Search for η

We measured missing mass spectrum of the ^{12}C(γ,p) reaction for the first time in coincidence with potential decay products from η^{'} bound nuclei. We tagged an (η+p) pair associated with the η^{'}N→ηN process in a nucleus. After applying kinematical selections to reduce backgrounds, no signal events were observed in the bound-state region. An upper limit of the signal cross section in the opening angle cosθ_{lab}^{ηp}<-0.9 was obtained to be 2.2 nb/sr at the 90% confidence level. It is compared with theoretical cross sections, whose normalization ambiguity is suppressed by measuring a quasifree η^{'} production rate. Our results indicate a small branching fraction of the η^{'}N→ηN process and/or a shallow η^{'}-nucleus potential.

Evaluating the impacts of agricultural land management practices on water resources: A probabilistic hydrologic modeling approach.

Evaluating the effectiveness of agricultural land management practices in minimizing environmental impacts using models is challenged by the presence of inherent uncertainties during the model development stage. One issue faced during the model development stage is the uncertainty involved in model parameterization. Using a single optimized set of parameters (one snapshot) to represent baseline conditions of the system limits the applicability and robustness of the model to properly represent future or alternative scenarios. The objective of this study was to develop a framework that facilitates model parameter selection while evaluating uncertainty to assess the impacts of land management practices at the watershed scale. The model framework was applied to the Lake Creek watershed located in southwestern Oklahoma, USA. A two-step probabilistic approach was implemented to parameterize the Agricultural Policy/Environmental eXtender (APEX) model using global uncertainty and sensitivity analysis to estimate the full spectrum of total monthly water yield (WYLD) and total monthly Nitrogen loads (N) in the watershed under different land management practices. Twenty-seven models were found to represent the baseline scenario in which uncertainty of up to 29% and 400% in WYLD and N, respectively, is plausible. Changing the land cover to pasture manifested the highest decrease in N to up to 30% for a full pasture coverage while changing to full winter wheat cover can increase the N up to 11%. The methodology developed in this study was able to quantify the full spectrum of system responses, the uncertainty associated with them, and the most important parameters that drive their variability. Results from this study can be used to develop strategic decisions on the risks and tradeoffs associated with different management alternatives that aim to increase productivity while also minimizing their environmental impacts.

Fibulin-2 is involved in early extracellular matrix development of the outgrowing mouse mammary epithelium.

Cell-matrix interactions control outgrowth of mammary epithelium during puberty and pregnancy. We demonstrate here that the glycoprotein fibulin-2 (FBLN2) is strongly associated with pubertal and early pregnant mouse mammary epithelial outgrowth. FBLN2 was specifically localized to the cap cells of the terminal end buds during puberty and to myoepithelial cells during very early pregnancy (days 2-3) even before morphological changes to the epithelium become microscopically visible, but was down-regulated thereafter. Exposure to exogenous oestrogen (E2) or E2 plus progesterone (P) increased Fbln2 mRNA expression in the pubertal gland, indicating hormonal control. FBLN2 was co-expressed and co-localised with the proteoglycan versican (VCAN) and co-localised with laminin (LN), while over-expression of FBLN2 in HC-11 cells increased cell adhesion to several extracellular matrix proteins including LN and fibronectin, but not collagens. Mammary glands from Fbln2 knockout mice showed no obvious phenotype but increased fibulin-1 (FBLN1) staining was detected, suggesting a compensatory mechanism by other fibulin family members. We hypothesise that similar to embryonic aortic smooth muscle development, FBLN2 and VCAN expression alters the cell-matrix interaction to allow mammary ductal outgrowth and development during puberty and to enable epithelial budding during pregnancy.

Structure and expression of fibulin-2, a novel extracellular matrix protein with multiple EGF-like repeats and consensus motifs for calcium binding.

