experimental sponge-induced granulomas in rats. After daily administration of 5 micrograms of EGF into the sponge, total RNA was extracted from the ingrown granulation tissue at days 4 and 7 and analysed by Northern hybridization for the contents of mRNAs for types I and III procollagens. EGF treatment increased procollagen mRNA, particularly at day 4. To determine whether this elevation was due to increased proliferation of collagen-producing fibroblasts or to activation of collagen-gene expression in these cells, fibroblast cultures were started from granulation tissue and treated with EGF. These experiments confirmed that EGF is a potent mitogen for granuloma fibroblasts in a dose-dependent manner. The effect of EGF treatment on radioactive hydroxyproline production in cultured cells was inhibitory. The decreased rate of collagen synthesis was also indicated by decreased amounts of procollagen mRNAs. The results suggest that the stimulation of wound healing and collagen production by EGF is due to increased fibroblast proliferation, and not to increased expression of type I and III procollagen genes.     

Subcellular localization of procollagen I and prolyl 4-hydroxylase in corneal endothelial cells

To investigate the molecular mechanism of intracellular degradation of type I collagen in normal corneal endothelial cells (CEC), we studied the role of prolyl 4-hydroxylase (P4-H) and protein disulfide-isomerase (PDI; the beta subunit of P4-H) during procollagen I biosynthesis. When the subcellular localization of P4-H and PDI was determined, P4-H demonstrated a characteristic diffuse endoplasmic reticulum (ER) pattern, whereas PDI showed a slightly more restricted distribution within the ER. When colocalization of procollagen I with the enzymes was examined, procollagen I and PDI showed a large degree of colocalization. P4-H and procollagen I were predominantly colocalized at the perinuclear site. When colocalization of type IV collagen with PDI and P4-H was examined, type IV collagen was largely colocalized with PDI, which showed a wider distribution than type IV collagen. Type IV collagen is similarly colocalized with P4-H, except in some perinuclear sites. The colocalization profiles of procollagen I with both PDI and P4-H were not altered in cells treated with alpha,alpha'-dipyridyl compared to those of the untreated cells. The underhydroxylated type IV collagen demonstrated a colocalization profile with PDI similar to that observed with procollagen I, while the underhydroxylated type IV collagen was predominantly colocalized with P4-H at the perinuclear sites. Immunoblot analysis showed no real differences in the amounts of the beta subunit/PDI and the catalytic alpha subunit of P4-H in CEC compared to those of corneal stromal fibroblasts (CSF). When protein-protein association was determined, procollagen I was associated with PDI much more in CEC than it was in CSF, whereas type IV collagen showed no differential association specificity to PDI in both cells. Limited proteolysis of the newly synthesized intracellular procollagen I with pepsin showed that procollagen I in CEC was degraded by pepsin, whereas CSF contained type I collagen composed of alpha1(I) and alpha2(I). These findings suggest that procollagen I synthesized in CEC is not in triple helical conformation and that the improperly folded procollagen I may be preferentially associated with PDI before targeting to the intracellular degradation.     

Separate cis-acting DNA elements of the mouse pro-alpha 1(I) collagen promoter direct expression of reporter genes to different type I collagen-producing cells in transgenic mice

The genes coding for the two type I collagen chains, which are active selectively in osteoblasts, odontoblasts, fibroblasts, and some mesenchymal cells, constitute good models for studying the mechanisms responsible for the cell-specific activity of genes which are expressed in a small number of discrete cell types. To test whether separate genetic elements could direct the activity of the mouse pro-alpha 1(I) collagen gene to different cell types in which it is expressed, transgenic mice were generated harboring various fragments of the proximal promoter of this gene cloned upstream of the Escherichia coli beta-galactosidase gene. During embryonic development, X-gal staining allows for the precise identification of the different cell types in which the beta-galactosidase gene is active. Transgenic mice harboring 900 bp of the pro-alpha 1(I) proximal promoter expressed the transgene at relatively low levels almost exclusively in skin. In mice containing 2.3 kb of this proximal promoter, the transgene was also expressed at high levels in osteoblasts and odontoblasts, but not in other type I collagen-producing cells. Transgenic mice harboring 3.2 kb of the proximal promoter showed an additional high level expression of the transgene in tendon and fascia fibroblasts. The pattern of expression of the lacZ transgene directed by the 0.9- and 2.3-kb pro-alpha 1(I) proximal promoters was confirmed by using the firefly luciferase gene as a reporter gene. The pattern of expression of this transgene, which can be detected even when it is active at very low levels, paralleled that of the beta-galactosidase gene. These data strongly suggest a modular arrangement of separate cell-specific cis-acting elements that can activate the mouse pro-alpha(I) collagen gene in different type I collagen-producing cells. At least three different types of cell- specific elements would be located in the first 3.2 kb of the promoter: (a) an element that confers low level expression in dermal fibroblasts; (b) a second that mediates high level expression in osteoblasts and odontoblasts; and (c) one responsible for high level expression in tendon and fascia fibroblasts. Our data also imply that other cis- acting cell-specific elements which direct activity of the gene to still other type I collagen-producing cells remain to be identified.     