A new protein, fibulin-2, was predicted from sequence analysis of cDNA clones obtained from a mouse fibroblast library. This protein consists of a 1195-residue polypeptide preceded by a 26-residue signal peptide. The COOH-terminal region of 787 amino acids contained three anaphylatoxin-related segments (domain I), 11 EGF-like repeats (domain II), 10 of which had a consensus motif for calcium-binding, and a 115-residue globular domain III. Except for two additional EGF-like repeats, this COOH-terminal region showed 43% sequence identity with the previously described fibulin-1 (BM-90). The NH2-terminal 408 residues, unique to fibulin-2, showed no sequence homology to other known proteins and presumably form two additional domains that differ in their cysteine content. Recombinant fibulin-2 was produced and secreted by human cell clones as a disulfide-bonded trimer. Rotary shadowing visualized the protein as three 40-45 nm long rods which are connected at one end in a globe-like structure. No significant immunological cross-reaction could be detected between fibulin-1 and fibulin-2. Production of the fibulin-2 was demonstrated by Northern blots and radioimmunoassay in fibroblasts but not in several tumor cell lines. Together with the observation that the serum level of fibulin-2 is 1,000-fold lower than that of fibulin-1, the data indicate that these two isoforms are not always coordinately expressed. This is also suggested by Northern blots of tissue mRNAs and by immunofluorescence localizations using mouse tissues. The latter studies also demonstrated an extracellular localization for fibulin-2 in basement membranes and other connective tissue compartments.

Exon skipping mutations in collagen VI are common and are predictive for severity and inheritance.

Mutations in the genes encoding collagen VI (COL6A1, COL6A2, and COL6A3) cause Bethlem myopathy (BM) and Ullrich congenital muscular dystrophy (UCMD), two related conditions of differing severity. BM is a relatively mild dominantly inherited disorder characterized by proximal weakness and distal joint contractures. UCMD was originally regarded as an exclusively autosomal recessive condition causing severe muscle weakness with proximal joint contractures and distal hyperlaxity. We and others have subsequently modified this model when we described UCMD patients with heterozygous in-frame deletions acting in a dominant-negative way. Here we report 10 unrelated patients with a UCMD clinical phenotype and de novo dominant negative heterozygous splice mutations in COL6A1, COL6A2, and COL6A3 and contrast our findings with four UCMD patients with recessively acting splice mutations and two BM patients with heterozygous splice mutations. We find that the location of the skipped exon relative to the molecular structure of the collagen chain strongly correlates with the clinical phenotype. Analysis by immunohistochemical staining of muscle biopsies and dermal fibroblast cultures, as well as immunoprecipitation to study protein biosynthesis and assembly, suggests different mechanisms each for exon skipping mutations underlying dominant UCMD, dominant BM, and recessive UCMD. We provide further evidence that de novo dominant mutations in severe UCMD occur relatively frequently in all three collagen VI chains and offer biochemical insight into genotype-phenotype correlations within the collagen VI-related disorders by showing that severity of the phenotype depends on the ability of mutant chains to be incorporated in the multimeric structure of collagen VI.

Type 1 neurofibromatosis: selective expression of extracellular matrix genes by Schwann cells, perineurial cells, and fibroblasts in mixed cultures.

Cutaneous neurofibromas, characteristic lesions of neurofibromatosis 1, are composed of an abundant extracellular matrix and nerve connective tissue-derived cell types: Schwann cells, perineurial cells, and fibroblasts. In this study, the extracellular matrix gene expression by these cells was examined under culture conditions that allowed them to be metabolically active and readily identifiable by morphologic and immunocytochemical criteria. Northern hybridizations demonstrated expression of genes for type I, III, IV, and VI collagens, as well as for fibronectin, laminin, and elastin. In situ hybridizations revealed that all three cell types expressed pro alpha 1 (I), pro alpha 2 (VI), and laminin B1 chain genes. However, fibroblasts did not contain [35S]cDNA-mRNA hybrids specific for type IV collagen, whereas both Schwann cells and perineurial cells expressed these genes. Perineurial cells and fibroblasts readily expressed the fibronectin gene whereas Schwann cells were essentially devoid of the corresponding mRNA. Perineurial cells also expressed the gene for laminin A chain. The results indicate that the extracellular matrix gene expression profiles of Schwann cells, perineurial cells, and fibroblasts are distinct: all three cell types are capable of expressing some of the genes for extracellular matrix components, such as type I and VI collagens, whereas Schwann cells and perineurial cells may have the primary role in synthesizing basement membrane zone components, type IV collagen and laminin. These observations potentially relate to the mechanisms of growth and development of human neurofibromas. The results attest to the applicability of the methodology utilized here to study other human tumors with mixed cell populations.

Collagen gene expression by cultured human skin fibroblasts. Abundant steady-state levels of type VI procollagen messenger RNAs.

Previous studies have suggested that procollagen types I and III are the major collagenous gene products of cultured human skin fibroblasts. In this study the expression of 10 different genes, encoding the subunit polypeptides for collagen types I-VI, by human skin fibroblasts in culture was analyzed by molecular hybridizations. Northern transfer analysis demonstrated the presence of specific mRNA transcripts for collagen types I, III, IV, V, and VI, but not for type II collagen. Quantitation of the abundance of these mRNAs by slot blot hybridizations revealed that type I, III, and VI procollagens were the major collagenous gene products of skin fibroblasts in culture. The mRNAs for type IV and V collagens represented only a small percentage of the total collagenous mRNA transcripts. Further analysis by in situ hybridization demonstrated that the majority of the cultured cells coexpressed the genes for type I, III, and VI procollagen pro-alpha chains. Further in situ hybridization analyses revealed the expression of type VI collagen genes in normal human skin. These data demonstrate that human skin fibroblast cultures can be used to study the transcriptional regulation of at least nine genetically distinct procollagen genes. The data further suggest that type VI collagen, in addition to types I and III, may be a major collagenous component of human skin.

Modulation of procollagen gene expression by retinoids. Inhibition of collagen production by retinoic acid accompanied by reduced type I procollagen messenger ribonucleic acid levels in human skin fibroblast cultures.

Recent clinical observations have suggested that retinoids, which are in frequent use in dermatology, can affect the connective tissue metabolism in skin and other tissues. In this study, we examined the effects of several retinoids on the metabolism of collagen by human skin fibroblasts in culture. Incubation of cultured fibroblasts with all-trans-retinoic acid or 13-cis-retinoic acid, in 10(-5) M or higher concentrations, markedly reduced the procollagen production, as measured by synthesis of radioactive hydroxyproline. The effect was selective in that little, if any, inhibition was noted in the incorporation of [3H]leucine into the noncollagenous proteins, when the cells were incubated with the retinoids in 10(-5) M concentration. Similar reduction in procollagen production was noted with retinol and retinal, whereas an aromatic analogue of retinoic acid ethyl ester (RO-10-9359) resulted in a slight increase in procollagen production in these cultures. The reduction in procollagen production by all-trans-retinoic acid was accompanied by a similar reduction in pro alpha 2(I) of type I procollagen specific messenger RNA (mRNA), as detected by dot blot and Northern blot hybridizations. Hybridizations with human fibronectin and beta-actin specific DNA probes indicated that the levels of the corresponding mRNAs were not affected by the retinoids, further suggesting selectivity in the inhibition of procollagen gene expression. Further control experiments indicated that all-trans-retinoic acid, under the culture conditions employed, did not affect the posttranslational hydroxylation of prolyl residues, the mannosylation of newly synthesized procollagen, the specific radioactivity of the intracellular prolyltransfer RNA pool, or DNA replication. All-trans-retinoic acid also elicited a reduction in trypsin-activatable collagenase, but not in the activity of prolyl hydroxylase or an elastaselike neutral protease in the fibroblast cultures. Incubation of three fibroblast lines established from human keloids with all-trans-retinoic acid or 13-cis-retinoic acid also resulted in a marked reduction in procollagen production. The results, therefore, suggest that further development of retinoids might provide a novel means of modulating collagen gene expression in patients with various diseases affecting the connective tissues.