Laminin and collagen modulate expression of the small leucine-rich proteoglycan fibromodulin in rat anterior pituitary gland

The anterior pituitary is a complex organ consisting of five types of hormone-producing cells, non–hormone-producing cells such as folliculostellate (FS) cells and vascular cells (endothelial cells and pericytes). We have previously shown that FS cells and pericytes produce fibromodulin, a small leucine-rich proteoglycan (SLRP). SLRPs are major proteoglycans of the extracellular matrix (ECM) and are important in regulating cell signaling pathways and ECM assembly. However, the mechanism regulating fibromodulin expression in the anterior pituitary has not been elucidated. Here, we investigate whether fibromodulin expression is modulated by major anterior pituitary ECM components such as laminin and type I collagen. Using transgenic rats expressing green fluorescent protein (GFP) specifically in FS cells, we examine fibromodulin expression in GFP-positive (FS cells) and GFP-negative cells (e.g., pericytes, endocrine cells and endothelial cells). Immunostaining and Western blot analysis were used to assess protein expression in the presence and absence of laminin or type I collagen. We confirmed fibromodulin expression in the pituitary and observed the up-regulation of fibromodulin in FS cells in the presence of ECM components. However, neither laminin nor type I collagen affected expression in GFP-negative cells. This suggests that laminin and type I collagen support the function of FS cells by increasing fibromodulin protein expression in the anterior pituitary.     

Basement membrane-like matrix inhibits proliferation and collagen synthesis by activated rat hepatic stellate cells: evidence for matrix-dependent deactivation of stellate cells.

During liver fibrosis hepatic stellate cells become activated, transforming into proliferative myofibroblastic cells expressing type I collagen and alpha-smooth muscle actin. They become the major producers of the fibrotic neomatrix in injured liver. This study examines if activated stellate cells are a committed phenotype, or whether they can become deactivated by extracellular matrix. Stellate cells isolated from normal rat liver proliferated and expressed mRNA for activation markers, alpha-smooth muscle actin, type I procollagen and tissue inhibitor of metalloproteinases-1 following 5-7 day culture on plastic, but culture on Matrigel suppressed proliferation and mRNA expression. Activated stellate cells were recovered from plastic by trypsinisation and replated onto plastic, type I collagen films or Matrigel. Cells replated on plastic and type I collagen films proliferated and remained morphologically myofibroblastic, expressing alpha-smooth muscle actin and type I procollagen. However, activated cells replated on Matrigel showed <30% of the proliferative rate of these cells, and this was associated with reduced cellular expression of proliferating cell nuclear antigen and phosphorylation of mitogen-activated protein kinase in response to serum. Activated HSC replated on Matrigel for 3-7 days progressively reduced their expression of mRNA for type I procollagen and alpha-smooth muscle actin and both became undetectable after 7 days. We conclude that basement membrane-like matrix induces deactivation of stellate cells. Deactivation represents an important potential mechanism mediating recovery from liver fibrosis in vivo where type I collagen is removed from the liver and stellate cells might re-acquire contact with their normal basement membrane-like pericellular matrix.     

Role of the pro-α2(I) COOH-terminal region in assembly of type I collagen: Disruption of two intramolecular disulfide bonds in pro-α2(I) blocks assembly of type I collagen

Collagen biosynthesis is a complex process that begins with the association of three procollagen chains. A series of conserved intra- and interchain disulfide bonds in the carboxyl-terminal region of the procollagen chains, or C-propeptide, has been hypothesized to play an important role in the nucleation and alignment of the chains. We tested this hypothesis by analyzing the ability of normal and cysteine-mutated pro-α2(I) chains to assemble into type I collagen heterotrimers when expressed in a cell line (D2) that produces only endogenous pro-α1(I). Pro-α2(I) chains containing single or double cysteine mutations that disrupted individual intra- or interchain disulfide bonds were able to form pepsin resistant type I collagen with pro-α1(I), indicating that individual disulfide bonds were not critical for assembly of the pro-α2(I) chain with pro-α1(I). Pro-α2(I) chains containing a triple cysteine mutation that disrupted both intrachain disulfide bonds were not able to form pepsin resistant type I collagen with pro-α1(I). Therefore, disruption of both pro-α2(I) intrachain disulfide bonds prevented the production and secretion of type I collagen heterotrimers. Although none of the individual disulfide bonds is essential for assembly of the procollagen chains, the presence of at least one intrachain disulfide bond may be necessary as a structural requirement for chain association or to stabilize the protein to prevent intracellular degradation. J.Cell. Biochem. 71:233–242, 1998. © 1998 Wiley-Liss, Inc.     