Perinatal lethality and endothelial cell abnormalities in several vessel compartments of fibulin-1-deficient mice.

The extracellular matrix protein fibulin-1 is a distinct component of vessel walls and can be associated with other ligands present in basement membranes, microfibrils, and elastic fibers. Its biological role was investigated by the targeted inactivation of the fibulin-1 gene in mice. This led to massive hemorrhages in several tissues starting at midgestation, ultimately resulting in the death of almost all homozygous embryos upon birth. Histological analysis demonstrated dilation and ruptures in the endothelial lining of various small vessels but not in that of larger vessels. Kidneys displayed a distinct malformation of glomeruli and disorganization of podocytes. A delayed development of lung alveoli suggested impairment in lung inflation. Immunohistology demonstrated the absence of fibulin-1 in its typical localizations but no aberrant patterns for several other extracellular matrix proteins. Electron microscopy revealed intact basement membranes but very irregular cytoplasmic processes of capillary endothelial cells in the organs that were most severely affected. Absence of fibulin-1 caused considerable blood loss but did not compromise blood clotting. The data indicate a strong but restricted abnormality in some endothelial compartments which, together with some kidney and lung defects, may be responsible for early death.

Automated genomic sequence analysis of the three collagen VI genes: applications to Ullrich congenital muscular dystrophy and Bethlem myopathy.

INTRODUCTION: Mutations in the genes encoding collagen VI (COL6A1, COL6A2, and COL6A3) cause Bethlem myopathy (BM) and Ullrich congenital muscular dystrophy (UCMD). BM is a relatively mild dominantly inherited disorder with proximal weakness and distal joint contractures. UCMD is an autosomal recessive condition causing severe muscle weakness with proximal joint contractures and distal hyperlaxity. METHODS: We developed a method for rapid direct sequence analysis of all 107 coding exons of the COL6 genes using single condition amplification/internal primer (SCAIP) sequencing. We have sequenced all three COL6 genes from genomic DNA in 79 patients with UCMD or BM. RESULTS: We found putative mutations in one of the COL6 genes in 62% of patients. This more than doubles the number of identified COL6 mutations. Most of these changes are consistent with straightforward autosomal dominant or recessive inheritance. However, some patients showed changes in more than one of the COL6 genes, and our results suggest that some UCMD patients may have dominantly acting mutations rather than recessive disease. DISCUSSION: Our findings may explain some or all of the cases of UCMD that are unlinked to the COL6 loci under a recessive model. The large number of single nucleotide polymorphisms which we generated in the course of this work may be of importance in determining the major phenotypic variability seen in this group of disorders.

Structure and multiple conformations of the kunitz-type domain from human type VI collagen alpha3(VI) chain in solution.

BACKGROUND: The Kunitz-type inhibitor motif is found at the C terminus of the human collagen alpha3(VI) chain. This 76-residue module (domain C5) was prepared in recombinant form and showed high stability against proteases; however, it lacked any inhibitory activity against trypsin, thrombin, kallikrein and several other proteases. We have undertaken the determination of the three-dimensional (3D) structure of domain C5 in solution, by nuclear magnetic resonance (NMR), in order to establish the structural basis for the properties of this protein. RESULTS: The 7 N-terminal and 12 C-terminal residues of domain C5 are disordered in the solution structure. The 55-residue core, which shows high homology to bovine pancreatic trypsin inhibitor, retains the characteristic fold of all members of the Kunitz-type inhibitor family. 24 residues of this main structural body show more than one resonance, symptomatic of multiple conformations slowly exchanging on the NMR time scale. In addition, significant proton chemical exchange line broadening is observed for residues in the vicinity of the disulfide bridge between residues 20 and 44: this indicates interconversion, on the micro- to millisecond time scale, between multiple conformations. CONCLUSION: The NMR study demonstrates that domain C5 is a highly dynamic molecule at temperatures studied (between 10 and 30 degrees C). Indeed, some 44% of the main body structure of C5 showed multiple conformations. The existence of multiple conformations was not necessarily expected in view of the conformational constraints imposed by the 3D structure of proteins as rigid as C5; it should therefore be considered in the interpretation of its structural and dynamical properties. The accessibility of the inhibitory binding loop (Gly18 [P4] to Leu25 [P4']) should be relatively unaffected by this conformational exchange and thus would not explain the unusual specificity of C5. Most serine proteinase inhibitors that, like C5, have an arginine at the P1 position inhibit trypsin; the lack of trypsin inhibition of C5 must therefore arise from the amino-acid side-chain composition of the adjoining positions in the binding loop.

Polymorphism in fowl serum albumin. VII. Distribution and activity of free and membrane-bound polysomes in developing fowl liver.

The quantity and activities of membrane-bound and free polysomes in livers from chick embryos at successive stages of development were compared in cell-free protein-synthesizing systems. Membrane-bound polysomes increased 2-fold between 8 and 18 days of development, while total ribosome content remained constant. Free polysome activity also remained constant during this period, while that of membrane-bound (total--free) polysomes decreased, possibly because of an increase in ribonuclease activity in this fraction. Serum albumin biosynthesis occurred primarily on membrane-bound polysomes. With liver development, increased secretion of serum proteins may be correlated with synthesis of serum albumin on increasing numbers of membrane bound polyribosomes.

Transcriptional and posttranscriptional regulation of fibulin-1 by estrogens leads to differential induction of messenger ribonucleic acid variants in ovarian and breast cancer cells.

Fibulin-1 is an extracellular matrix protein overexpressed in epithelial ovarian and breast cancers. In estrogen receptor (ER)-positive ovarian and breast cancer cell lines, fibulin-1 mRNA levels are markedly increased by estrogens. Transfection experiments using fibulin-1 promoter constructs indicate that 17beta-estradiol (E2) increases fibulin-1 gene transcription and that ERalpha is more potent than ERbeta to mediate E2 regulation of the transfected fibulin-1 promoter. Using SL2 cells devoid of specificity protein 1 (Sp1) and site-directed mutagenesis of GC boxes, we evidenced that the E2 regulation occurs through a proximal specificity protein 1 binding site. In addition, we show that fibulin-1C and -1D mRNAs, the two major fibulin-1 splicing variants, are differentially induced by E2. The induction of both mRNAs variants is direct and independent of a newly synthesized protein intermediate. Interestingly, actinomycin D chase experiments demonstrate that E2 treatment selectively shortens the fibulin-1D mRNA half-life. This indicates that estrogens affect differentially the stability of fibulin-1 variants and may explain the lower accumulation of fibulin-1D mRNA on E2 treatment. In conclusion, our data show that estrogens, via ERalpha, are key regulators of fibulin-1 expression at both the transcriptional and posttranscriptional levels. The preferential induction of the fibulin-1C variant, which is overexpressed in ovarian and breast cancer, might play an important role in estrogen-promoted carcinogenesis.

Binding of mouse and human fibulin-2 to extracellular matrix ligands.

Recombinant mouse and human fibulin-2 were obtained as disulfide-bonded trimers from transfected kidney cell clones and used in solid phase, biosensor and radioligand binding assays. Strong binding occurred with fibronectin and required calcium. A distinct interaction was also observed with nidogen but this was only partially blocked by EDTA. Distinctly weaker affinities were detected for collagen IV, perlecan and the N-terminal globule of collagen VI alpha 3 chain, while no or only little binding activity could be detected for several other collagen types, laminin-1, BM-40, fibulin-1 and vitronectin. This weaker binding reactions were also dependent on calcium. Surface plasmon resonance assays demonstrated for fibulin-2 binding to nidogen and fibronectin high equilibrium dissociation constants (0.5 to 1 microM) due to a rapid initial dissociation of the complexes. This is apparently followed by a slower stabilizing reaction. The fibulin-2 binding site of nidogen could be localized to its C-terminal globular domain G3, which also possesses a high-affinity binding site for laminin-1. Several tests demonstrated competition between the two ligands, probably due to steric hindrance. Binding of nidogen to immobilized fibulin-2 allowed the formation of ternary complexes with collagen IV, perlecan and fibulin-1, which, as shown previously, bind independently of the G3 domain. This indicated multifunctional binding properties for fibulin-2 and several alternative routes for its integration into basement membranes and other extracellular structures.