Angiogenic potential of microvessel fragments established in three-dimensional collagen gels

Summary During angiogenesis, the microvasculature displays both vessel remodeling and expansion under the control of both cellular and extracellular influences. We have evaluated the role of angiogenic and angiostatic molecules on angiogenesis in anin vitro model that more appropriately duplicates the cellular and extracellular components of this process. Freshly isolated microvessel fragments from rat adipose tissue (RFMF) were cultured within three-dimensional collagen I gels. These fragments were characterized at the time of isolation and were composed of vessel segments observed in the microvasculature of fatin situ (i.e., arterioles, venules, and capillaries). Fragments also exhibited characteristic ablumenally associated cells including smooth muscle cells and pericytes. Finally, fragments were encased in an extracellular matrix composed of collagen type IV and collagen type I/III. The elongation of microvascular elements was subsequently evaluated using morphologic and immunocytochemical techniques. The proliferation, migration, and elongation of cellular elements in microvessel fragments from rat adipose tissue was dependent on initial fragment density, matrix density, and required serum. Inclusion of endothelial cell growth factors to microvessel fragments from rat adipose tissue 3-D cultures resulted in the accelerated elongation of tube structures and the expression of von Willebrand factor in cells constituting these tubes. Molecules with reported angiostatic capacity (e.g., transforming growth factor and hydrocortisone) inhibited vessel tube elongation. In vitro methods have been developed to evaluate numerous mechanisms associated with angiogenesis, including endothelial cell proliferation, migration, and phenotypic modulation. Microvascular endothelial cell fragments described in this study represent anin vitro population of cells that accurately duplicate thein vivo microcirculatory elements of fat. The proliferation of cells and elongation of microvascular elements subsequently observed in three-dimensional cultures provides anin vitro model of angiogenesis. Microvascular formation in this system results from pre-existing microvessel fragments unlike tube formation observed when cultured endothelial cells are placed in three-dimensional gels. This form of tube formation from cultured endothelium is more characteristic of vasculogenesis. Thus, the formation of microvascular elements from microvessel fragments provides the opportunity to examine the mechanisms regulating angiogenesis in anin vitro system amenable to precise experimental manipulation.     

Quantitative analysis of collagen expression in embryonic chick chondrocytes having different developmental fates

A quantitative determination of collagen expression was carried out in cultured chondrocytes obtained from a tissue that undergoes endochondral bone replacement (ventral vertebra) and one that does not (caudal sterna). The "short chain" collagen, type X is only expressed in the former while the other "short chain" collagen type IX, was primarily expressed in the latter. These two tissues also differ in that vertebral chondrocytes express moderate levels of both type I procollagen mRNAs which were translated into full length procollagen chains both in vivo and in vitro, while caudal sternal chondrocytes did not. The percent of collagen synthesis was about 50% in both cell types, but sternal cells expressed twice as much collagen as vertebral cells even though type II procollagen was more efficiently processed to alpha-chains in vertebral chondrocytes than in sternal chondrocytes. The number of type II procollagen mRNA molecules/cell was found to be about 2300 in vertebral chondrocytes and about 8000 in sternal cells, in good agreement with the results reported by Kravis and Upholt (Kravis, D., and Upholt, W. B. (1985) Dev. Biol. 108, 164-172). There were about 630 copies of type I procollagen mRNAs with an alpha 1/alpha 2 ratio of 1.6 in vertebral chondrocytes compared with 5100 copies and an alpha 1/alpha 2 ratio of 2.2 in osteoblasts, and less than 40 copies in sternal cells. Since the rate of type I collagen chain synthesis was 50 times greater in osteoblasts than in vertebral cells, type I procollagen mRNAs were about six times less efficiently translated in vertebral cells than in osteoblasts. The type I mRNAs in vertebral chondrocytes were polyadenylated and had 5' ends that were identical in osteoblasts, fibroblasts, and myoblasts. Moreover, type I mRNAs isolated from vertebral chondrocytes were translated into full length preprocollagen chains in vitro in rabbit reticulocyte lysates. Thus, chondrocytes isolated from cartilage tissues with different developmental fates differed quantitatively and qualitatively in total collagen synthesis, procollagen processing, and distribution of collagen types.     