Structural characterization of two variants of fibulin-1 that differ in nidogen affinity.

Two C-terminal variants C and D of mouse fibulin-1 were purified from the culture medium of stably transfected human kidney cell clones. They showed, after rotary shadowing, a dumbbell-like structure of about 33 nm in length. Pepsin digestion demonstrated stability of the disulfide-bonded domains 1 (anaphylatoxin-like) and II (multiple EGF-like motifs) but not for domain III which is different in the variants. A close similarity of the variants was observed in immunochemical assays indicating that domain III epitopes are not very antigenic. Binding analysis in solid phase assays demonstrated for variant C a 100-fold stronger binding to the basement membrane protein nidogen than for variant D. Both interactions were sensitive to EDTA. Surface plasmon resonance assays confirmed this difference and showed KD = 60 nM for variant C and KD > 1 microM for variant D. Lower binding activities and smaller differences between both variants were observed for the calcium-dependent binding to fibronectin, laminin-1 and collagen IV. Self aggregation into nest-like oligomers was observed at high concentrations of fibulin-1 which was not sensitive to EDTA.

Binding of fibulin-1 to nidogen depends on its C-terminal globular domain and a specific array of calcium-binding epidermal growth factor-like (EG) modules.

The calcium-binding basement membrane protein fibulin-1C was shown to bind nidogen in a calcium-dependent fashion. Fibulin-1C consists of small N (domain 1) and C-terminal (domain III) globular structures connected by a central rod (domain II) composed of nine epidermal growth factor (EG) modules, eight of which possess a consensus sequence for calcium binding. Several point and deletion mutants and chimeric protein constructs were used to define the nidogen binding epitope of fibulin-1C by surface plasmon resonance and solid phase assays. All recombinant products were obtained from transfected kidney cells in a folded form as shown by CD spectroscopy, electron microscopy and proteolysis. They were used to demonstrate that calcium-binding is essentially due to the EG modules possessing the consensus binding sequence. Deletion of domain III caused a 30-fold reduction in nidogen binding, whereas deletion of domain I had no effect, yet domain III alone was also inactive. Successive deletions of two to seven EG modules of domain II also caused partial of complete inactivation of binding depending on how many were deleted or their position relative to domain III. Site-directed mutagenesis within the calcium binding consensus sequences demonstrated a similar dependence. Replacement of seven of the calcium-binding modules by a similar tandem array from a related protein showed a distinct (fibulin-2) to almost complete loss of binding (fibrillin-1). This indicates a complex epitope structure involving domains II and III, which each may provide binding epitopes or stabilize each other.

Effects of phenobarbital on leukocyte activation: membrane potential, actin polymerization, chemotaxis, respiratory burst, cytokine production, and lymphocyte proliferation.