Expression of contractile proteins α-actin and myosin of smooth muscle cells and of type IV collagen in human placenta at placental insufficiency in III trimester of pregnancy

Changes of expression of contractile proteins (smooth muscle cell α-actin and myosin) and of type IV collagen in villous stroma of human placenta were studied at the diagnosed placental insufficiency (PI) in III trimester of pregnancy. The study revealed pronounced disturbances of expression of contractile proteins and type IV collagen at PI. It is shown that in perivascular sheaths of vessels of stem and intermediate villi there is present a much greater amount of cells expressing smooth muscle actin and myosin. These cells are arranged by the denser concentric layers and more compactly than in norm and fill the intervascular space inside the villi. The width of perivascular sheaths of vessels is higher, while vascular lumens are lower than in norm. In terminal villi the capillary walls are thickened and the number of pericytes immunopositive against the smooth muscle cell α-actin and myosin as well as type IV collagen is increased. The change of synthesis of the cytoskeletal contractile proteins and type IV collagen is shown to lead to structural disturbances of villi of different types and of perivascular areas and vessels, which doubtlessly indicates their participation in pathogenesis of placental dysfunction and of disturbance of placental hemodynamics.     

Alteration of the kinetics of type I procollagen synthesis in human osteosarcoma cells by 1,25-dihydroxyvitamin D3

The kinetics of type I procollagen synthesis in a human osteosarcoma cell line, MG 63, were investigated after treatment with 1,25-dihydroxyvitamin D₃ (1,25-(OH)₂ D₃), a hormonal inducer of phenotypic differentiation. Pulse label and chase experiments demonstrated greatly enhanced production and more rapid reduction of intracellular procollagen molecules in the 1,25-(OH)₂ D₃–treated cells as compared to the nontreated case. After a chase for 1 h, labeled procollagen was reduced by nine-tenths in 1,25-(OH)₂ D₃–treated cells, while half of the radioactivity still remained in nontreated cells. The expression rate of type I collagen, which was examined by pulse label experiment, was elevated in association with an increase in the mRNA coding for the type I collagen α1 chain by 1,25-(OH)₂ D₃ treatment. However, the amount of intracellular procollagen present after 4 h continuous labeling was almost the same, independent of the 1,25-(OH)₂ D₃ treatment. Thus, we conclude that strage of the molecule was not affected. The results therefore suggest an increase in both the synthesis and secretion of type I collagen. The 1,25-(OH)₂ D₃ treatment was also found to induce the α subunit of prolyl 4-hydroxylase and to be associated with an elevated level of hydroxyproline in the procollagen. Moreover, gelatinase B–resistant procollagen molecules, indicative of intracellular procollagen molecules in the stable triple helical form, were detected only in the 1,25-(OH)₂ D₃–treated cells. These data suggest more efficient proline hydroxylation is involved in rapid secretion of procollagen after hormone administration. The present evidence points to posttranslational control of procollagen synthesis. J. Cell. Biochem. 65:542–549. © 1997 Wiley-Liss Inc.     

Desmin expression in fibroblasts of murine periovular granuloma during liver Schistosoma mansoni infection

We have studied the expression of the desmin gene, a muscle-specific intermediate filament protein in the granuloma cells of mouse liver infected with Schistosoma mansoni. In situ hybridization using a desmin DNA probe showed that fibroblastic cells in the granuloma strongly expressed desmin mRNAs, while in normal liver these cells did not express this mRNA to a detectable degree. The quantitative analysis of total RNAs demonstrated that the proportion of specific desmin mRNA increased from 14 to 18 weeks after infection and decreased at 20 weeks. The analysis of collagen gene expression indicated that the amount of type III collagen mRNAs was still increasing after 18 weeks from infection; in contrast, the amount of type I collagen mRNAs remained unchanged at that stage. A good correlation was observed between the detection of the specific mRNAs and the detection of both desmin and collagen molecules. Therefore, these data point to a coordinate induction of desmin and collagen gene expression during Schistosomal granuloma formation. They also suggest that the expression of the myofibroblast phenotype involves the induction of both genes.     

Analogues of dealanylalahopcin are inhibitors of human HIF prolyl hydroxylases

Analogues of the naturally occurring cyclic hydroxamate dealanylalahopcin, which is an inhibitor of procollagen prolyl-4-hydroxylase, were synthesised and shown to be inhibitors of the human hypoxia-inducible factor prolyl hydroxylases.