Leukocyte activation is known to involve cell membrane potential changes. Phenobarbital, an anesthetic and anticonvulsant that can inhibit neuronal membrane depolarization, may also affect leukocyte activation. Measuring membrane potential, actin polymerization, chemotaxis, superoxide production, lymphocyte proliferation, intracellular calcium concentration, and cytokine production, we found that phenobarbital at a concentration of 15-30 micrograms/ml, which is considered a therapeutic serum level for controlling seizures, did not affect polymorphonuclear neutrophil (PMN) activation. At levels higher than 100 micrograms/ml, phenobarbital significantly suppressed formylmethionyl-leucyl-phenylalanine (fMLP)-induced chemotaxis. Concentrations greater than 300 micrograms/ml also inhibited phorbol myristate acetate-stimulated membrane potential change. In contrast, 30 micrograms/ml phenobarbital significantly inhibited lymphocyte proliferation stimulated by phytohemagglutinin (PHA) and pokeweed mitogen. This concentration of phenobarbital also suppressed the increase of intracellular free calcium induced by PHA. However, only a higher concentration of phenobarbital (300 micrograms/ml) was able to inhibit PHA-induced interleukin-2 (IL-2) production and suppress the proliferation of PHA-induced IL-2 receptor-bearing lymphocytes. These results suggest that concentrations of phenobarbital associated with anticonvulsive levels do not affect PMN activation but suppress lymphocyte activation, possibly by affecting intracellular signal transduction.

Expression of basement membrane zone genes coding for type IV procollagen and laminin by human skin fibroblasts in vitro: elevated alpha 1 (IV) collagen mRNA levels in lipoid proteinosis.

Basement membrane zone gene expression by fibroblast cultures established from adult human skin was examined by molecular hybridizations with human sequence-specific cDNAs corresponding to pro-alpha 1 (IV) chain of type IV collagen and B2 chain of laminin. Northern transfer analysis of poly(A)+RNA isolated from fibroblast cultures clearly revealed the presence of specific mRNA transcripts, indicating the expression of the genes coding for pro-alpha 1 (IV) and laminin B2 polypeptides. Quantitative estimates of the relative levels of these mRNAs coding for basement membrane zone components indicated that they are of relatively low abundance in control fibroblasts, as compared with mRNAs coding for fibronectin or pro-alpha 1 (I) chain of type I procollagen. In fibroblast cultures established from the lesional skin of a 32-year-old patient with lipoid proteinosis, the levels of mRNA coding for pro-alpha 1 (IV) polypeptides were increased approximately 4.5-fold, as compared with age- and passage-matched control cultures. This increase was selective in that the levels of fibronectin, pro-alpha 1 (I), and beta-actin mRNAs were unaltered in the same cultures. The increase in pro-alpha 1 (IV) mRNA level was also uncoordinate with the expression of the laminin B2 chain gene, which was unaltered in lipoid proteinosis. The selective increase in pro-alpha 1 (IV) mRNA may have relevance to the accumulation of this basement membrane component in the skin in lipoid proteinosis.

Regulation of collagen gene expression in cutaneous diseases with dermal fibrosis: evidence for pretranslational control.

Dermal fibrosis, characterized by collagen accumulation, is the hallmark of several cutaneous diseases. To examine the mechanisms of collagen deposition in fibrotic skin diseases, fibroblast cultures were established from the skin of patients with progressive systemic sclerosis, morphea, scleredema, familial cutaneous collagenoma, connective tissue nevi of the collagen type, or keloids; these patients served as prototypes of fibrotic skin diseases with varying clinical features and potentially different etiologic factors. Collagen production was assayed by the synthesis of [3H]hydroxyproline, and types I and III procollagen messenger RNA (mRNA) levels were determined by dot blot hybridizations using human type I and type III procollagen-specific cDNA probes. The collagen production in fibroblast cultures from the fibrotic diseases was increased up to 6-fold over the controls, and a relatively good correlation between the collagen production and type I collagen mRNA levels was noted. The type I/III procollagen mRNA ratio in control fibroblast cultures was 5.9 +/- 1.6 (mean +/- SD). The corresponding ratio in keloid cell culture was markedly increased, while slightly decreased values were noted in the case of morphea and familial cutaneous collagenoma; the values in other cultures were within the normal range. The results suggest that procollagen production in fibroblast cultures derived from fibrotic skin diseases reflects elevated levels of the corresponding procollagen mRNA. The increased mRNA abundance, suggesting pretranslational control, may result from enhanced transcriptional activity of the corresponding gene or alternatively reflects increased stability of the mRNA molecule.